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Mitochondrial import and the twin-pore translocase
Abstract
The mitochondrial inner membrane is rich in multispanning integral membrane proteins, most of which mediate the vital transport of molecules between the matrix and the intermembrane space. The correct transport and membrane insertion of such proteins is essential for maintaining the correct exchange of molecules between mitochondria and the rest of the cell. Mitochondria contain several specific complexes — known as translocases — that translocate precursor proteins. Recent analysis of the inner-membrane, twin-pore protein translocase (TIM22 complex) allows a glimpse of the molecular mechanisms by which this machinery triggers protein insertion using the membrane potential as an external driving force.
... Successful localization of proteins to the IMM and matrix requires functionally and kinetically coupled reactions of the TOM and TIM complexes (2,3,63,68,96,102). Once the polypeptide traverses the OMM, a perfect temporal and spatial coupling of TOM-TIM23 complexes occurs sequentially for the import of preproteins with an N-terminal targeting signal and destined for IMM or matrix, and the TOM-TIM22 complex for carrier proteins of the IMM with an internal targeting sequence (2,3,5,6,8,9,62,68,96,103). Here, we describe the various preprotein import mechanisms of the TOM complex. ...
... After traversing the Tom40 pore, the preprotein is handed over to the hexameric IMS chaperones Tim9-Tim10 (Fig. 5B). Next, Tim12 (of the TIM22 complex) additionally forms a complex with Tim9-Tim10 by displacing one of the copies of Tim10 from Tim9-Tim10 (3,54,103). The chaperone complex additionally interacts with Tim54 of the TIM22 complex to complete transfer of the preprotein to the Tim22 channel of the TIM22 complex (Fig. 5B) (54,102,(124)(125)(126)(127)(128)(129)(130). ...
... Additional mechanistic details behind Tom70-MIM communication, and the precise signal sequence of OMM helices that is recognized by this complex, are yet to be deduced (3,5,9,103,142,158). . TOM-TIM interaction for protein import. ...
The human mitochondrial outer membrane is biophysically unique as it is the only membrane possessing transmembrane β-barrel proteins (mitochondrial outer membrane proteins, mOMPs) in the cell. The most vital of the three mOMPs is the core protein of the translocase of the outer mitochondrial membrane (TOM) complex. Identified first as MOM38 in Neurospora in 1990, the structure of Tom40, the core 19-stranded β-barrel translocation channel, was solved in 2017, after nearly three decades. Remarkably, the past four years have witnessed an exponential increase in structural and functional studies of yeast and human TOM complexes. In addition to being conserved across all eukaryotes, the TOM complex is the sole ATP–independent import machinery for nearly all of the ∼1000–1500 known mitochondrial proteins. Recent cryo–EM structures have provided detailed insight into both possible assembly mechanisms of the TOM core complex and organizational dynamics of the import machinery, and now reveal novel regulatory interplay with other mOMPs. Functional characterization of the TOM complex using biochemical and structural approaches has also revealed mechanisms for substrate recognition and at least five defined import pathways for precursor proteins. In this review, we discuss the discovery, recently solved structures, molecular function, and regulation of the TOM complex and its constituents, along with the implications these advances have for human diseases.
... Overall, the TIM22 carrier import pathway can be divided into five distinct and consecutive stages ( Figure 1) with different energy requirements, producing perceivable transport intermediates to be monitored in vitro [102]. The stages are described in yeast below but are thought to be very similar in humans. ...
... Next, ATP binding to the cytosolic chaperone triggers the release of the precursor and progression through the Tom40 channel. Importantly, the precursor can be arrested in Stage II by ATP depletion [102]. Interestingly, it is thought that carrier proteins are inserted into the Tom40 channel with both termini remaining in the cytosol, in a looplike formation [107]. ...
... However, experimental data where Δψ was dissipated showed the accumulation of two distinct populations, suggesting that the following stages, namely insertion, are Δψ-dependent, and that Stage III is further divided in two sub-stages. Stage IIIa represents the precursor deeply inserted in the TOM complex and protected from exogenous proteases [102]. Stage IIIb represents a fully translocated precursor across the OMM, tethered to the TIM22-bound TIM chaperone complex (Tim9-Tim10-Tim12) via hydrophobic interactions [102]. ...
The fact that >99% of mitochondrial proteins are encoded by the nuclear genome and synthesised in the cytosol renders the process of mitochondrial protein import fundamental for normal organelle physiology. In addition to this, the nuclear genome comprises most of the proteins required for respiratory complex assembly and function. This means that without fully functional protein import, mitochondrial respiration will be defective, and the major cellular ATP source depleted. When mitochondrial protein import is impaired, a number of stress response pathways are activated in order to overcome the dysfunction and restore mitochondrial and cellular proteostasis. However, prolonged impaired mitochondrial protein import and subsequent defective respiratory chain function contributes to a number of diseases including primary mitochondrial diseases and neurodegeneration. This review focuses on how the processes of mitochondrial protein translocation and respiratory complex assembly and function are interlinked, how they are regulated, and their importance in health and disease.
... Import of these proteins takes place in a well-orchestrated manner -first, the internal import signals of the precursor proteins are recognised by Tom70 and transferred to the central receptor of the TOM complex, Tom22. Second, the precursors translocate across the OMM through the Tom40 channel in a hairpin loop conformation, in a way such that the middle portion passes through it while both termini still remain in the cytosol [Wiedemann et al., 2001;Endo et al., 2003;Koehler, 2004;Rehling et al., 2004]. The N-terminus of the Tom40 channel recruits small TIM chaperones of the IMS to the (A) Cleavable pre-sequence-containing precursors are transferred from TOM to the pre-sequence translocase of the IMM (TIM23 complex). ...
... These chaperones associate with the TIM22 complex via Tim12 [Rehling et al., 2003;Zhang et al., 2020]. TIM22 receives the carrier precursors from the small TIM chaperones and subsequently conciliates protein insertion into the IMM [Endo et al., 2003;Rehling et al., 2004]. The difference in membrane potential across the IMM acts as the driving force that promotes the insertion process through the TIM22 complex [Jensen and Dunn, 2002;Koehler, 2004;Rehling et al., 2004;Brandner et al., 2005]. ...
... TIM22 receives the carrier precursors from the small TIM chaperones and subsequently conciliates protein insertion into the IMM [Endo et al., 2003;Rehling et al., 2004]. The difference in membrane potential across the IMM acts as the driving force that promotes the insertion process through the TIM22 complex [Jensen and Dunn, 2002;Koehler, 2004;Rehling et al., 2004;Brandner et al., 2005]. While Tim22 is the pore-forming core subunit of the translocase, other proteins of this complex, like Tim54 interacts with Tim9-Tim10-Tim12 complex. ...
Mitochondria are organelles involved in various functions related to cellular metabolism and homeostasis. Though mitochondria contain own genome, their nuclear counterparts encode most of the different mitochondrial proteins. These are synthesized as precursors in the cytosol and have to be delivered into the mitochondria. These organelles hence have elaborate machineries for the import of precursor proteins from cytosol. The protein import machineries present in both mitochondrial membrane and aqueous compartments show great variability in pre‐protein recognition, translocation and sorting across or into it. Mitochondrial protein import machineries also interact transiently with other protein complexes of the respiratory chain or those involved in the maintenance of membrane architecture. Hence mitochondrial protein translocation is an indispensable part of the regulatory network that maintains protein biogenesis, bioenergetics, membrane dynamics and quality control of the organelle. Various stress conditions and diseases that are associated with mitochondrial import defects lead to changes in cellular transcriptomic and proteomic profiles. Dysfunction in mitochondrial protein import also causes over‐accumulation of precursor proteins and their aggregation in the cytosol. Multiple pathways may be activated for buffering these harmful consequences. Here we present a comprehensive picture of import machinery and its role in cellular quality control in response to defective mitochondrial import. We also discuss the pathological consequences of dysfunctional mitochondrial protein import in neurodegeneration and cancer.
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... were grouped in a particular MQC mechanism, as in Figure 1, [13][14][15][16] although they may participate in several mechanisms. Samples of myocardium from human left ventricles were first homogenized in TRIzol (Thermo Fisher Scientific) and the RNA was extracted according to the manufacturer's instructions. ...
... UPRmt can block mitochondrial protein import via TIM, 8 which is made of two translocons, TIM23 and TIM22 ( Figure 5A). 16 Except for TIMM50, whose mRNA expresison was not altered in ICM and DCM, all other tested TIM mRNAs displayed significantly lower expression in ICM and DCM than in control samples ( Figure 5). We observed decreased expression of mRNAs in the TIM23 complex: TIMM23, TIMM17A, TIMM17B, TIMM44 and PAM16 (codingTIM16), as well as TIM22 complex: TIMM22, TIMM9 and TIMM10, including both channel-forming elements, that is, TIMM23 and TIMM22 ( Figure 5B,H). ...
Background:
Mitochondrial dysfunction is one of key factors causing heart failure. We performed a comprehensive analysis of expression of mitochondrial quality control (MQC) genes in heart failure.
Methods:
Myocardial samples were obtained from patients with ischemic and dilated cardiomyopathy in a terminal stage of heart failure and donors without heart disease. Using quantitative real-time PCR, we analysed a total of 45 MQC genes belonging to mitochondrial biogenesis, fusion-fission balance, mitochondrial unfolded protein response (UPRmt), translocase of the inner membrane (TIM) and mitophagy. Protein expression was analysed by ELISA and immunohistochemistry.
Results:
The following genes were downregulated in ischemic and dilated cardiomyopathy: COX1, NRF1, TFAM, SIRT1, MTOR, MFF, DNM1L, DDIT3, UBL5, HSPA9, HSPE1, YME1L, LONP1, SPG7, HTRA2, OMA1, TIMM23, TIMM17A, TIMM17B, TIMM44, PAM16, TIMM22, TIMM9, TIMM10, PINK1, PARK2, ROTH1, PARL, FUNDC1, BNIP3, BNIP3L, TPCN2, LAMP2, MAP1LC3A and BECN1. Moreover, MT-ATP8, MFN2, EIF2AK4 and ULK1 were downregulated in heart failure from dilated, but not ischemic cardiomyopathy. VDAC1 and JUN were only genes that exhibited significantly different expression between ischemic and dilated cardiomyopathy. Expression of PPARGC1, OPA1, JUN, CEBPB, EIF2A, HSPD1, TIMM50 and TPCN1 was not significantly different between control and any form of heart failure. TOMM20 and COX proteins were downregulated in ICM and DCM.
Conclusions:
Heart failure in patients with ischemic and dilated cardiomyopathy is associated with downregulation of large number of UPRmt, mitophagy, TIM and fusion-fission balance genes. This indicates multiple defects in MQC and represents one of potential mechanisms underlying mitochondrial dysfunction in patients with heart failure.
... In mitochondria, mortalin performs two specific roles: as a chaperone and stress-survival factor, it assists in protein quality control by (re)folding or degrading non-functional proteins [138][139][140], and as an essential component of the presequence translocase-associated motor (PAM), it binds precursor proteins to promote their unidirectional [116,138,141,142] translocation across the two mitochondrial membranes and into the mitochondrial matrix [142]. An excellent review by Pfanner et al. [143] provides a more detailed characterization of the biogenesis of mitochondrial proteins. ...
... In mitochondria, mortalin performs two specific roles: as a chaperone and stress-survival factor, it assists in protein quality control by (re)folding or degrading non-functional proteins [138][139][140], and as an essential component of the presequence translocase-associated motor (PAM), it binds precursor proteins to promote their unidirectional [116,138,141,142] translocation across the two mitochondrial membranes and into the mitochondrial matrix [142]. An excellent review by Pfanner et al. [143] provides a more detailed characterization of the biogenesis of mitochondrial proteins. ...
Since their discovery, heat shock proteins (HSPs) have been identified in all domains of life, which demonstrates their importance and conserved functional role in maintaining protein homeostasis. Mitochondria possess several members of the major HSP sub-families that perform essential tasks for keeping the organelle in a fully functional and healthy state. In humans, the mitochondrial HSP70 chaperone system comprises a central molecular chaperone, mtHSP70 or mortalin (HSPA9), which is actively involved in stabilizing and importing nuclear gene products and in refolding mitochondrial precursor proteins, and three co-chaperones (HSP70-escort protein 1—HEP1, tumorous imaginal disc protein 1—TID-1, and Gro-P like protein E—GRPE), which regulate and accelerate its protein folding functions. In this review, we summarize the roles of mitochondrial molecular chaperones with particular focus on the human mtHsp70 and its co-chaperones, whose deregulated expression, mutations, and post-translational modifications are often considered to be the main cause of neurological disorders, genetic diseases, and malignant growth.
... To this end, the mitochondria are equipped with a very elaborate and highly specific protein import machinery hardwired on translocase complexes embedded in the OMM and IMM. These translocases work along five different protein import pathways [80,82,95]. Most mitochondrial precursor proteins have a targeting sequence necessary for their mitochondrial entry and correct addressing to their final mitochondrial compartments [80,82,95]. ...
... These translocases work along five different protein import pathways [80,82,95]. Most mitochondrial precursor proteins have a targeting sequence necessary for their mitochondrial entry and correct addressing to their final mitochondrial compartments [80,82,95]. The TOM40 channel, the core subunit of the translocase of the outer mitochondrial membrane (TOM) complex, is the mitochondrial entry gate [96][97][98]. ...
SAM50, a 7-8 nm diameter β-barrel channel of the mitochondrial outer membrane, is the central channel of the sorting and assembly machinery (SAM) complex involved in the biogenesis of β-barrel proteins. Interestingly, SAM50 is not known to have channel translocase activity; however, we have recently found that this channel is necessary and sufficient for mitochondrial entry of cytotoxic proteases. Cytotoxic lymphocytes eliminate cells that pose potential hazards, such as virus- and bacteria-infected cells as well as cancer cells. They induce cell death following the delivery of granzyme cytotoxic proteases into the cytosol of the target cell. Although granzyme A and granzyme B (GA and GB), the best characterized of the five human granzymes, trigger very distinct apoptotic cascades, they share the ability to directly target the mitochondria. GA and GB do not have a mitochondrial targeting signal, yet they enter the target cell mitochondria to disrupt respiratory chain complex I and induce mitochondrial reactive oxygen species (ROS)-dependent cell death. We found that granzyme mitochondrial entry requires SAM50 and the translocase of the inner membrane 22 (TIM22). Preventing granzymes' mitochondrial entry compromises their cytotoxicity, indicating that this event is unexpectedly an important step for cell death. Although mitochondria are best known for their roles in cell metabolism and energy conversion, these double-membrane organelles are also involved in Ca2+ homeostasis, metabolite transport, cell cycle regulation, cell signaling, differentiation, stress response, redox homeostasis, aging, and cell death. This multiplicity of functions is matched with the complexity and plasticity of the mitochondrial proteome as well as the organelle's morphological and structural versatility. Indeed, mitochondria are extremely dynamic and undergo fusion and fission events in response to diverse cellular cues. In humans, there are 1,500 different mitochondrial proteins, the vast majority of which are encoded in the nuclear genome and translated by cytosolic ribosomes, after which they must be imported and properly addressed to the right mitochondrial compartment. To this end, mitochondria are equipped with a very sophisticated and highly specific protein import machinery. The latter is centered on translocase complexes embedded in the outer and inner mitochondrial membranes working along five different import pathways. We will briefly describe these import pathways to put into perspective our finding regarding the ability of granzymes to enter the mitochondria.
... Whereas the mitochondrial genome encodes only a few proteins (8 in S. cerevisiae [24], 13 in humans [25,26] and 21 in N. crassa [27]), over 1000 proteins [28,29] are required for mitochondrial function, so most have to be imported from the cytosol. The TOM complex has been shown to play a central role in the recognition of most mitochondrial proteins which are nuclear-encoded [30] and serve as the main entry gate for proteins to be targeted to either the OMM, IMS, IMM or mitochondrial matrix [29,[31][32][33]. TOM complex function is also strictly regulated [34,35]. ...
To date, there is no general physical model of the mechanism by which unfolded polypeptide chains with different properties are imported into the mitochondria. At the molecular level, it is still unclear how transit polypeptides approach, are captured by the protein translocation machinery in the outer mitochondrial membrane, and how they subsequently cross the entropic barrier of a protein translocation pore to enter the intermembrane space. This deficiency has been due to the lack of detailed structural and dynamic information about the membrane pores. In this review, we focus on the recently determined sub-nanometer cryo-EM structures and our current knowledge of the dynamics of the mitochondrial two-pore outer membrane protein translocation machinery (TOM core complex), which provide a starting point for addressing the above questions. Of particular interest are recent discoveries showing that the TOM core complex can act as a mechanosensor, where the pores close as a result of interaction with membrane-proximal structures. We highlight unusual and new correlations between the structural elements of the TOM complexes and their dynamic behavior in the membrane environment.
... However, real-world systems are subject to biological activity and external disturbance, resulting in timevarying environments. For instance, the twin-pore translocase complex in the inner membrane of mitochondria [125] and the nuclear pore complex [126] exhibit periodic changes in channel width during the translocation process. In the realm of biological respiration, the environments hosting active polymers often undergo periodic fluctuations. ...
Active matter systems, which convert internal chemical energy or energy from the environment into directed motion, are ubiquitous in nature and exhibit a range of emerging non-equilibrium behaviors. However, most of the current works on active matter have been devoted to particles, and the study of active polymers has only recently come into the spotlight due to their prevalence within living organisms. The intricate interplay between activity and conformational degrees of freedom gives rise to novel structural and dynamical behaviors of active polymers. Research in active polymers remarkably broadens diverse concepts of polymer physics, such as molecular architecture, dynamics, scaling and so on, which is of significant importance for the development of new polymer materials with unique performance. Furthermore, active polymers are often found in strongly interacting and crowded systems and in complex environments, so that the understanding of this behavior is essential for future developments of novel polymer-based biomaterials. This review thereby focuses on the study of active polymers in complex and crowded environments, and aims to provide insights into the fundamental physics underlying the adaptive and collective behaviors far from equilibrium, as well as the open challenges that the field is currently facing.
... There are different scenarios in biological nanopores such as the twin pore complex in the inner membrane of mitochondria [42] and the nuclear pore complex (NPC) [43], where the pore size can change during protein translocation. The NPC serves as the gatekeeper between the cell nucleus and the cytoplasm, and its cylindrical structure is highly dynamic both spatially and temporally. ...
We study the driven translocation of a semiflexible polymer through an attractive extended pore with a periodically oscillating width. Similar to its flexible counterpart, a stiff polymer translocates through an oscillating pore more quickly than a static pore whose width is equal to the oscillating pore's mean width. This efficiency quantified as a gain in the translocation time, highlights a considerable dependence of the translocation dynamics on the stiffness of the polymer and the attractive nature of the pore. The gain characteristics for various polymer stiffness exhibit a trend reversal when the stickiness of the pore is changed. The gain reduces with increasing stiffness for a lower attractive strength of the pore, whereas it increases with increasing stiffness for higher attractive strengths. Such a dependence leads to the possibility of a high degree of robust selectivity in the translocation process.
... Proteins need to be unfolded when translocated through the pores in cellular membranes. The radii of mitochondrial or proteasome pores are generally larger than 6 Å, [125,126] while the radius of gyration of a tight trefoil knot is estimated to be 7−8 Å and more complex knots are even larger. [27] As a consequence, knotted proteins could get stuck during this process. ...
The translocation of a polymer through a pore that is much smaller than its size is a fundamental and actively researched topic in polymer physics. An understanding of the principles governing polymer translocation provides important guidance for various practical applications, such as the separation and purification of polymers, nanopore-based single-molecule deoxyribonucleic acid/ribonucleic acid(DNA/RNA) sequencing, transmembrane transport of DNA or RNA, and infection of bacterial cells by bacteriophages. The past several decades have seen great progresses on the study of polymer translocation. Here we present an overview of theoretical, experimental, and simulational stduies on polymer translocation, focusing on the roles played by several important factors, including initial polymer conformations, external fields, polymer topology and architectures, and confinement degree. We highlight the physical mechanisms of different types of polymer translocations, and the main controversies about the basic rules of translocation dynamics. We compare and contrast the behaviors of force-induced versus flow-induced translocations and the effects of unknotted versus knotted polymers. Finally, we mention several opportunities and challenges in the study of polymer translocation.
... Los complejos TOM y TIM22 trabajan en forma conjunta en cinco pasos que se describen a continuación (Figura 2, flechas lavanda; Pfanner & Neupert, 1987;Rehling, Brandner & Pfanner, 2004): i) el precursor mitocondrial es llevado hasta el complejo TOM por chaperonas presentes en el citosol (Young, Hoogenraad & Hartl, 2003) donde ii) interacciona con el receptor Tom70, que reconoce las secuencias internas en los precursores (Brix et al., 2000;Wiedemann, Pfanner & Ryan, 2001;Young et al., 2003), iii) al cruzar la membrana externa mitocondrial, el sustrato es recibido por las chaperonas Tim9 3 -Tim10 3 que Vol. 24 https://doi.org/10.22201/fesz.23958723e.2021.370 ...
Las mitocondrias son organelos fascinantes, no solo porque son el sitio en donde se genera la energía de las células, sino por su relevancia en procesos y patologías de interés médico. La gran mayoría de las proteínas que constituyen el proteoma mitocondrial están codificadas en el núcleo y se traducen por ribosomas citosólicos, por lo que deben ser identificadas correctamente para ser distribuidas e insertadas en cada uno de los subcompartimentos mitocondriales. En este artículo realizamos una descripción de las las maquinarias de importación mitocondrial, además de las diferentes respuestas celulares que contrarrestan las alteraciones en los procesos de transporte de las proteínas o cuando existe una disfunción mitocondrial. Finalmente, mencionamos las enfermedades causadas por mutaciones en los complejos transportadores y de distribución de las proteínas de este organelo, que se han identificado hasta la fecha.
... On the contrary, the inner membrane (IM) contains two translocases: the TIM23 complex ( presequence translocase) and the TIM22 complex (carrier translocase) (Chacinska et al., 2009;Schmidt et al., 2010;Dudek et al., 2013). The TIM23 complex mediates the sorting of proteins with presequences into the IM and the matrix, whereas the TIM22 complex integrates polytopic membrane proteins into the IM (Sirrenberg et al., 1996;Rehling et al., 2004;van der Laan et al., 2010;Schendzielorz et al., 2018;Kumar et al., 2022). ...
The TIM22 pathway cargos are essential for sustaining mitochondrial homeostasis as an excess of these proteins leads to proteostatic stress and cell death. Yme1 is an inner membrane metalloprotease that regulates protein quality control with chaperone-like and proteolytic activities. Although the mitochondrial translocase and protease machinery are critical for organelle health, their functional association remains unexplored. The present study unravels a novel genetic connection between the TIM22 complex and YME1 machinery in Saccharomyces cerevisiae required for maintaining mitochondrial health. Our genetic analyses indicate that impairment in the TIM22 complex rescues the respiratory growth defects of cells without Yme1. Further, Yme1 is essential for the stability of the TIM22 complex and regulates the proteostasis of the TIM22 pathway substrates. Moreover, impairment in the TIM22 complex suppressed the mitochondrial structural and functional defects of Yme1 devoid cells. In summary, excessive levels of the TIM22 pathway substrates could be one of the reasons for respiratory growth defects of cells lacking Yme1, and compromising the TIM22 complex can compensate for the imbalance in mitochondrial proteostasis caused by the loss of Yme1.
... The radius of gyration of the tight knot has been estimated to be around 7-8 Å for the simplest protein knot (a trefoil) and correspondingly larger for more complicated knots. On the other hand, the smallest constrictions in mitochondrial pores or proteasome openings are 6-7 Å in radius [27,28], thus proteins with knotted backbones might have problems navigating them. ...
Knotted proteins, when forced through the pores, can get stuck if the knots in their backbone tighten under force. Alternatively, the knot can slide off the chain, making translocation possible. We construct a simple energy landscape model of this process with a time-periodic potential that mimics the action of a molecular motor. We calculate the translocation time as a function of the period of the pulling force, discuss the asymptotic limits and biological relevance of the results.
... In humans, COX biogenesis involves the coordinated assembly of 13 subunits, the largest 3 of which, I, II, and III, are catalytic subunits encoded by mitochondrial DNA (mtDNA) and synthesized within the mitochondria. The remaining 10 structural subunits are encoded by nuclear DNA and synthesized on cytosolic ribosomes, subsequently being translocated into the mitochondrial inner membrane mainly through mitochondrial targeting and import [2][3][4]. The mitochondrial-encoded subunits act in electron transfer, whereas the nuclear-encoded subunits appear to be involved in the structural integrity, regulation, and dimerization of the enzyme. ...
Cytochrome c oxidase subunit VIc (COX6c) is one of the most important subunits of the terminal enzyme of the respiratory chain in mitochondria. Numerous studies have demonstrated that COX6c plays a critical role in the regulation of oxidative phosphorylation (OXPHOS) and energy production. The release of COX6c from the mitochondria may be a hallmark of the intrinsic apoptosis pathway. Moreover, The changes in COX6c expression are widespread in a variety of diseases and can be chosen as a potential biomarker for diagnosis and treatment. In light of its exclusive effects, we present the elaborate roles that COX6c plays in various diseases. In this review, we first introduced basic knowledge regarding COX6c and its functions in the OXPHOS and apoptosis pathways. Subsequently, we described the regulation of COX6c expression and activity in both positive and negative ways. Furthermore, we summarized the elaborate roles that COX6c plays in various diseases, including cardiovascular disease, kidney disease, brain injury, skeletal muscle injury, and tumors. This review highlights recent advances and provides a comprehensive summary of COX6c in the regulation of OXPHOS in multiple diseases and may be helpful for drug design and the prediction, diagnosis, treatment, and prognosis of diseases.
... The carrier translocase (TIM22 complex) facilities the insertion of carrier proteins into the inner membrane. These substrates contain several transmembrane spans in addition to an internal targeting sequence [31,161]. Typically, TIM22 cargo proteins included six transmembrane span-containing carrier proteins and four transmembrane span channelforming subunits of the TIM23 complex or TIM22 itself [4,[162][163][164][165][166][167][168]. ...
In human mitochondria, mtDNA encodes for only 13 proteins, all components of the OXPHOS system. The rest of the mitochondrial components, which make up approximately 99% of its proteome, are encoded in the nuclear genome, synthesized in cytosolic ribosomes and imported into mitochondria. Different import machineries translocate mitochondrial precursors, depending on their nature and the final destination inside the organelle. The proper and coordinated function of these molecular pathways is critical for mitochondrial homeostasis. Here, we will review molecular details about these pathways, which components have been linked to human disease and future perspectives on the field to expand the genetic landscape of mitochondrial diseases.
... The inner and outer mitochondrial membranes play important roles in the transport of cytosolic pre-proteins into and out of the mitochondrial matrix (Rehling, Brandner, & Pfanner, 2004;Wiedemann & Pfanner, 2017). TIMM50, the translocase of inner mitochondrial membrane 50, is a protein encoded by the Timm50 gene in mice. ...
Mitochondria are involved in a variety of developmental processes and neurodegenerative diseases. The translocase complexes of the outer and inner mitochondrial membranes (TOM and TIM) are protein complexes involved in transporting protein precursors across mitochondrial membranes. Although rabbits are important animal models for neurodegenerative diseases, the expression of TOM and TIM complexes has yet to be examined in the rabbit brain. In the present study, we quantitatively evaluated the protein expression of the translocase of outer mitochondrial membrane 40 (TOMM40) and inner mitochondrial membrane 50 (TIMM50) complexes, two of the TOM/TIM complexes, in the cerebral, cerebellar, and hippocampal cortices of the New Zealand white rabbit brain, using immunohistochemistry. Sections from brain specimens were initially stained for cytochrome c oxidase (COX), a well‐known mitochondrial marker, which was found to be homogeneously expressed in the cerebrum, but localized to the Purkinje and pyramidal neurons of the cerebellum and hippocampus, respectively. TOMM40 and TIMM50 proteins consistently revealed a similar expression pattern, although at different ratios. In the cerebrum, TOMM40 and TIMM50 immunoreactions were homogeneously distributed within the cytoplasm of various neurons. Meanwhile, Purkinje cells in the cerebellum and pyramidal neurons in the hippocampus displayed higher intensities in their cytoplasm. The specific cellular localization of TOMM40 and TIMM50 proteins in various regions of the rabbit brain suggests a distinct function of each protein in these regions. Further analysis will be required to evaluate the molecular functions of these proteins.
... This is the case for all proteins of the outer membrane and most components of the intermembrane space (IMS) that use several distinct pathways (Drwesh and Rapaport, 2020;Wiedemann and Pfanner, 2017;Finger and Riemer, 2020;Edwards et al., 2020;Doan et al., 2020). In addition, many mitochondrial inner membrane proteins, in particular the members of the metabolite carrier family (carriers for short), lack presequences and are imported by a distinct ''carrier pathway'' (Horten et al., 2020;Rehling et al., 2004). From studies using the ATP/ADP carrier (i.e., Pet9 in yeast) as a model protein, it was proposed that the carrier pathway differs from the import route of matrix-destined proteins already on the surface of mitochondria, where carriers bind the ''carrier receptor'' Tom70 that would insert them into the universal protein-conducting channel of the TOM complex. ...
Most mitochondrial proteins are synthesized as precursors in the cytosol and post-translationally transported into mitochondria. The mitochondrial surface protein Tom70 acts at the interface of the cytosol and mitochondria. In vitro import experiments identified Tom70 as targeting receptor, particularly for hydrophobic carriers. Using in vivo methods and high-content screens, we revisit the question of Tom70 function and considerably expand the set of Tom70-dependent mitochondrial proteins. We demonstrate that the crucial activity of Tom70 is its ability to recruit cytosolic chaperones to the outer membrane. Indeed, tethering an unrelated chaperone-binding domain onto the mitochondrial surface complements most of the defects caused by Tom70 deletion. Tom70-mediated chaperone recruitment reduces the proteotoxicity of mitochondrial precursor proteins, particularly of hydrophobic inner membrane proteins. Thus, our work suggests that the predominant function of Tom70 is to tether cytosolic chaperones to the outer mitochondrial membrane, rather than to serve as a mitochondrion-specifying targeting receptor.
... Most of these proteins within the organelles, even endosymbiotic-derived organelles that contain their own genome, originate from nuclear genes, and are initially translated in the cytosol. A total of 99% of mitochondrial proteins (Rehling et al., 2004) and more than 95% of chloroplast proteins (Soll, 2002) are encoded by nuclear DNA and imported into the organelles. From this cytosolic pool, it is imperative that a protein reaches the correct compartment, and a multitude of mechanisms exist to ensure this specific targeting takes place. ...
Membrane-targeting sequences, connected targeting mechanisms, and co-factors orchestrate primary targeting of proteins to membranes.
... SFXNs are TIM22 complex substrates MCs in the SLC25 family possess internal targeting elements and are integral polytopic proteins characterized by 6 TMDs. Their import and integration into the IMM are mediated by the TIM22 complex (Chacinska et al., 2009;Koehler, 2004;Neupert and Herrmann, 2007;Rehling et al., 2004). Recent studies have highlighted the substrate preference of TIM22 complex subunits and identified AGK as a metazoan-specific component essential for the import of MCs in the SLC25 family (Callegari et al., 2016;Kang et al., 2016Kang et al., , 2017Vukotic et al., 2017). ...
Mitochondrial carriers (MCs) mediate the passage of small molecules across the inner mitochondrial membrane (IMM), enabling regulated crosstalk between compartmentalized reactions. Despite MCs representing the largest family of solute carriers in mammals, most have not been subjected to a comprehensive investigation, limiting our understanding of their metabolic contributions. Here, we functionally characterize SFXN1, a member of the non-canonical, sideroflexin family. We find that SFXN1, an integral IMM protein with an uneven number of transmembrane domains, is a TIM22 complex substrate. SFXN1 deficiency leads to mitochondrial respiratory chain impairments, most detrimental to complex III (CIII) biogenesis, activity, and assembly, compromising coenzyme Q levels. The CIII dysfunction is independent of one-carbon metabolism, the known primary role for SFXN1 as a mitochondrial serine transporter. Instead, SFXN1 supports CIII function by participating in heme and α-ketoglutarate metabolism. Our findings highlight the multiple ways that SFXN1-based amino acid transport impacts mitochondrial and cellular metabolic efficiency.
... Until recently, only two classes of TIM22 substrates were known: SLC25 carrier proteins, which mediate transport of metabolites across the inner membrane, and TIM proteins (Tim17/Tim23/Tim22), which are subunits of the inner membrane translocase complexes. These substrates with six and four transmembrane domains, respectively, have shaped dogma in the field as to how the TIM22 complex mediates insertion of substrates in a hairpin loop conformation (Rehling et al., 2003;Rehling et al., 2004). Based on this, the SFXN proteins with their five transmembrane domains, do not fit with these previous models of TIM22 membrane insertion. ...
Acylglycerol Kinase (AGK) is a mitochondrial lipid kinase that contributes to protein biogenesis as a subunit of the TIM22 complex at the inner mitochondrial membrane. Mutations in AGK cause Sengers syndrome, an autosomal recessive condition characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy and lactic acidosis. We mapped the proteomic changes in Sengers patient fibroblasts and AGK KO cell lines to understand the effects of AGK dysfunction on mitochondria. This uncovered downregulation of a number of proteins at the inner mitochondrial membrane, including many SLC25 carrier family proteins, which are predicted substrates of the complex. We also observed downregulation of SFXN proteins, which contain five transmembrane domains, and show that they represent a novel class of TIM22 complex substrate. Perturbed biogenesis of SFXN proteins in cells lacking AGK reduces the proliferative capabilities of these cells in the absence of exogenous serine, suggesting that dysregulation of one carbon metabolism is a molecular feature in the biology of Sengers syndrome.
... Mitochondrial protein is mostly encoded by the nuclear genome. These are imported from cytosol into mitochondria via translocator, translocase of inner mitochondrial membrane 44 homolog (TRMM44) [88]. Variations in the inner mitochondrial membrane transporter TIMM44 have been observed in patients with oncocytic thyroid tumors. ...
Simple Summary
Hürthle cell carcinoma (HCC) represents 3–4% of thyroid carcinoma cases. It is characterized by its large, granular and eosinophilic cytoplasm, due to an excessive number of mitochondria. Hürthle cells can be identified only after fine needle aspiration cytology biopsy or by histological diagnosis after the surgical operation. Published studies on HCC indicate its putative high aggressiveness. In this article, current knowledge of HCC focusing on clinical features, cytopathological features, genetic changes, as well as pitfalls in diagnosis are reviewed in order to improve clinical management.
Abstract
Hürthle cell carcinoma (HCC) represents 3–4% of thyroid carcinoma cases. It is considered to be more aggressive than non-oncocytic thyroid carcinomas. However, due to its rarity, the pathological characteristics and biological behavior of HCC remain to be elucidated. The Hürthle cell is characterized cytologically as a large cell with abundant eosinophilic, granular cytoplasm, and a large hyperchromatic nucleus with a prominent nucleolus. Cytoplasmic granularity is due to the presence of numerous mitochondria. These mitochondria display packed stacking cristae and are arranged in the center. HCC is more often observed in females in their 50–60s. Preoperative diagnosis is challenging, but indicators of malignancy are male, older age, tumor size > 4 cm, a solid nodule with an irregular border, or the presence of psammoma calcifications according to ultrasound. Thyroid lobectomy alone is sufficient treatment for small, unifocal, intrathyroidal carcinomas, or clinically detectable cervical nodal metastases, but total thyroidectomy is recommended for tumors larger than 4 cm. The effectiveness of radioactive iodine is still debated. Molecular changes involve cellular signaling pathways and mitochondria-related DNA. Current knowledge of Hürthle cell carcinoma, including clinical, pathological, and molecular features, with the aim of improving clinical management, is reviewed.
... Mitochondrial targeting by Agno was also supported by our recent proteomic studies, which revealed the interaction of Agno with a substantial number of mitochondrial proteins (nearly 50), some of which are the structural components of the mitochondrial protein import pathways, electron transport chaincomplexes and metabolic enzymes (Saribas et al., 2020). Regarding the mitochondrial protein import pathways, we found the interaction of Agno with components of the outer (TOM) and inner (TIM) membrane translocase complexes, which are essential for the mitochondrial maturation and survival (Becker et al., 2012;Harbauer et al., 2014;Rehling et al., 2004). Particularly, its interaction with TOM70 and TOM22 in the TOM complex and with several components of TIM23 complex suggests that Agno may also dysregulate the mitochondrial functions by targeting its protein import pathways. ...
JC virus encodes an important regulatory protein, known as Agnoprotein (Agno). We have recently reported Agno's first protein-interactome with its cellular partners revealing that it targets various cellular networks and organelles, including mitochondria. Here, we report further characterization of the functional consequences of its mitochondrial targeting and demonstrated its co-localization with the mitochondrial networks and with the mitochondrial outer membrane. The mitochondrial targeting sequence (MTS) of Agno and its dimerization domain together play major roles in this targeting. Data also showed alterations in various mitochondrial functions in Agno-positive cells; including a significant reduction in mitochondrial membrane potential, respiration rates and ATP production. In contrast, a substantial increase in ROS production and Ca²⁺ uptake by the mitochondria were also observed. Finally, findings also revealed a significant decrease in viral replication when Agno MTS was deleted, highlighting a role for MTS in the function of Agno during the viral life cycle.
... Transport of the AAC across the mitochondrial outer membrane is membrane potential-independent while subsequent insertion into the mitochondrial inner membrane is essentially driven by the mitochondrial membrane potential [41]. We therefore expected that targeting of the AAC to the mitochondrial outer membrane should be independent of positively charged residues. ...
Background
The uptake of newly synthesized nuclear-encoded mitochondrial proteins from the cytosol is mediated by a complex of mitochondrial outer membrane proteins comprising a central pore-forming component and associated receptor proteins. Distinct fractions of proteins initially bind to the receptor proteins and are subsequently transferred to the pore-forming component for import. The aim of this study was the identification of the decisive elements of this machinery that determine the specific selection of the proteins that should be imported.
Results
We identified the essential internal targeting signal of the members of the mitochondrial metabolite carrier proteins, the largest protein family of the mitochondria, and we investigated the specific recognition of this signal by the protein import machinery at the mitochondrial outer surface. We found that the outer membrane import receptors facilitated the uptake of these proteins, and we identified the corresponding binding site, marked by cysteine C141 in the receptor protein Tom70. However, in tests both in vivo and in vitro, the import receptors were neither necessary nor sufficient for specific recognition of the targeting signals. Although these signals are unrelated to the amino-terminal presequences that mediate the targeting of other mitochondrial preproteins, they were found to resemble presequences in their strict dependence on a content of positively charged residues as a prerequisite of interactions with the import pore.
Conclusions
The general import pore of the mitochondrial outer membrane appears to represent not only the central channel of protein translocation but also to form the decisive general selectivity filter in the uptake of the newly synthesized mitochondrial proteins.
... Translocation across the inner membrane requires the force generated by translocase-associated motor (PAM) proteins coupled to TIM23 and the ATP hydrolysis [47,[56][57][58] (Fig. 3A). ...
The ubiquitin-proteasome system constitutes a major pathway for protein degradation in the cell. Therefore the crosstalk of this pathway with mitochondria is a major topic with direct relevance to many mitochondrial diseases. Proteasome dysfunction triggers not only protein toxicity, but also mitochondrial dysfunction. The involvement of proteasomes in the regulation of protein transport into mitochondria contributes to an increase in mitochondrial function defects. On the other hand, mitochondrial impairment stimulates reactive oxygen species production, which increases protein damage, and protein misfolding and aggregation leading to proteasome overload. Concurrently, mitochondrial dysfunction compromises cellular ATP production leading to reduced protein ubiquitination and proteasome activity. In this review we discuss the complex relationship and interdependence of the ubiquitin-proteasome system and mitochondria. Furthermore, we describe pharmacological inhibition of proteasome activity as a novel strategy to treat a group of mitochondrial diseases.
... There are some biological examples of such fluctuating environment in translocation are the nuclear pore complex, which plays an essential role in nucleocytoplasmic transport in eukaryotes [51]. Another example is the exchange of molecules between mitochondria and the rest of cell which is controlled by the twin-pore protein translocate (TIM22 complex) in the inner membrane of the mitocondria [52]. Moreover, using an alternating electric field (time-dependent driving force) in the nanopore has been implied as a source for DNA sequencing [53]. ...
We study the effect of fluctuating environment in protein transport dynamics. In particular, we investigate the translocation of a structured biomolecule (protein) across a temporally modulated nano-pore. We allow the radius of the cylindrical pore to oscillate harmonically with certain frequency and amplitude about an average radius. The protein is imported inside the pore whose dynamics is influences by the fluctuating nature of the pore. We investigate the dynamic and thermodynamical properties of the translocation process by revealing the statistics of translocation time as a function of the pulling inward force acting along the axis of the pore, and the frequency of the time dependent radius of the channel. We also examine the distribution of translocation time in the intermediate frequency regime. We observe that the shaking mechanism of pore leads to accelerate the translocation process as compared to the static channel that has a radius equal to the mean radius of oscillating pore. Moreover, the translocation time shows a global maximum as a function of frequency of the oscillating radius, hence revealing a resonant activation phenomenon in the dynamics of protein translocation.
... As mtDNA codes only for thirteen proteins in mitochondria, constant import of nuclear-encoded proteins into the mitochondria is vital not only for healthy and functional mitochondria, but also for maintaining cellular homeostasis. Mitochondrial proteins utilize a variety of dynamic import machinery for their efficient import into mitochondria [21,[37][38][39]. This review article presents current knowledge and recent updates in the mitochondrial import pathways and its regulation, followed by the discussion on mitochondrial stress stimuli and compensatory mechanisms exhibited by the cell upon defective protein import into mitochondria. ...
Mitochondria are involved in several vital functions of the eukaryotic cell. The majority of mitochondrial proteins are coded by nuclear DNA. Constant import of proteins from the cytosol is a prerequisite for the efficient functioning of the organelle. The protein import into mitochondria is mediated by diverse import pathways and is continuously under watch by quality control systems. However, it is often challenged by both internal and external factors, such as oxidative stress or energy shortage. The impaired protein import and biogenesis leads to the accumulation of mitochondrial precursor proteins in the cytosol and activates several stress response pathways. These defense mechanisms engage a network of processes involving transcription, translation, and protein clearance to restore cellular protein homeostasis. In this review, we provide a comprehensive analysis of various factors and processes contributing to mitochondrial stress caused by protein biogenesis failure and summarize the recovery mechanisms employed by the cell.
... Taken together, these results allow us to propose the following model: IL-1β stimulation leads to MyD88/IRAK4/IRAK2 Myddosome complex formation and translocation to the outer mitochondrial membrane, where IRAK2 may then dissociate from the complex and translocate into mitochondrial intermembrane space and inner membranes. In support of this model, through mass spectrometric analysis of IRAK2-interacting proteins, we found that IRAK2 was able to interact with TOM20 and TIMM50, which are translocators on mitochondrial outer and inner membranes, respectively [36][37][38] . We validated IL-1β-induced IRAK2's interaction with TOM20 and TIMM50 by co-immunoprecipitation. (Extended Data Fig. 4e). ...
Chronic inflammation is a common feature of obesity, with elevated cytokines such as interleukin-1 (IL-1) in the circulation and tissues. Here, we report an unconventional IL-1R–MyD88–IRAK2–PHB/OPA1 signaling axis that reprograms mitochondrial metabolism in adipocytes to exacerbate obesity. IL-1 induced recruitment of IRAK2 Myddosome to mitochondria outer membranes via recognition by TOM20, followed by TIMM50-guided translocation of IRAK2 into mitochondria inner membranes, to suppress oxidative phosphorylation and fatty acid oxidation, thereby attenuating energy expenditure. Adipocyte-specific MyD88 or IRAK2 deficiency reduced high-fat-diet-induced weight gain, increased energy expenditure and ameliorated insulin resistance, associated with a smaller adipocyte size and increased cristae formation. IRAK2 kinase inactivation also reduced high-fat diet-induced metabolic diseases. Mechanistically, IRAK2 suppressed respiratory super-complex formation via interaction with PHB1 and OPA1 upon stimulation of IL-1. Taken together, our results suggest that the IRAK2 Myddosome functions as a critical link between inflammation and metabolism, representing a novel therapeutic target for patients with obesity. Obesity is often accompanied by chronic inflammation. Li and colleagues show that, in mice fed high-fat diets, IL-1 signaling in adipocytes induces an unconventional IRAK2 translocation to mitochondria and suppresses respiratory super-complex formation to alter mitochondrial function, and exacerbates obesity.
... In the context of a long-term study using Drosophila towards understanding the e↵ect of ectopic expression of testis proteins in the soma, we have found that Tiny tim 2 (Ttm2) and Tomboy20, have tumorigenic e↵ects when expressed in somatic epithelia. Ttm2 and Tomboy20 are testis-specific components of the translocases of outer (TOM) and inner (TIM23) membrane complexes that translocate nuclearly encoded mitochondrial proteins into mitochondria [18][19][20]. Larval neuroepithelial cells expressing ttm2 overproliferate and fail to di↵erentiate into medulla neuroblasts (NBs). Imaginal wing disc epithelial cells expressing ttm2 or tomboy20 invade and induce non-autonomous massive overgrowth of the nearest wild-type epithelium. ...
We have undertaken a study towards understanding the effect of ectopic expression of testis proteins in the soma in Drosophila. Here, we show that in the larval neuroepithelium, ectopic expression of the germline-specific component of the inner mitochondrial translocation complex tiny tim 2 (ttm2) brings about cell autonomous hyperplasia and extension of G2 phase. In the wing discs, cells expressing ectopic ttm2 upregulate Jun N-terminal kinase (JNK) signaling, present extended G2, become invasive, and elicit non-cell autonomous G2 extension and overgrowth of the wild-type neighboring tissue. Ectopic tomboy20, a germline-specific member of the outer mitochondrial translocation complex is also tumorigenic in wing discs. Our results demonstrate the tumorigenic potential of unscheduled expression of these two testis proteins in the soma. They also show that a unique tumorigenic event may trigger different tumor growth pathways depending on the tissular context.
... Previous studies have reported that mitocx43 plays an important role in the balance of reactive oxygen species (ROS) for redox signalling (5,6). Mitocx43 is also involved in cardioprotection, as it has been demonstrated that it is overexpressed in ischemic pre-conditioning (7), as well as in other forms of cardioprotection, through the control of the initiation of apoptosis (8). The authors have previously demonstrated an increase in mitocx43 expression in H9c2 cells (9) and in hearts of mice treated with doxorubicin (doxo) (10). ...
Oxidative stress is widely accepted as a key factor of doxorubicin (Doxo)‑induced cardiotoxicity. There is evidence to indicate that nitrosative stress is involved in this process, and that Doxo interacts by amplifying cell damage. Mitochondrial connexin 43 (mitoCx43) can confer cardioprotective effects through the reduction of mitochondrial reactive oxygen species production during Doxo‑induced cardiotoxicity. The present study aimed to evaluate the involvement of mitoCx43 in Doxo‑induced nitrosative stress. Rat H9c2 cardiomyoblasts were treated with Doxo in the absence or presence of radicicol, an inhibitor of Hsp90, the molecular chaperone involved in Cx43 translocation to the mitochondria that underlies its role in cardioprotection. FACS analysis and RT‑qPCR revealed that Doxo increased superoxide dismutase, and catalase gene and protein expression. As shown by hypodiploid nuclei and confirmed by western blot analysis, Doxo increased caspase 9 expression and reduced procaspase 3 levels, which induced cell death. Moreover, a significant increase in the activation of the NF‑κB signaling pathway was observed. It is well known that the increased expression of inducible nitric oxide synthase results in nitric oxide overproduction, which then rapidly reacts with hydrogen peroxide or superoxide generated by the mitochondria, to form highly reactive and harmful peroxynitrite, which ultimately induces nitrotyrosine formation. Herein, these interactions were confirmed and increased effects were observed in the presence of radicicol. On the whole, the data of the present study indicate that an interplay between oxidative and nitrosative stress is involved in Doxo‑induced cardiotoxicity, and that both aspects are responsible for the induction of apoptosis. Furthermore, it is demonstrated that the mechanisms that further increase mitochondrial superoxide generation (e.g., the inhibition of Cx43 translocation into the mitochondria) significantly accelerate the occurrence of cell death.
... This import is via pore-forming multi-protein complexes called translocases which are located in both the inner and outer membranes. Cytosolic proteins move though the OMM, across the inter membrane space and the IMM, and are either inserted into the appropriate membrane leaflet or retained in the matrix (Voos et al. 1999;Rehling et al. 2004). ...
The neuronal ceroid lipofuscinoses (Batten disease) are a group of inherited neurological disorders which predominantly affect children. They are characterized by the accumulation of autofluorescent storage material in lysosomes and occur with a frequency between 1 in 12 500 and 1 in 100 000 births. To date, six genes underlying different sub-types of the disease have been cloned and many studies in cell culture and mouse models performed. However the mechanism of disease pathogenesis remains poorly understood. The simple nematode worm, Caenorhabditis elegans, has a fully sequenced genome, completely mapped cell lineage and nervous system, is easy to maintain and manipulate, and is an organism about which much information, including the results of many genome wide studies, is available. It is thus a good model organism for the study of genes that underlie human neurological disorders. The aim of this work was to investigate whether C. elegans could be used as a model system for investigating the function of NCL genes and the pathological mechanisms that underlie disease manifestation. Homologues to PPT1 and CLN3 were identified in C. elegans and their expression confirmed. Mutation of PPT1 underlies the most severe NCL type, infantile NCL (INCL). Further analysis of CePPT-1 determined a high level of sequence and structural similarity to the human enzyme and demonstrated that it could perform the same catalytic reaction under the same conditions. Analysis of a ppt-1 null mutant (MN1) identified a phenotype of developmental delay, defective egg laying and grossly abnormal mitochondrial morphology. A homologue to the enzyme, acyl-protein thioesterase-1 was also identified (ATH-1) and expression confirmed. The role of PPT-1 in the cell and how this may relate to the pathogenesis of INCL and other NCL types is presented.
... The IM possesses two distinct types of machinery; the presequence translocase (TIM23 complex) and the carrier translocase (TIM22 complex). The TIM23 complex mediates import of presequence-containing proteins into the matrix or the IM, while the TIM22 complex facilitates the IM insertion of more complex multispanning membrane proteins that have internal hydrophobic targeting sequences (Sirrenberg et al., 1996;Rehling et al., 2004;Chacinska et al., 2009;Schmidt et al., 2010;van der Laan et al., 2010;Dudek et al., 2013;Schendzielorz et al., 2018). Substrates of the TIM22 pathway include members of mitochondrial metabolite carriers as well as subunits of the translocases, such as Tim23, Tim22, and Tim17 Paschen et al., 2000). ...
Mitochondrial biogenesis requires efficient sorting of various proteins into different mitochondrial sub-compartments mediated by dedicated protein machinery present in the outer and inner membrane. Among them, the TIM22 complex enables the integration of complex membrane proteins with internal targeting signals into the inner membrane. Although the Tim22 forms the core of the complex, the dynamic recruitment of subunits to the channel is still enigmatic. The present study first-time highlights that IMS and TM4 regions of Tim22 are critically required for the interaction of the membrane-embedded subunits including, Tim54, Tim18, and Sdh3, thereby maintain the functional architecture of TIM22 translocase. On the other hand, TM1 and TM2 regions of Tim22 are important for the Tim18 association, while TM3 is exclusively required for the Sdh3 interaction. Moreover, the impairment in TIM22 complex assembly influences its translocase activity, mitochondrial network, and the viability of cells lacking mitochondrial DNA. Overall our findings provide compelling evidence to highlight the significance of conserved regions of Tim22 that are important for the maintenance of the TIM22 complex and mitochondrial integrity.
... The mtHsp70 protein was described above in the first localization experiment. Probing with antibodies for the Translocase of the Outer Membrane (TOM20), found anchored to the outer membrane but facing the cytosol (Rehling et al., 2004), and cytochrome c, loosely associated with the mitochondrial inner membrane (MIM) proteins, revealed PK treatments were highly successful in degradation of outer membrane proteins exposed to the cytosol and that the outer mitochondrial membrane (OMM) was intact. Probing for OPA1 and mtHsp70 reveals bands are not clearly separated into pellet and supernatant as would be expected. ...
This study was aimed at establishing the localization of NipSnapl and to explore its possible functions within the cell. Using fluorescent microscopy, NipSnapl-GFP constructs transfected into COS-7 cells showed a co-localization with mitochondrial specific tracking dyes. In addition, import analysis, proteinase K treatments, sodium carbonate extraction and mitochondrial swelling were utilized to determine sub-mitochondrial localization. Data from those assays suggest that NipSnapl contains a cleavable mitochondrial targeting signal directing it to the inner membrane. Overexpression of NipSnapl-GFP did not reveal any obvious aberrations in mitochondrial morphology. However, these experiments indicate that NipSnapl may not play a role in this process. Preliminary siRNA experiments were also performed to observe mitochondria during knockdown of NipSnapl-GFP. In addition NipSnapl antibody production is planned for future work. For this, NipSnapl was cloned into an expression vector for future purification of a GST-tagged version of the protein, although it has proved insoluble.
... Classical mitochondrial protein imports are carried out by the outer (TOM) and inner (TIM) membrane translocase complexes, which are essential for the mitochondrial maturation and survival. Majority of the mitochondrial proteins were synthesized along with MTS in the cytosol and imported into mitochondria via TOM/TIM complexes (Becker et al., 2012;Harbauer et al., 2014;Rehling et al., 2004). Our data further revealed that Agno particularly targets the TOM70 and TOM22 in the TOM complex and several components of (Coric et al., 2014(Coric et al., , 2017. ...
JC virus (JCV) Agnoprotein (Agno) plays critical roles in successful completion of the viral replication cycle. Understanding its regulatory roles requires a complete map of JCV-host protein interactions. Here, we report the first Agno interactome with host cellular targets utilizing "Two-Strep-Tag" affinity purification system coupled with mass spectroscopy (AP/MS). Proteomics data revealed that Agno primarily targets 501 cellular proteins, most of which contain "coiled-coil" motifs. Agno-host interactions occur in several cellular networks including those involved in protein synthesis and degradation; and cellular transport; and in organelles, including mitochondria, nucleus and ER-Golgi network. Among the Agno interactions, Rab11B, Importin and Crm-1 were first validated biochemically and further characterization was done for Crm-1, using a HIV-1 Rev-M10-like Agno mutant (L33D + E34L), revealing the critical roles of L33 and E34 residues in Crm-1 interaction. This comprehensive proteomics data provides new foundations to unravel the critical regulatory roles of Agno during the JCV life cycle.
... Most mitochondrial proteins are nuclear encoded and need to be imported into mitochondria post-translation. The classical pathway for mitochondrial import is through the translocase of the outer membrane (TOM) followed by the translocase of the inner membrane (TIM) [32]. ...
The nuclear factor-κB (NF-κB) family of transcription factors can directly or indirectly regulate many important areas of biology, including immunity, inflammation and cell survival. One intriguing aspect of NF-κB crosstalk with other cell signalling pathways is its regulation of mitochondrial biology, including biogenesis, metabolism and apoptosis. In addition to regulating the expression of mitochondrial genes encoded in the nucleus, NF-κB signalling components are also found within mitochondria themselves and associated with mitochondrial DNA. However, complete biochemical analysis of mitochondrial and sub-mitochondrial localisation of all NF-κB subunits has not been undertaken. Here, we show that only the RelA NF-κB subunit and its inhibitor IκBα reside within mitochondria, whilst p50 is found in the endoplasmic reticulum (ER). Fractionation of mitochondria revealed that only RelA was found in the mitoplast, the location of the mtDNA. We demonstrate that hypoxia leads to a very rapid but transient accumulation of RelA and IκBα in mitochondria. This effect required reactive oxygen species (ROS) but was not dependent on the hypoxia sensing transcription factor subunit HIF1α or intracellular Ca2+ release. We also observed rapid mitochondrial localisation of transcription factor STAT3 following hypoxia. Inhibition of STAT3 blocked RelA and IκBα mitochondrial localisation revealing a previously unknown aspect of crosstalk between these key cellular regulators.
Mitochondrial membrane proteins play an essential role in all major mitochondrial functions. The respiratory complexes of the inner membrane are key for the generation of energy. The carrier proteins for the influx/efflux of essential metabolites to/from the matrix. Many other inner membrane proteins play critical roles in the import and processing of nuclear encoded proteins (∼99% of all mitochondrial proteins). The outer membrane provides another lipidic barrier to nuclear-encoded protein translocation and is home to many proteins involved in the import process, maintenance of ionic balance, as well as the assembly of outer membrane components. While many aspects of the import and assembly pathways of mitochondrial membrane proteins have been elucidated, many open questions remain, especially surrounding the assembly of the respiratory complexes where certain highly hydrophobic subunits are encoded by the mitochondrial DNA and synthesised and inserted into the membrane from the matrix side. This review will examine the various assembly pathways for inner and outer mitochondrial membrane proteins while discussing the most recent structural and biochemical data examining the biogenesis process.
Significance
The coordinated motion and conformational changes are fundamental for the function of many macromolecules. However, obtaining direct evidence of coordinated changes between different subdomains within an individual molecule remains challenging. Here, we apply a single-molecule three-color Förster resonance energy transfer to simultaneously measure three distances within a single molecule. We utilized this method to analyze the coordinated motion of heat-shock proteins (Hsps). Excitingly, our study provides direct evidence for key differences in the conformational cycle of different Hsp70s despite their high evolutionary conservation. This work paves the way to relate coordinated motion and allosteric effects to molecular function.
The role of translocases was underappreciated and was not included as a separate class in the enzyme commission until August 2018. The recent research interests in proteomics of orphan enzymes, ionomics, and metallomics along with high throughput sequencing technologies generated overwhelming data and revamped this enzyme into a separate class. This offers a great opportunity to understand the role of new or orphan enzymes in general, and translocases in specific. The enzymes belonging to translocases regulate/permeate the transfer of ions or molecules across the membranes. These enzyme entries were previously associated with other enzyme classes, which are now transferred to a new enzyme class 7 (EC 7). The entries that are reclassified are important to extend the enzyme list and it is the need of the hour. Accordingly, there are upgradation of entries of this class of enzymes in several databases. This review is a concise compilation of translocases with reference to the number of entries currently available in the databases. This review also focuses on function as well as dysfunction of translocases during normal and disordered states, respectively. This article is protected by copyright. All rights reserved
Les sidéroflexines forment une famille de transporteurs mitochondriaux encore peu étudiés. Leur forte conservation chez les eucaryotes suggère un rôle important dans la régulation des fonctions mitochondriales, mais ce rôle est encore mal compris. Récemment, il a été montré qu’elles étaient impliquées dans la voie du métabolisme à un carbone et que leur dérégulation impactait l’homéostasie du fer et la respiration mitochondriale. En outre, de smutations de SFXN4 sont responsables d’une maladie mitochondriale rare, le syndrome COXPD18. Pour mieux comprendre les fonctions de SFXN, j’ai développé mes travaux de recherche selon deux axes prioritaires : 1) la recherche de partenaires de SFXN1 en cellules humaines ; 2) l’étude des activités des sidéroflexines de drosophile (dSfxn) vis-à-vis de l’apoptose, processus de mort cellulaire régulé par la mitochondrie.Notre recherche de partenaires physiques de SFXN1 révèle que cette protéine interagit avec des sous-unités de la chaîne respiratoire et les protéines TIM50, HSD10 et ATAD3A. Ces interactions pourraient expliquer les altérations de la respiration mitochondriale observées dans certaines études et permettent d’envisager un rôle des SFXN dans d’autres processus mitochondriaux tels que la synthèse des ARNt mitochondriaux et l’organisation mitochondriale, processus dans lesquels sont impliqués HSD10 et ATAD3A, respectivement.Chez la drosophile, j’ai pu montrer que la modulation des niveaux de dSfxn1/3 et dSfxn2 affectait l’apoptose induite par Rbf1, orthologue de Rb et Debcl, membre pro-apoptotique de la famille Bcl2. Enfin, j’ai généré des résultats suggérant un rôle essentiel de ces protéines dans la régulation de la physiologie neuronale. En effet, la déplétion des dSfxn dans les neurones in vivo chez la drosophile se traduit par une diminution de la longévité des drosophiles et unemodification de leur comportement locomoteur.
The formation of disulfide bonds is probably the most influential modification of peptides and proteins. An elaborate set of cellular machinery exists to catalyze and guide this process. In recent years, significant developments have been made in both our understanding of the in vivo situation and the in vitro manipulation of disulfide bonds. This is the first monograph to provide a comprehensive overview of this exciting and rapidly developing area. It offers in-depth insights into the mechanisms of in vivo and in vitro oxidative folding of proteins as well as mono- and multiple-stranded peptides. Procedures applied for laboratory and industrial purposes are also discussed by top experts in the field. The book describes the enzymes involved in the correct oxidative folding of cysteine-containing proteins in prokaryotes and eukaryotes. It then goes on to discuss the mimicking of these enzymes for successful in vitro folding of proteins (including synthetic replicates) and to deal with important issues concerning cysteine-rich peptides. The ability of natural bioactive peptides to fold correctly, and in high yields, to form defined structural motifs using cysteine sequence patterns is still puzzling. With this in mind, synthetic procedures for establishing native cysteine frameworks are discussed using selected examples, such as the potential of selenocysteines. The biotechnological and pharmaceutical relevance of proteins, peptides, their variants and synthetic replicates is continuously increasing. Consequently, this book is invaluable for peptide and protein chemists involved in related research and production.
The sections in this article are
Introduction
Mitochondrial‐Targeting Signals
Cytosolic Factors
Sorting of Precursors between Mitochondria and Chloroplasts
Translocation Machinery
Proteolytic Events
Evolution of Protein Import Components
Genomic Perspective of Mitochondrial Protein Import Components
Concluding Remarks
Acknowledgements
In this paper we discuss a simple theoretical approach, taken from the theory of stochastic processes to understand the basic phenomenology of protein translocation through a flickering pore. In this theoretical approach we investigate the dynamics of Brownian particle driven by a periodically driving force. This toy model is further extended by considering the Langevin equation with constants drift and time dependent variance. Using the first passage time theory we derived the formalism for probability density function to comprehend the translocation process occurring in the presence of fluctuating environment.
Most mitochondrial proteins are synthesized as precursors in the cytosol and post-translationally transported into mitochondria. The mitochondrial surface protein Tom70 acts at the interface of the cytosol and mitochondria. In vitro import experiments identified Tom70 as targeting receptor, particularly for hydrophobic carriers. Using in vivo methods and high content screens, we revisited the question of Tom70 function and considerably expanded the set of Tom70-dependent mitochondrial proteins. We demonstrate that the crucial activity of Tom70 is its ability to recruit cytosolic chaperones to the outer membrane. Indeed, tethering an unrelated chaperone-binding domain onto the mitochondrial surface complements most of the defects caused by Tom70 deletion. Tom70-mediated chaperone recruitment reduces the proteotoxicity of mitochondrial precursor proteins, in particular of hydrophobic inner membrane proteins. Thus, our work suggests that the predominant function of Tom70 is to tether cytosolic chaperones to the outer mitochondrial membrane, rather than to serve as a mitochondria-specifying targeting receptor.
: Metabolite carriers of the mitochondrial inner membrane are crucial for cellular physiology since mitochondria contribute essential metabolic reactions and synthesize the majority of the cellular ATP. Like almost all mitochondrial proteins, carriers have to be imported into mitochondria from the cytosol. Carrier precursors utilize a specialized translocation pathway dedicated to the biogenesis of carriers and related proteins, the carrier translocase of the inner membrane (TIM22) pathway. After recognition and import through the mitochondrial outer membrane via the translocase of the outer membrane (TOM) complex, carrier precursors are ushered through the intermembrane space by hexameric TIM chaperones and ultimately integrated into the inner membrane by the TIM22 carrier translocase. Recent advances have shed light on the mechanisms of TOM translocase and TIM chaperone function, uncovered an unexpected versatility of the machineries, and revealed novel components and functional crosstalk of the human TIM22 translocase.
Mitochondrial carriers (MC) mediate the passage of small molecules across the inner mitochondrial membrane (IMM) enabling regulated crosstalk between compartmentalized reactions. Despite MCs representing the largest family of solute carriers in mammals, most have not been subjected to a comprehensive investigation, limiting our understanding of their metabolic contributions. Here, we functionally characterized SFXN1, a member of the non-canonical, sideroflexin MC family. We find that SFXN1, an integral membrane protein in the IMM with an uneven number of transmembrane domains, is a novel TIM22 substrate. SFXN1 deficiency specifically impairs Complex III (CIII) biogenesis, activity, and assembly, compromising coenzyme Q levels. This CIII dysfunction is independent of one-carbon metabolism, the known primary role for SFXN1 as a mitochondrial serine transporter. Instead, SFXN1 supports CIII function by participating in heme and central carbon metabolism. Our findings highlight the multiple ways that SFXN1-based amino acid transport impacts mitochondrial and cellular metabolic efficiency.
In mitochondria, the carrier translocase (TIM22 complex) facilitates membrane insertion of multi-spanning proteins with internal targeting signals into the inner membrane [1-3]. Tom70, a subunit of TOM complex, represents the major receptor for these precursors [2, 4-6]. After transport across the outer membrane, the hydrophobic carriers engage with the small TIM protein complex composed of Tim9 and Tim10 for transport across the intermembrane space (IMS) toward the TIM22 complex [7-12]. Tim22 represents the pore-forming core unit of the complex [13, 14]. Only a small subset of TIM22 cargo molecules, containing four or six transmembrane spans, have been experimentally defined. Here, we used a tim22 temperature-conditional mutant to define the TIM22 substrate spectrum. Along with carrier-like cargo proteins, we identified subunits of the mitochondrial pyruvate carrier (MPC) as unconventional TIM22 cargos. MPC proteins represent substrates with atypical topology for this transport pathway. In agreement with this, a patient affected in TIM22 function displays reduced MPC levels. Our findings broaden the repertoire of carrier pathway substrates and challenge current concepts of TIM22-mediated transport processes.
The evolution of mitochondrial protein import and the systems that mediate it marks the boundary between the endosymbiotic ancestor of mitochondria and a true organelle that is under the control of the nucleus. Protein import has been studied in great detail in Saccharomyces cerevisiae. More recently it has also been extensively investigated in the parasitic protozoan Trypanosoma brucei making it arguably the second best studied system. Here I provide a comparative analysis of the protein import complexes of yeast and trypanosomes. Together with data from other systems, this allows to reconstruct the ancestral features of import complexes that were present in the last eukaryotic common ancestor (LECA) and to identify which subunits were added later in evolution. I discuss how these data can be translated into plausible scenarios providing insights into the evolution of (i) outer membrane protein import receptors, (ii) proteins involved in biogenesis of α-helically anchored outer membrane proteins, and (iii) of the intermembrane space import and assembly system. Finally, I show that the unusual presequence-associated import motor of trypanosomes suggests a scenario of how the two ancestral inner membrane protein translocases present in LECA evolved into the single bifunctional one found in extant trypanosomes.
In the past three decades, significant advances have been made in providing the biochemical background of TOM-mediated protein translocation into mitochondria. In the light of recent cryoelectron microscopy-derived structures of TOM isolated from Neurospora crassa and Saccharomyces cerevisiae, the interpretation of biochemical and biophysical studies of TOMmediated protein transport into mitochondria now rests on a solid basis. In this review, we compare the sub-nanometer structure of N. crassa TOM core complex with that of yeast. Both structures reveal remarkably well-conserved symmetrical dimers of ten membrane protein subunits. The structural data also validate predictions of weakly stable regions in the transmembrane β-barrel domains of the protein-conducting subunit Tom40, which signal the existence of β-strands located in interfaces of protein-protein interactions.
Background:
Tim21, a subunit of a highly dynamic translocase of the inner mitochondrial membrane (TIM23) complex, translocates proteins by interacting with subunits in the translocase of the outer membrane (TOM) complex and Tim23 channel in the TIM23 complex. A loop segment in Tim21, which is in close proximity of the binding site of Tim23, has different conformations in X-ray, NMR and new crystal contact-free space (CCFS) structures. MD simulations can provide information on the structure and dynamics of the loop in solution.
Methods:
The conformational ensemble of the loop was characterized using loop modeling and molecular dynamics (MD) simulations.
Results:
MD simulations confirmed mobility of the loop. Multidimensional scaling and clustering were used to characterize the dynamic conformational ensemble of the loop. Free energy landscape showed that the CCFS crystal structure occupied a low energy region as compared to the conventional X-ray crystal structure. Analysis of crystal packing indicates that the CCFS provides larger conformational space for the motions of the loop.
Conclusions:
Our work reported the conformational ensemble of the loop in solution, which is in agreement with the structure obtained from CCFS approach. The combination of the experimental techniques and computational methods is beneficial for studying highly flexible regions of proteins.
General significance:
Computational methods, such as loop modeling and MD simulations, have proved to be useful for studying conformational flexibility of proteins. These methods in integration with experimental techniques such as CCFS has the potential to transform the studies on flexible regions of proteins.
We have identified a new protein, Tim54p, located in the yeast mitochondrial inner membrane. Tim54p is an essential import component, required for the insertion of at least two polytopic proteins into the inner membrane, but not for the translocation of precursors into the matrix. Several observations suggest that Tim54p and Tim22p are part of a protein complex in the inner membrane distinct from the previously characterized Tim23p-Tim17p complex. First, multiple copies of the TIM22 gene, but not TIM23 or TIM17, suppress the growth defect of a tim54-1 temperature-sensitive mutant. Second, Tim22p can be coprecipitated with Tim54p from detergent-solubilized mitochondria, but Tim54p and Tim22p do not interact with either Tim23p or Tim17p. Finally, the tim54-1 mutation destabilizes the Tim22 protein, but not Tim23p or Tim17p. Our results support the idea that the mitochondrial inner membrane carries two independent import complexes: one required for the translocation of proteins across the inner membrane (Tim23p–Tim17p), and the other required for the insertion of proteins into the inner membrane (Tim54p–Tim22p).
The preprotein translocase of the outer mitochondrial membrane (TOM complex) contains one essential subunit, the channel Tom40. The assembly pathway of the precursor of Tom40 involves the TOM complex and the sorting and assembly machinery (SAM complex) with the non-essential subunit Mas37. We have identified Sam50, the second essential protein of the mitochondrial outer membrane. Sam50 contains a beta-barrel domain conserved from bacteria to man and is a subunit of the SAM complex. Yeast mutants of Sam50 are defective in the assembly pathways of Tom40 and the abundant outer membrane protein porin, while the import of matrix proteins is not affected. Thus the protein sorting and assembly machinery of the mitochondrial outer membrane involves an essential, conserved protein.
We analysed the import pathway of Tim23 and of Tim17, components of the mitochondrial import machinery for matrix-targeted preproteins. Tim23 contains two independent import signals. One is located within the first 62 amino acid residues of the hydrophilic domain that, in the assembled protein, is exposed to the intermembrane space. This signal mediates translocation of Tim23 across the outer membrane independently of the membrane potential, DeltaPsi. A second import signal is located in the C-terminal membrane-integrated portion of Tim23. It mediates translocation across the outer membrane and insertion into the inner membrane in a strictly DeltaPsi-dependent fashion. Structurally, Tim17 is related to Tim23 but lacks a hydrophilic domain. It contains an import signal in the C-terminal half and its import requires DeltaPsi. The DeltaPsi-dependent import signals of Tim23 and Tim17 are located at corresponding sites in these two homologous proteins. They exhibit features reminiscent of the positively charged N-terminal presequences of matrix-targeted precursors. Import of Tim23 and its insertion into the inner membrane requires Tim22 but not functional Tim23. Thus, biogenesis of the Tim23.17 complex depends on the Tim22 complex, which is the translocase identified as mediating the import of carrier proteins.
The transport of precursor proteins into mitochondria requires an energized inner membrane. We report here that the import of various precursor proteins showed a differential sensitivity to treatment of the mitochondria with the uncoupler carbonyl cyanide m-chlorophenylhydrazone. The differential inhibition by carbonyl cyanide m-chlorophenylhydrazone was not influenced by the length of the precursor, the presence of mature protein parts, or the folding state of the precursor but was specific for the presequence. Moreover, only the membrane potential delta psi and not the total proton motive force was required for the transport of precursors, indicating that protein translocation across the inner membrane is not driven by a movement of protons. We conclude that delta psi (negative inside) is needed for the translocation of the positively charged presequences, possibly via an electrophoretic effect.
We have probed the environment of a precursor protein stuck in mitochondrial import sites using cleavable bifunctional crosslinking reagents. The stuck precursor was crosslinked to a 70 kd protein which, by immunological techniques, was shown to be a matrix protein. The protein was purified to homogeneity by ATP-Sepharose chromatography and partially sequenced. Fourteen of its 15 N-terminal amino acids were identical to residues 24-38 of the protein encoded by the nuclear gene SSC1, which had been proposed to encode a dnaK-like 70 kd mitochondrial stress protein. Our data imply that this mitochondrial hsp70 is made with a cleavable matrix-targeting sequence composed of 23 residues. The complex containing stuck precursor, mitochondrial hsp70, and ISP42 could be solubilized from mitochondria by the non-ionic detergent Triton X-100 even without crosslinking, suggesting tight association of these three components. As the stuck precursor is arrested at an early stage of translocation, mitochondrial hsp70 may initiate the events that lead to refolding of imported precursors in the matrix space.
The precursor of the mitochondrial inner membrane protein ADP/ATP carrier is cytoplasmically synthesized without an amino-terminal peptide extension. We constructed a truncated precursor lacking the 103 amino acids from the amino terminus (about a third of the protein). Import of the truncated precursor into mitochondria showed the import characteristics of the authentic precursor, including nucleoside triphosphate dependence, requirement for a protease-sensitive component on the mitochondrial surface, two-step specific binding to the outer membrane, and membrane potential-dependent translocation into the inner membrane. We conclude that, in contrast to all other mitochondrial precursor proteins studied so far, domains of the ADP/ATP carrier distant from the amino terminus can carry specific targeting information for transport into mitochondria.
The ADP/ATP carrier of yeast (309 amino acids) is an abundant transmembrane protein of the mitochondrial inner membrane whose import involves well-defined steps (Pfanner, N., and Neupert, W. (1987) J. Biol. Chem. 262, 7528-7536). Analysis of the in vitro import of gene fusion products containing ADP/ATP carrier (AAC) sequences at the amino terminus and mouse dihydrofolate reductase (DHFR) at the carboxyl terminus indicates that the first 72 amino acids of the soluble carrier protein, a hydrophilic region of the protein, are not by themselves sufficient for initial binding to the AAC receptor on the mitochondrial surface. However, an AAC-DHFR gene fusion containing the first 111 residues of the ADP/ATP carrier protein exhibited binding to mitochondria at low temperature (2 degrees C) and internalization at 25 degrees C to a mitochondrial space protected from proteinase K in the same manner as the wild-type ADP/ATP carrier protein. The AAC-DHFR protein, in contrast to the wild-type AAC protein imported into mitochondria under optimal conditions, remained extractable at alkaline pH and appeared to be blocked at an intermediate step in the AAC import pathway. Based on its extraction properties, this AAC-DHFR hybrid is proposed to be associated with a proteinaceous component of the import apparatus within mitochondria. These data indicate that the import determinants for the AAC protein are not located at its extreme amino terminus and that protein determinants distal to the first 111 residues of the carrier may be necessary to move the protein beyond the alkali-extractable step in the biogenesis of a functional AAC protein.
Transport of the precursor to the ADP/ATP carrier from the cytosol into the mitochondrial inner membrane was resolved into several consecutive steps. The precursor protein was trapped at distinct stages of the import pathway and subsequently chased to the mature form. In a first reaction, the precursor interacts with a protease-sensitive component on the mitochondrial surface. It then reaches intermediate sites in the outer membrane which are saturable and where it is protected against proteases. This translocation intermediate can be extracted at alkaline pH. We suggest that it is anchored to the membrane by a so far unknown proteinaceous component. The membrane potential delta psi-dependent entrance of the ADP/ATP carrier into the inner membrane takes place at contact sites between outer and inner membranes. Completion of translocation into the inner membrane can occur in the absence of delta psi. A cytosolic component which is present in reticulocyte lysate and which interacts with isolated mitochondria is required for the specific binding of the precursor to mitochondria.
Most mitochondrial proteins are encoded in the nucleus and synthesized in the cytoplasm as larger precursors containing NH2-terminal 'leader' peptides. To test whether a leader peptide is sufficient to direct mitochondrial import, we fused the cloned nucleotide sequence encoding the leader peptide of the mitochondrial matrix enzyme ornithine transcarbamylase (OTC) with the sequence encoding the cytosolic enzyme dihydrofolate reductase (DHFR). The fused sequence, joined with SV40 regulatory elements, was introduced along with a selectable marker into a mutant CHO cell line devoid of endogenous DHFR. In stable transformants, the predicted 26-K chimeric precursor protein and two additional proteins, 22 K and 20 K, were detected by immunoprecipitation with anti-DHFR antiserum. In the presence of rhodamine 6G, an inhibitor of mitochondrial import, only the chimeric precursor was detected. Immunofluorescent staining of stably transformed cells with anti-DHFR antiserum produced a pattern characteristic of mitochondrial localization of immunoreactive material. When the chimeric precursor was synthesized in a cell-free system and incubated post-translationally with isolated rat liver mitochondria, it was imported and converted to a major product of 20 K that associated with mitochondria and was resistant to proteolytic digestion by externally added trypsin. Thus, both in intact cells and in vitro, a leader sequence is sufficient to direct the post-translational import of a chimeric precursor protein by mitochondria.
The precursor form of Neurospora crassa mitochondrial ADP/ATP carrier synthesized in a cell-free protein-synthesizing system can be imported into isolated mitochondria. If the mitochondrial transmembrane potential is abolished, import does not occur but the precursor binds to the mitochondrial surface. Upon reestablishment of the membrane potential, the bound precursor is imported. This occurs without dissociation of the bound precursor from the mitochondrial surface. We conclude that the binding observed represents an interaction with receptor sites and thus is an early step in the import pathway.
The import of preproteins into mitochondria involves translocation of the polypeptide chains through putative channels in the outer and inner membranes. Preprotein-binding proteins are needed to drive the unidirectional translocation of the precursor polypeptides. Two of these preprotein-binding proteins are the peripheral inner membrane protein MIM44 and the matrix heat shock protein hsp70. We report here that MIM44 is mainly exposed on the matrix side, and a fraction of mt-hsp70 is reversibly bound to the inner membrane. Mt-hsp70 binds to MIM44 in a 1:1 ratio, suggesting that mt-hsp70 is localizing to the membrane via its interaction with MIM44. Formation of the complex requires a functional ATPase domain of mt-hsp70. Addition of Mg-ATP leads to dissociation of the complex. Overexpression of mt-hsp70 rescues the protein import defect of mutants in MIM44; conversely, overexpression of MIM44 rescues protein import defects of mt-hsp70 mutants. In addition, yeast strains with conditional mutations in both MIM44 and mt-hsp70 are barely viable, showing a synthetic growth defect compared to strains carrying single mutations. We propose that MIM44 and mt-hsp70 cooperate in translocation of preproteins. By binding to MIM44, mt-hsp70 is recruited at the protein import sites of the inner membrane, and preproteins arriving at MIM44 may be directly handed over to mt-hsp70.
The protein import system of the yeast mitochondrial inner membrane includes at least three membrane proteins that presumably form a transmembrane channel as well as several chaperone proteins that mediate the import and refolding of precursor proteins. We show that one of the membrane proteins, Isp45, spans the mitochondrial inner membrane yet is extracted from this membrane at high pH. Solubilization of mitochondria with a nonionic detergent releases Isp45 as a complex with the chaperones mitochondrial hsp70 (mhsp70) and GrpEp. Both chaperones reversibly dissociate from Isp45 upon addition of ATP or adenosine 5'-[gamma-thio]triphosphate, suggesting that dissociation requires the binding of ATP. Control experiments indicate that the interaction between mhsp70 and Isp45 occurs in the intact mitochondria. We propose that Isp45 lines the inside of a proteinaceous channel across the inner membrane and that it is the membrane anchor for an ATP-driven "import motor" composed of mhsp70 and GrpEp. This arrangement is reminiscent of the protein transport systems of the yeast endoplasmic reticulum and the bacterial plasma membrane.
To identify new components that mediate mitochondrial protein import, we analyzed mas6, an import mutant in the yeast Saccharomyces cerevisiae. mas6 mutants are temperature sensitive for viability, and accumulate mitochondrial precursor proteins at the restrictive temperature. We show that mas6 does not correspond to any of the presently identified import mutants, and we find that mitochondria isolated from mas6 mutants are defective at an early stage of the mitochondrial protein import pathway. MAS6 encodes a 23-kD protein that contains several potential membrane spanning domains, and yeast strains disrupted for MAS6 are inviable at all temperatures and on all carbon sources. The Mas6 protein is located in the mitochondrial inner membrane and cannot be extracted from the membrane by alkali treatment. Antibodies to the Mas6 protein inhibit import into isolated mitochondria, but only when the outer membrane has been disrupted by osmotic shock. Mas6p therefore represents an essential import component located in the mitochondrial inner membrane.
Preprotein import into mitochondria is mediated by translocases located in the outer and inner membranes (Tom and Tim) and a matrix Hsp70-Tim44 driving system. By blue native electrophoresis, we identify an approximately 90K complex with assembled Tim23 and Tim17 as the core of the inner membrane import site for presequence-containing preproteins. Preproteins spanning the two membranes link virtually all Tim core complexes with one in four Tom complexes in a stable 600K supercomplex. Neither mtHsp70 nor Tim44 are present in stoichiometric amounts in the 600K complex. Preproteins in transit stabilize the Tim core complex, preventing an exchange of subunits. Our studies define a central role for the Tim core complexes in mitochondrial protein import; they are not passive diffusion channels, but can stably interact with preproteins and determine the number of translocation contact sites. We propose the hypothesis that mtHsp70 functions in protein import not only by direct interaction with preproteins, but also by exerting a regulatory effect on the Tim channel.
Mitochondrial protein import is thought to involve the sequential interaction of preproteins with binding sites on cis and trans sides of the membranes. For translocation across the outer membrane, preproteins first interact with the cytosolic domains of import receptors (cis) and then are translocated through a general import pore, in a process proposed to involve binding to a trans site on the intermembrane space (IMS) side. Controversial results have been reported for the role of the IMS domain of the essential outer membrane protein Tom22 in formation of the trans site. We show with different mutant mitochondria that a lack of the IMS domain only moderately reduces the direct import of preproteins with N-terminal targeting sequences. The dependence of import on the IMS domain of Tom22 is significantly enhanced by removing the cytosolic domains of import receptors or by performing import in two steps, i.e., accumulation of a preprotein at the outer membrane in the absence of a membrane potential (delta psi) and subsequent import after reestablishment of a delta psi. After the removal of cytosolic receptor domains, two-step import of a cleavable preprotein strictly requires the IMS domain. In contrast, preproteins with internal targeting information do not depend on the IMS domain of Tom22. We conclude that the negatively charged IMS domain of Tom22 functions as a trans binding site for preproteins with N-terminal targeting sequences, in agreement with the acid chain hypothesis of mitochondrial protein import.
We have identified a new protein, Tim54p, located in the yeast mitochondrial inner membrane. Tim54p is an essential import component, required for the insertion of at least two polytopic proteins into the inner membrane, but not for the translocation of precursors into the matrix. Several observations suggest that Tim54p and Tim22p are part of a protein complex in the inner membrane distinct from the previously characterized Tim23p-Tim17p complex. First, multiple copies of the TIM22 gene, but not TIM23 or TIM17, suppress the growth defect of a tim54-1 temperature-sensitive mutant. Second, Tim22p can be coprecipitated with Tim54p from detergent-solubilized mitochondria, but Tim54p and Tim22p do not interact with either Tim23p or Tim17p. Finally, the tim54-1 mutation destabilizes the Tim22 protein, but not Tim23p or Tim17p. Our results support the idea that the mitochondrial inner membrane carries two independent import complexes: one required for the translocation of proteins across the inner membrane (Tim23p-Tim17p), and the other required for the insertion of proteins into the inner membrane (Tim54p-Tim22p).
In order to reach the inner membrane of the mitochondrion, multispanning carrier proteins must cross the aqueous intermembrane
space. Two essential proteins of that space, Tim10p and Tim12p, were shown to mediate import of multispanning carriers into
the inner membrane. Both proteins formed a complex with the inner membrane protein Tim22p. Tim10p readily dissociated from
the complex and was required to transport carrier precursors across the outer membrane; Tim12p was firmly bound to Tim22p
and mediated the insertion of carriers into the inner membrane. Neither protein was required for protein import into the other
mitochondrial compartments. Both proteins may function as intermembrane space chaperones for the highly insoluble carrier
proteins.
Mitochondrial precursor proteins with basic targeting signals may be transported across the outer membrane by sequential binding to acidic receptor sites of increasing affinity. To test this 'acid chain' hypothesis, we assayed the interaction of mitochondrial precursors with three acidic receptor domains: the cytosolic domain of Tom20 and the intermembrane space domain of Tom22 and Tim23. The apparent affinity and salt resistance of precursor binding increased in the order Tom20<Tom22 (internal)<Tim23. Precursor binding to the three acidic receptor domains and to the pure cytosolic domain of Tom70 was inhibited by excess targeting peptide, but not by an equally basic control peptide. In this membrane-free and defined system, a precursor pre-bound to the Tom70 or Tom20 domain was transferred efficiently to the Tim23 domain. Transfer was stimulated by the internal Tom22 domain and was much less efficient in the reverse direction. Precursors destined for the outer membrane bound only to Tom20, but not to the internal Tom22 or the Tim23 domain, and a precursor destined for the inner membrane bound only to the Tom20 and the internal Tom22 domain, but not to the Tim23 domain. These results suggest that specific and sequential binding of a targeting signal to strategically situated acidic receptors delivers a precursor across the outer membrane and contributes to intramitochondrial sorting of imported proteins.
The Tim23 protein is an essential inner membrane (IM) component of the yeast mitochondrial protein import pathway. Tim23p does not carry an amino-terminal presequence; therefore, the targeting information resides within the mature protein. Tim23p is anchored in the IM via four transmembrane segments and has two positively charged loops facing the matrix. To identify the import signal for Tim23p, we have constructed several altered versions of the Tim23 protein and examined their function and import in yeast cells, as well as their import into isolated mitochondria. We replaced the positively charged amino acids in one or both loops with alanine residues and found that the positive charges are not required for import into mitochondria, but at least one positively charged loop is required for insertion into the IM. Furthermore, we find that the signal to target Tim23p to mitochondria is carried in at least two of the hydrophobic transmembrane segments. Our results suggest that Tim23p contains separate import signals: hydrophobic segments for targeting Tim23p to mitochondria, and positively charged loops for insertion into the IM. We therefore propose that Tim23p is imported into mitochondria in at least two distinct steps.
Tim10p, a protein of the yeast mitochondrial intermembrane space, was shown previously to be essential for the import of multispanning carrier proteins from the cytoplasm into the inner membrane. We now identify Tim9p, another essential component of this import pathway. Most of Tim9p is associated with Tim10p in a soluble 70 kDa complex. Tim9p and Tim10p co-purify in successive chromatographic fractionations and co-immunoprecipitated with each other. Tim9p can be cross-linked to a partly translocated carrier protein. A small fraction of Tim9p is bound to the outer face of the inner membrane in a 300 kDa complex whose other subunits include Tim54p, Tim22p, Tim12p and Tim10p. The sequence of Tim9p is 25% identical to that of Tim10p and Tim12p. A Ser67-->Cys67 mutation in Tim9p suppresses the temperature-sensitive growth defect of tim10-1 and tim12-1 mutants. Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane proteins across the aqueous intermembrane space.
We have identified Tim9, a new component of the TIM22.54 import machinery, which mediates transport of proteins into the inner membrane of mitochondria. Tim9, an essential protein of Saccharomyces cerevisiae, shares sequence similarity with Tim10 and Tim12. Tim9 is located in the mitochondrial intermembrane space and is organized into two distinct hetero-oligomeric assemblies with Tim10 and Tim12. One complex contains Tim9 and Tim10. The other complex contains Tim9, Tim10 and Tim12 and is tightly associated with Tim22 in the inner membrane. The TIM9.10 complex is more abundant than the TIM9.10.12 complex and mediates partial translocation of mitochondrial carriers proteins across the outer membrane. The TIM9.10.12 complex assists further translocation into the inner membrane in association with TIM22.54.
Preproteins destined for mitochondria either are synthesized with amino-terminal signal sequences, termed presequences, or possess internal targeting information within the protein. The preprotein translocase of the outer mitochondrial membrane (designated Tom) contains specific import receptors. The cytosolic domains of three import receptors, Tom20, Tom22, and Tom70, have been shown to interact with preproteins. Little is known about the internal targeting information in preproteins and the distribution of binding sequences for the three import receptors. We have studied the binding of the purified cytosolic domains of Tom20, Tom22, and Tom70 to cellulose-bound peptide scans derived from a presequence-carrying cleavable preprotein, cytochrome c oxidase subunit IV, and a non-cleavable preprotein with internal targeting information, the phosphate carrier. All three receptor domains are able to bind efficiently to linear 13-mer peptides, yet with different specificity. Tom20 preferentially binds to presequence segments of subunit IV. Tom22 binds to segments corresponding to the carboxyl-terminal part of the presequence and the amino-terminal part of the mature protein. Tom70 does not bind efficiently to any region of subunit IV. In contrast, Tom70 and Tom20 bind to multiple segments within the phosphate carrier, yet the amino-terminal region is excluded. Both charged and uncharged peptides derived from the phosphate carrier show specific binding properties for Tom70 and Tom20, indicating that charge is not a critical determinant of internal targeting sequences. This feature contrasts with the crucial role of positively charged amino acids in presequences. Our results demonstrate that linear peptide segments of preproteins can serve as binding sites for all three receptors with differential specificity and imply different mechanisms for translocation of cleavable and non-cleavable preproteins.
Members of the mitochondrial carrier family such as the ADP/ATP carrier (AAC) are composed of three structurally related modules. Here we show that each of the modules contains a mitochondrial import signal recognized by Tim10 and Tim12 in the intermembrane space. The first and the second module are translocated across the outer membrane independently of the membrane potential, DeltaDeltapsipsi, but they are not inserted into the inner membrane. The third module interacts tightly with the TOM complex and thereby prevents complete translocation of the precursor across the outer membrane. At this stage, binding of a TIM9.10 complex confers a topology to the translocation intermediate which reflects the modular structure of the AAC. The precursor is then transferred to the TIM9.10.12 complex, still interacting with the TOM complex. Release of the precursor from the TOM complex and insertion into the inner membrane by the TIM22.54 complex requires a DeltaDeltapsipsi-responsive signal in the third module.
We performed a comprehensive approach to determine the proteome of Saccharomyces cerevisiae mitochondria. The proteins of highly pure yeast mitochondria were separated by several independent methods and analyzed by tandem MS. From >20 million MS spectra, 750 different proteins were identified, indicating an involvement of mitochondria in numerous cellular processes. All known components of the oxidative phosphorylation machinery, the tricarboxylic acid cycle, and the stable mitochondria-encoded proteins were found. Based on the mitochondrial proteins described in the literature so far, we calculate that the identified proteins represent approximate to90% of all mitochondrial proteins. The function of a quarter of the identified proteins is unknown. The mitochondrial proteome will provide an important database for the analysis of new mitochondrial and mitochondria-associated functions and the characterization of mitochondrial diseases.
Chloroplasts are organelles of endosymbiotic origin, and they transferred most of their genetic information to the host nucleus during this process. They therefore have to import more than 95% of their protein complement post-translationally from the cytosol. In vivo results from the model plant Arabidopsis thaliana — together with biochemical, biophysical and structural data from other plants — now allow us to outline the mechanistic details of the molecular machines that facilitate this translocation. It has become clear that chloroplasts evolved a unique translocation system, which is inherited, in part, from their bacterial ancestors.
Mitochondrial heat shock protein 70 (mtHsp70) functions in unfolding, translocation, and folding of imported proteins. Controversial models of mtHsp70 action have been discussed: (1) physical trapping of preproteins is sufficient to explain the various mtHsp70 functions, and (2) unfolding of preproteins requires an active motor function of mtHsp70 (“pulling”). Intragenic suppressors of a mutant mtHsp70 separate two functions: a nonlethal folding defect caused by enhanced trapping of preproteins, and a conditionally lethal unfolding defect caused by an impaired interaction of mtHsp70 with the membrane anchor Tim44. Even enhanced trapping in wild-type mitochondria does not generate a pulling force. The motor function of mtHsp70 cannot be explained by passive trapping alone but includes an essential ATP-dependent interaction with Tim44 to generate a pulling force and unfold preproteins.
Protein translocation systems consist of complex molecular machines whose activities are not limited to unidirectional protein targeting. Protein translocons and their associated receptor systems can be viewed as dynamic modular units whose interactions, and therefore functions, are regulated in response to specific signals. This flexibility allows translocons to interact with multiple signal receptor systems to manage the targeting of topologically distinct classes of proteins, to mediate targeting to different suborganellar compartments, and to respond to stress and developmental cues. Furthermore, the activities of translocons are tightly coordinated with downstream events, thereby providing a direct link between targeting and protein maturation.
The genome of Saccharomyces cerevisiae encodes 35 putative members of the mitochondrial carrier family. Known members of this family transport substrates and products across the inner membranes of mitochondria. We are attempting to identify the functions of the yeast mitochondrial transporters via high-yield expression in Escherichia coli and/or S. cerevisiae, purification and reconstitution of their protein products into liposomes, where their transport properties are investigated. With this strategy, we have already identified the functions of seven S. cerevisiae gene products, whose structural and functional properties assigned them to the mitochondrial carrier family. The functional information obtained in the reconstituted system and the use of knock-out yeast strains can be usefully exploited for the investigation of the physiological role of individual transporters. Furthermore, the yeast carrier sequences can be used to identify the orthologous proteins in other organisms, including man.
Selection for regain-of-function mutations in the yeast ADP/ATP carrier AAC2 has revealed an unexpected series of charge-pairs. Four of the six amino acids involved are found in the mitochondrial energy transfer motifs used to define this family of proteins. As such, the results found with the ADP/ATP carrier may apply to the family as a whole. Mitochondrial carriers are built from three homologous domains, each with the conserved motif PX(D,E)XX(K,R). Neutralization of the conserved positive charges at K48, R152 or R252 in these motifs results in respiration defective yeast. Neutralization of the negative charges at D149 and D249 also make respiration defective yeast, though E45G or E45Q mutants are able to grow on glycerol. Regain of function occurs when a complementary charge is lost from another site in the molecule. This phenomenon has been observed independently eight times and thus is strong evidence for charge-pairs existing between the affected residues. Five different charge-pairs have been detected in the yeast AAC2 by this method and three more can be predicted based on homology between the domains. The highly conserved charge-pairs occurring within or between the three mitochondrial energy transfer signatures seem to be a critical feature of mitochondrial carrier structure, independent of the substrates transported. Conformational switching between alternative charge-pairs may constitute part of the basis for transport.
A discontinuous electrophoretic system for the isolation of membrane proteins from acrylamide gels has been developed using equipment for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Coomassie dyes were introduced to induce a charge shift on the proteins and aminocaproic acid served to improve solubilization of membrane proteins. Solubilized mitochondria or extracts of heart muscle tissue, lymphoblasts, yeast, and bacteria were applied to the gels. From cells containing mitochondria, all the multiprotein complexes of the oxidative phosphorylation system were separated within one gel. The complexes were resolved into the individual polypeptides by second-dimension Tricine-SDS-PAGE or extracted without SDS for functional studies. The recovery of all respiratory chain complexes was almost quantitative. The percentage recovery of functional activity depended on the respective protein complex studied and was zero for some complexes, but almost quantitative for others. The system is especially useful for small scale purposes, e.g., separation of radioactively labeled membrane proteins, N-terminal protein sequencing, preparation of proteins for immunization, and diagnostic studies of inborn neuromuscular diseases.
Mitochondrial precursor proteins are known to be imported at sites of close contact between mitochondrial outer and inner membranes. We have identified translocation intermediates exposed to the intermembrane space, including the precursor of the ADP/ATP carrier accumulated at the general insertion site GIP, and the precursor of F1-ATPase subunit beta accumulated on its import pathway at low levels of ATP. These results suggest that mitochondrial contact sites are not sealed structures, but that polypeptides pass (at least partly) through the intermembrane space on their route from the outer membrane to the inner membrane.
We have identified a mitochondrial outer membrane protein of 72 kd (MOM72) that exhibits the properties of an import receptor for the ADP/ATP carrier (AAC), the most abundant mitochondrial protein. Monospecific antibodies and Fab fragments against MOM72 selectively inhibit import of AAC at the level of specific binding to the mitochondria. AAC bound to the mitochondrial surface is coprecipitated with antibodies against MOM72 after lysis of mitochondria with detergent. MOM72 thus has a complementary function to that of MOM19, which acts as an import receptor for the majority of mitochondrial proteins studied so far but not for the AAC. The import pathway of the precursor of MOM72 appears to involve MOM19 as receptor.
By analysis of a temperature-sensitive yeast mutant, a heat-shock protein in the matrix of mitochondria, mitochondrial hsp70 (Ssc1p), is found to be involved both in translocation of nuclear-encoded precursor proteins across the mitochondrial membranes and in (re)folding of imported proteins in the matrix.
The role of nucleoside triphosphates (NTPs) in mitochondrial protein import was investigated with the precursors of N. crassa ADP/ATP carrier, F1-ATPase subunit beta, F0-ATPase subunit 9, and fusion proteins between subunit 9 and mouse dihydrofolate reductase. NTPs were necessary for the initial interaction of precursors with the mitochondria and for the completion of translocation of precursors from the mitochondrial surface into the mitochondria. Higher levels of NTPs were required for the latter reactions as compared with the early stages of import. Import of precursors having identical presequences but different mature protein parts required different levels of NTPs. The sensitivity of precursors in reticulocyte lysate to proteases was decreased by removal of NTPs and increased by their readdition. We suggest that the hydrolysis of NTPs is involved in modulating the folding state of precursors in the cytosol, thereby conferring import competence.
The transfer of cytoplasmically synthesized precursor proteins into or across the inner mitochondrial membrane is dependent on energization of the membrane. To investigate the role of this energy requirement, a buffer system was developed in which efficient import of ADP/ATP carrier into mitochondria from the receptor-bound state occurred. This import was rapid and was dependent on divalent cations, whereas the binding of precursor proteins to the mitochondrial surface was slow and was independent of added divalent cations. Using this buffer system, the import of ADP/ATP carrier could be driven by a valinomycin-induced potassium diffusion potential. The protonophore carbonylcyanide m-chlorophenyl-hydrazone was not able to abolish this import. Imposition of a delta pH did not stimulate the import. We conclude that the membrane potential delta psi itself and not the total protonmotive force delta p is the required energy source.
The cleavable prepiece of the precursor to yeast cytochrome c oxidase subunit IV (an imported mitochondrial protein) was attached to the amino-terminus of mouse dihydrofolate reductase (a cytosolic protein) by gene fusion. The resulting fusion protein was imported into the matrix of isolated, energized yeast mitochondria and cleaved to a polypeptide whose size was similar to that of authentic dihydrofolate reductase.
The mitochondrial ADP/ATP carrier is an integral transmembrane protein of the inner membrane. It is synthesized on cytoplasmic ribosomes. Kinetic data suggested that this protein is transferred into mitochondria in a posttranslational manner. The following results provide further evidence for such a mechanism and provide information on its details.
1. In homologous and heterologous translation systems the newly synthesized ADP/ATP carrier protein is present in the postribosomal supernatant.
2. Analysis by density gradient centrifugation and gel filtration shows, that the ADP/ATP carrier molecules in the postribosomal fraction are present as soluble complexes with apparent molecular weights of about 120000 and 500000 or larger. The carrier binds detergents such as Triton X-100 and deoxycholate forming mixed micelles with molecular weights of about 200000–400000.
3. Incubation of a postribosomal supernatant of a reticulocyte lysate containing newly synthesized ADP/ATP carrier with mitochondria isolated from Neurospora spheroplasts results in efficient transfer of the carrier into mitochondria. About 20–30% of the transferred carrier are resistant to proteinase in whole mitochondria. The authentic mature protein is also largely resistant to proteinase in whole mitochondria and sensitive after lysis of mitochondria with detergent. Integrity of mitochondria is a prerequisite for translocation into proteinase resistant position.
4. The transfer in vitro into a proteinase-resistant form is inhibited by the uncoupler carbonyl-cyanide m-chlorophenylhydrazone but not the proteinase-sensitive binding.
These observations suggest that the posttranslational transfer of ADP/ATP carrier occurs via the cytosolic space through a soluble oligomeric precursor form. This precursor is taken up by intact mitochondria into an integral position in the membrane. These findings are considered to be of general importance for the intracellular transfer of insoluble membrane proteins. They support the view that such proteins can exist in a water-soluble form its precursors and upon integration into the membrane undergo a conformational change. Uptake into the membrane may involve the cleavage of an additional sequence in some proteins, but this appears not to be a prerequisite as demonstrated by the ADP/ATP carrier protein.
Protein translocation into mitochondria requires the mitochondrial protein Hsp70. This molecular chaperone of the mitochondrial matrix is recruited to the protein import machinery by MIM44, a component associated with the inner membrane of the mitochondria. Formation of the mt-Hsp70/MIM44 complex is regulated by ATP. MIM44 and mt-Hsp 70 interact in a sequential manner with incoming segments of unfolded preproteins and thereby facilitate stepwise vectorial translocation of proteins across the mitochondrial membranes. The complex appears to act as a molecular ratchet which is energetically driven by the hydrolysis of ATP.
Three proteins of the mitochondrial inner membrane are known that are essential for the viability of yeast and seem to be involved in import of preproteins; the integral membrane proteins MIM17 and MIM23 and the peripheral membrane protein MIM44, MIM17 and MIM23 are homologous to each other in their hydrophobic domain, expose their termini to the intermembrane space, and span the inner membrane up to four times, each. A preprotein in transit across the mitochondrial membrane is specifically cross-linked to MIM17, MIM23, MIM44, and matrix hsp70. We conclude that MIM17 and MIM23 are integral parts of a preprotein translocation channel and cooperate with MIM44 and hsp70 at the same protein import site.
Mitochondrial precursor proteins made in the cytosol bind to a hetero-oligomeric protein import receptor on the mitochondrial surface and then pass through the translocation channel across the outer membrane. This translocation step is accelerated by an acidic domain of the receptor subunit Mas22p, which protrudes into the intermembrane space. This 'trans' domain of Mas22p specifically binds functional mitochondrial targeting peptides with a Kd of < 1 microM and is required to anchor the N-terminal targeting sequence of a translocation-arrested precursor in the intermembrane space. If this Mas22p domain is deleted, respiration-driven growth of the cells is compromised and import of different precursors into isolated mitochondria is inhibited 3- to 8-fold. Binding of precursors to the mitochondrial surface appears to be mediated by cytosolically exposed acidic domains of the receptor subunits Mas20p and Mas22p. Translocation of a precursor across the outer membrane thus appears to involve sequential binding of the precursor's basic and amphiphilic targeting signal to acidic receptor domains on both sides of the membrane.
Tim23, an essential component of the protein import machinery of the inner membrane of mitochondria (TIM complex), forms dimers that display a dynamic behavior. Dimer formation is promoted by the membrane potential delta psi. Binding of a matrix targeting sequence to Tim23 triggers dimer dissociation. Monomeric Tim23 is present when a preprotein chain is in transit across the TIM complex. Dimerization of Tim23 is dependent on the second half of its N-terminal hydrophilic domain, which is exposed to the intermembrane space. This segment contains a heptad leucine repeat motif with a predicted capacity for dimer formation. We propose that Tim23 exerts a key function in protein import: Tim23 dimers formed in response to delta psi act as receptors for matrix targeting sequences on the surface of the inner membrane. The ensuring dissociation of Tim23 dimer triggers opening of the TIM channel and insertion of the preprotein.
The mitochondrial outer membrane contains import receptors for nuclear-encoded preproteins and a general import pore responsible for membrane translocation of preproteins. Receptors and the general import pore have been suggested to assemble into a loose complex. However, biochemical characterization of the complex has been limited so far. We report that blue native electrophoresis separates two complexes. One complex of approximately 400 kDa contains the receptor Tom22 and the general import pore component Tom40, the other complex of approximately 120 kDa contains the receptor Tom70. A preprotein accumulated at the general import pore apparently co-migrates with the larger complex, suggesting the functionality of the complex. We conclude that the translocase of the outer membrane consists of at least two subcomplexes and that blue native electrophoresis will be a powerful tool for biochemical analysis of the complexes.
Translocation of mitochondrial preproteins across the inner membrane is facilitated by the TIM machinery. Tim23 binds to matrix targeting signals and initiates membrane potential-dependent import. Tim23 and Tim17 are constituents of a translocation channel across the inner membrane. Tim44 is associated with this channel at the matrix side, and Tim44 recruits mitochondrial Hsp70 and its co-chaperone Mgel, which drive protein translocation into the matrix using ATP as an energy source. Tim22 is a new component of the import machinery of mitochondria, which shares sequence similarity with both Tim23 and Tim17. Here we report that Tim22 is required for the import of proteins of the mitochondrial ADP/ATP carrier (AAC) family into the inner membrane. Members of the yeast AAC family are synthesized without matrix targeting signals. Tim22 is in an assembly of high relative molecular mass that is distinct from the Tim23-Tim17 complex. Import of proteins of the AAC family is independent of Tim23, and import of matrix targeting signals containing preproteins is independent of Tim22.
Mitochondria import many hundreds of different proteins that are encoded by nuclear genes. These proteins are targeted to the mitochondria, translocated through the mitochondrial membranes, and sorted to the different mitochondrial subcompartments. Separate translocases in the mitochondrial outer membrane (TOM complex) and in the inner membrane (TIM complex) facilitate recognition of preproteins and transport across the two membranes. Factors in the cytosol assist in targeting of preproteins. Protein components in the matrix partake in energetically driving translocation in a reaction that depends on the membrane potential and matrix-ATP. Molecular chaperones in the matrix exert multiple functions in translocation, sorting, folding, and assembly of newly imported proteins.
Import of nuclear-encoded precursor proteins into mitochondria and their subsequent sorting into mitochondrial subcompartments is mediated by translocase enzymes in the mitochondrial outer and inner membranes. Precursor proteins carrying amino-terminal targeting signals are translocated into the matrix by the integral inner membrane proteins Tim23 and Tim17 in cooperation with Tim44 and mitochondrial Hsp70. We describe here the discovery of a new pathway for the transport of members of the mitochondrial carrier family and other inner membrane proteins that contain internal targeting signals. Two related proteins in the intermembrane space, Tim10/Mrs11 and Tim12/Mrs5, interact sequentially with these precursors and facilitate their translocation across the outer membrane, irrespective of the membrane potential. Tim10 and Tim12 are found in a complex with Tim22, which takes over the precursor and mediates its membrane-potential-dependent insertion into the inner membrane. This interaction of Tim10 and Tim12 with the precursors depends on the presence of divalent metal ions. Both proteins contain a zinc-finger-like motif with four cysteines and bind equimolar amounts of zinc ions.
The preprotein translocase of the outer membrane of mitochondria (TOM complex) facilitates the recognition, insertion, and translocation of nuclear-encoded mitochondrial preproteins. We have purified the TOM complex from Neurospora crassa and analyzed its composition and functional properties. The TOM complex contains a cation-selective high-conductance channel. Upon reconstitution into liposomes, it mediates integration of proteins into and translocation across the lipid bilayer. TOM complex particles have a diameter of about 138 A, as revealed by electron microscopy and image analysis; they contain two or three centers of stain-filled openings, which we interpret as pores with an apparent diameter of about 20 A. We conclude that the structure reported here represents the protein-conducting channel of the mitochondrial outer membrane.
The mitochondrial outer membrane contains machinery for the import of preproteins encoded by nuclear genes. Eight different Tom (translocase of outer membrane) proteins have been identified that function as receptors and/or are related to a hypothetical general import pore. Many mitochondrial membrane channel activities have been described, including one related to Tim23 of the inner-membrane protein-import system; however, the pore-forming subunit(s) of the Tom machinery have not been identified until now. Here we describe the expression and functional reconstitution of Tom40, an integral membrane protein with mainly beta-sheet structure. Tom40 forms a cation-selective high-conductance channel that specifically binds to and transports mitochondrial-targeting sequences added to the cis side of the membrane. We conclude that Tom40 is the pore-forming subunit of the mitochondrial general import pore and that it constitutes a hydrophilic, approximately 22 A wide channel for the import of preproteins.
The human deafness dystonia syndrome results from the mutation of a protein (DDP) of unknown function. We show now that DDP is a mitochondrial protein and similar to five small proteins (Tim8p, Tim9p, Tim10p, Tim12p, and Tim13p) of the yeast mitochondrial intermembrane space. Tim9p, Tim10p, and Tim12p mediate the import of metabolite transporters from the cytoplasm into the mitochondrial inner membrane and interact structurally and functionally with Tim8p and Tim13p. DDP is most similar to Tim8p. Tim8p exists as a soluble 70-kDa complex with Tim13p and Tim9p, and deletion of Tim8p is synthetically lethal with a conditional mutation in Tim10p. The deafness dystonia syndrome thus is a novel type of mitochondrial disease that probably is caused by a defective mitochondrial protein-import system.
Mitochondrial heat shock protein 70 (mtHsp70) functions in unfolding, translocation, and folding of imported proteins. Controversial models of mtHsp70 action have been discussed: (1) physical trapping of preproteins is sufficient to explain the various mtHsp70 functions, and (2) unfolding of preproteins requires an active motor function of mtHsp70 ("pulling"). Intragenic suppressors of a mutant mtHsp70 separate two functions: a nonlethal folding defect caused by enhanced trapping of preproteins, and a conditionally lethal unfolding defect caused by an impaired interaction of mtHsp70 with the membrane anchor Tim44. Even enhanced trapping in wild-type mitochondria does not generate a pulling force. The motor function of mtHsp70 cannot be explained by passive trapping alone but includes an essential ATP-dependent interaction with Tim44 to generate a pulling force and unfold preproteins.