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Deletion of an organellar peptidasome PreP affects early development in Arabidopsis thaliana
Abstract and Figures
A novel peptidasome PreP is responsible for degradation of targeting peptides and other unstructured peptides in mitochondria and chloroplasts. Arabidopsis thaliana contains two PreP isoforms, AtPreP1, and AtPreP2. Here we have characterized single and double prep knockout mutants. Immunoblot analysis of atprep1 and atprep2 mutants showed that both isoforms are expressed in all tissues with the highest expression in flowers and siliques; additionally, AtPreP1 accumulated to a much higher level in comparison to AtPreP2. The atprep2 mutant behaved like wild type, whereas deletion of AtPreP1 resulted in slightly pale-green seedlings. Analysis of the atprep1 atprep2 double mutant revealed a chlorotic phenotype in true leaves with diminished chlorophyll a and b content, but unchanged Chl a/b ratio indicating a proportional decrease of both PSI and PSII complexes. Mitochondrial respiratory rates (state 3) were lower and the mitochondria were partially uncoupled. EM pictures on cross sections of the first true leaves showed aberrant chloroplasts, including less grana stacking and less starch granules. Mitochondria with extremely variable size could also be observed. At later developmental stages the plants appeared almost normal. However, all through the development there was a statistically significant decrease of approximately 40% in the accumulated biomass in the double mutant plants in comparison to wild type. In mitochondria, deletion of AtPreP was not compensated by activation of any peptidolytic activity, whereas chloroplast membranes contained a minor peptidolytic activity not related to AtPreP. In summary, the AtPreP peptidasome is required for efficient plant growth and organelle function particularly during early development.
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... However, additional chloroplast aminopeptidases, such as the ORGANELLAR OLIGIPEPTIDASE (OOP), AT1G63770 (MA(E)-M1 family) and AT4G30920 (M17-20 or LAP2) (MF-M17 family) are required to complete degradation of cTP fragments into individual amino acids (Teixeira et al., 2017;Kmiec et al., 2018b). Double Arabidopsis T-DNA insertion mutants for PREP1 (prep1) and PREP2 (prep2) showed visibly reduced growth and delayed development (Cederholm et al., 2009). Complementation of prep1prep2 with a modified form of PREP1 that only sorts to mitochondria showed that most of the visible phenotype resulted from a lack of PREP1 activity in chloroplasts. ...
... To test the genetic and functional interactions between the chloroplast CLP system and the PREP peptidases, we obtained T-DNA insertion null alleles for PREP1 (prep1-1) and PREP2 (prep2-1) (Cederholm et al., 2009;Kmiec et al., 2018a) and we generated and genotyped the homozygous double mutant prep1-1prep2-1 (Fig. S2). We then crossed the clpr1-2 null mutant (with c. 30-50% CLP core capacity) , clpr2-1 (with c. 20% of CLPPR capacity) (Rudella et al., 2006) and clpt1-2clpt2-1 (with c. 10-15% CLPPR capacity) to prep1-1, prep2-1 and prep1-1prep2-1. ...
... The synergistic effect between clpr2 and prep2 was stronger than between clpr2 and prep1, as indicated by the much paler and smaller plants (Fig. 2c). This is surprising because PREP1 levels are several-fold higher than PREP2 (Cederholm et al., 2009), suggesting at least partially specific roles for each PREP homolog. A comparison between homozygous prep2clpr2 and a prep2clpr2 line heterozygous for the PREP2 T-DNA insertion showed a clear gene dosage effect on the growth phenotype (Fig. 2d). ...
A network of peptidases governs proteostasis in plant chloroplasts and mitochondria. This study reveals strong genetic and functional interactions in Arabidopsis between the chloroplast stromal CLP chaperone‐protease system and the PREP1,2 peptidases, which are dually localized to chloroplast stroma and the mitochondrial matrix.
Higher order mutants defective in CLP or PREP proteins were generated and analyzed by quantitative proteomics and N‐terminal proteomics (terminal amine isotopic labeling of substrates (TAILS)).
Strong synergistic interactions were observed between the CLP protease system (clpr1‐2, clpr2‐1, clpc1‐1, clpt1, clpt2) and both PREP homologs (prep1, prep2) resulting in embryo lethality or growth and developmental phenotypes. Synergistic interactions were observed even when only one of the PREP proteins was lacking, suggesting that PREP1 and PREP2 have divergent substrates. Proteome phenotypes were driven by the loss of CLP protease capacity, with little impact from the PREP peptidases. Chloroplast N‐terminal proteomes showed that many nuclear encoded chloroplast proteins have alternatively processed N‐termini in prep1prep2, clpt1clpt2 and prep1prep2clpt1clpt2.
Loss of chloroplast protease capacity interferes with stromal processing peptidase (SPP) activity due to folding stress and low levels of accumulated cleaved cTP fragments. PREP1,2 proteolysis of cleaved cTPs is complemented by unknown proteases. A model for CLP and PREP activity within a hierarchical chloroplast proteolysis network is proposed.
... Due to organellar peptide accumulation, the inhibition of proteases involved in targeting peptide degradation results in detrimental effects to organelles and mild defects to plant growth. Inactivation of two PreP homologues (PreP1 and PreP2) in Arabidopsis altered photosynthetic and respiratory capabilities, reflecting disadvantageous effects on both chloroplasts and mitochondria (Nilsson Cederholm et al., 2009). While prep2 single T-DNA insertion lines show no observable impact on overall plant phenotype, prep1 produced slightly pale seedlings (Nilsson Cederholm et al., 2009). ...
... Inactivation of two PreP homologues (PreP1 and PreP2) in Arabidopsis altered photosynthetic and respiratory capabilities, reflecting disadvantageous effects on both chloroplasts and mitochondria (Nilsson Cederholm et al., 2009). While prep2 single T-DNA insertion lines show no observable impact on overall plant phenotype, prep1 produced slightly pale seedlings (Nilsson Cederholm et al., 2009). Unsurprisingly the double knockout lines prep1::prep2 displayed a more severe phenotype, especially in early plant development. ...
... Unsurprisingly the double knockout lines prep1::prep2 displayed a more severe phenotype, especially in early plant development. Besides delayed growth, prep1::prep2 also exhibit chlorotic leaves, attributed to the diminished Chl a and b content (Nilsson Cederholm et al., 2009). The prep1::prep2 mutant also displayed ultrastructural irregularities in both organelles, with non-uniform mitochondria and chloroplasts showing decreased grana stacks and starch granules (Nilsson Cederholm et al., 2009). ...
The endosymbiotic origin of the mitochondrion and the subsequent transfer of its genome to the host nucleus has resulted in intricate mechanisms of regulating mitochondrial biogenesis and protein content. The majority of mitochondrial
proteins are nuclear encoded and synthesized in the cytosol, thus requiring specialized and dedicated machinery for the correct targeting import and sorting of its proteome. Most proteins targeted to the mitochondria utilize N-terminal targeting signals called presequences that are cleaved upon import. This cleavage is carried out by
a variety of peptidases, generating free peptides that can be detrimental to organellar and cellular activity. Research over the last few decades has elucidated a range of mitochondrial peptidases that are involved in the initial removal of the targeting signal and its sequential degradation, allowing for the recovery of single amino acids. The significance of these processing pathways goes beyond presequence degradation after protein import, whereby the deletion of processing peptidases induces plant stress responses, compromises mitochondrial respiratory capability, and alters overall plant growth and development. Here, we review the multitude of plant mitochondrial peptidases that are known to be involved in protein import and processing of targeting signals to detail how their activities can affect organellar protein homeostasis and overall plant growth.
... knockout of PreP1 and PreP2 oligopeptidases (Nilsson Cederholm et al., 2009;Kmiec et al., 2014), which lacks PreP, but maintains OOP activity. The detection method used for the peptidomic analysis allowed for identification of peptides ranging primarily between 6 and 30 aa ( Figure S2a). ...
... Arabidopsis thaliana (Columbia ecotype) double prep1 prep2 knockout and prep1 prep2 oop (tko-1) line were described before (Nilsson Cederholm et al., 2009;Kmiec et al., 2013). The tko-2 line was generated by crossing between the oop-1 knockout line (SALK_058439, (Kmiec et al., 2013)) and the double prep1 prep2 line. ...
The stepwise degradation of peptides to amino acids in plant mitochondria and chloroplasts is catalyzed by a network of oligopeptidases (presequence protease PreP, organellar oligopeptidase OOP) and aminopeptidases. In the present report, we show that the lack of oligopeptidase activity in Arabidopsis thaliana results in the accumulation of endogenous free peptides, mostly of chloroplastic origin (targeting peptides and degradation products). Using mRNA sequencing and deep coverage proteomics, allowing for the identification of 17000 transcripts and 11000 proteins, respectively, we uncover a peptide‐stress response occurring in plants lacking PreP and OOP oligopeptidase activity. The peptide‐stress response results in the activation of the classical plant defense pathways in the absence of pathogenic challenge. The constitutive activation of the pathogen defense pathways impose a strong growth penalty and a reduction of the plants reproductive fitness. Our results indicate that the absence of organellar oligopeptidases PreP1/2 and OOP results in the accumulation of peptides that are perceived as pathogenic effectors and activate the signaling pathways of plant defense response.
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... The two PrePs share high sequence similarities but their cleavage site preferences and peptide product sizes are different . They also differ in protein abundances; PreP1 is the major constituent (Nilsson Cederholm et al., 2009). ...
... The prep1 and prep2 single mutants have subtle or no apparent phenotypes. The prep1/2 double knockout has a pale-green phenotype and few stacked grana thylakoids (Nilsson Cederholm et al., 2009). Complementation of the mitochondrial PreP1 function in the double mutant does not restore the chlorotic phenotype, and thus chloroplast PreP1/2 may have compensatory actions (Kmiec et al., 2014a). ...
Plastids are unique organelles that can alter their structure and function in response to environmental and developmental stimuli. Chloroplasts are one type of plastid and are the sites for various metabolic processes, including photosynthesis. For optimal photosynthetic activity, the chloroplast proteome must be properly shaped and maintained through regulated proteolysis and protein quality control mechanisms. Enzymatic functions and activities are conferred by protein maturation processes involving consecutive proteolytic reactions. Protein abundances are optimized by balanced protein synthesis and degradation, depending on the metabolic status. Malfunctioning proteins are promptly degraded. Twenty chloroplast proteolytic machineries have been characterized to date. Specifically, processing peptidases and energy-driven processive proteases are the major players in chloroplast proteome biogenesis, remodeling, and maintenance. Recently identified putative proteases are potential regulators for photosynthetic functions. Here we provide an updated, comprehensive overview of chloroplast protein degradation machineries and discuss their importance for photosynthesis. Wherever possible, we also provide structural insights into chloroplast proteases that implement regulated proteolysis of substrate proteins/peptides.
... The polypeptide maturation and protein quality control are important for proper plastid differentiation and chloroplast development [75]. It has been found that chaperones and proteases, such as cpHsp70 [76,77], ClpB3 [78], caseinolytic protease, subunit C (ClpC) [79], FtsH8 [80], and PreP1 [81] were involved in leaf variegation. The Arabidopsis mutants of these genes shared some similar characteristics with the variegated leaves of Hosta "Gold Standard", such as leaf variegation, decreased pigment content, and reduced chloroplast development [76][77][78][79][80][81]. ...
... It has been found that chaperones and proteases, such as cpHsp70 [76,77], ClpB3 [78], caseinolytic protease, subunit C (ClpC) [79], FtsH8 [80], and PreP1 [81] were involved in leaf variegation. The Arabidopsis mutants of these genes shared some similar characteristics with the variegated leaves of Hosta "Gold Standard", such as leaf variegation, decreased pigment content, and reduced chloroplast development [76][77][78][79][80][81]. In our results, the abundances of ClpB3, Hsp93-V (ClpC1), PreP1, and FtsH8 showed obvious changes in various regions of variegated leaves (Table 1, Figure 9). ...
Leaf color change of variegated leaves from chimera species is regulated by fine-tuned molecular mechanisms. Hosta “Gold Standard” is a typical chimera Hosta species with golden-green variegated leaves, which is an ideal material to investigate the molecular mechanisms of leaf variegation. In this study, the margin and center regions of young and mature leaves from Hosta “Gold Standard”, as well as the leaves from plants after excess nitrogen fertilization were studied using physiological and comparative proteomic approaches. We identified 31 differentially expressed proteins in various regions and development stages of variegated leaves. Some of them may be related to the leaf color regulation in Hosta “Gold Standard”. For example, cytosolic glutamine synthetase (GS1), heat shock protein 70 (Hsp70), and chloroplastic elongation factor G (cpEF-G) were involved in pigment-related nitrogen synthesis as well as protein synthesis and processing. By integrating the proteomics data with physiological results, we revealed the metabolic patterns of nitrogen metabolism, photosynthesis, energy supply, as well as chloroplast protein synthesis, import and processing in various leaf regions at different development stages. Additionally, chloroplast-localized proteoforms involved in nitrogen metabolism, photosynthesis and protein processing implied that post-translational modifications were crucial for leaf color regulation. These results provide new clues toward understanding the mechanisms of leaf color regulation in variegated leaves.
... Loss of SPP function causes a short growth, incomplete chloroplast development, and a slower root growth (Yue et al. 2010). In Arabidopsis, AtPreP1 participates in the early development of chloroplast and mitochondria, and its mutation results in abnormal morphological structure of chloroplast and mitochondria and decreases the accumulation of biomass (Nilsson Cederholm et al. 2009). The most studies have focused on FtsH under various conditions, which mainly play important roles in maintaining the electron transfer rates of photosystem II (PSII) and photosystem I (PSI) and thylakoid formation (Chen et al. 2018;Kato et al. 2012). ...
Chloroplast is one of the most sensitive organelles to heat stress in plants. In chloroplasts, various proteases affect photosynthesis by degrading proteins under stress conditions. Tomato Lutescent2 (SlL2), a chloroplast zinc metalloprotease, was previously reported to alter chloroplast development and delay fruit ripening. However, its enzyme activity and roles in plant response to abiotic stress are still unclear. Here, we confirmed that the SlL2 protein which localized on thylakoid membrane was an ATP-independent hydrolase, and SlL2 gene responded to heat stress. Phenotype analysis showed that SlL2 plays a negative role in the heat-response mechanism. Under heat stress, the transgenic plants overexpressing SlL2 (OE) grew worse than the wild type (WT), as reflected by their decreased membrane stability, osmotic-regulating substance, and antioxidative enzyme activities, as well as increased reactive oxygen species (ROS) accumulation. By contrast, l2 mutant line showed the opposite phenotype and corresponding physiological indices under heat stress. In addition, overexpression of SlL2 decreased the photosynthetic activities, especially photosystem II. Moreover, SlL2 was found to interact with chloroplast-located chaperone protein SlCDJ1, decreasing its content under heat stress. These results indicate that SlL2 reduces the thermotolerance of tomato by reducing the content of SlCDJ1.
... PreP1 and PreP2 were found to degrade peptides in the range of 10-65 amino acid residues, corresponding to the average size of chloroplast transit peptides (Moberg et al., 2003;Bhushan et al., 2005). Analysis of Arabidopsis prep1/prep2 double knockout mutants revealed strong phenotypes characterized by a slow growth rate, reduced biomass and chlorosis in young leaves (Nilsson Cederholm et al., 2009). These three proteins were also overrepresented at different extents in the ClpP6 and ClpC RNAi mutant proteomes, which could well reflect overlapping substrates with the Clp system that would be most needed under conditions of chloroplast biogenesis and rapid protein import . ...
Protein degradation in the chloroplast is carried out by a set of proteases, which are in charge of
degrading misfolded, damaged or superfluous proteins. More than fifteen types of proteases are found
in plastids, with Clp, FtsH, Lon and Deg being the most important (major) proteases. The ATPdependent
caseinolytic protease (Clp) is the most complex protease in plastids. It is composed of 14
subunits and has been implicated mostly in stromal protein degradation.
To determine the effects of down-regulation of specific Clp subunits on plant growth and
development and to identify possible Clp targets, constitutive and ethanol-inducible RNAi lines
against different Clp subunits were generated in tobacco. Constitutively down-regulated lines
displayed a wide spectrum of phenotypes, including pigment deficiency, alterations in leaf
development, leaf variegation and impaired photosynthesis. In order to distinguish between primary
and secondary effects and determine time-resolved changes in proteins stability, proteomic
experiments were performed with the inducible RNAi lines. Time-resolved proteomic analyses
allowed the identification of putative substrates of the Clp protease complex and chloroplast processes
affected by the repression of Clp activity (e.g., photosynthesis and several metabolic pathways and
protein homeostasis).
Our work provides first insights into the role of specific subunits of the Clp protease complex in
protein degradation in tobacco plastids, and also revealed a set of putative target proteins of this
protease. In adittion, to determine the specific role of the Clp protease in the N-end rule, constitutive
and inducible RNAi lines were crossed to transplastomic lines expressing unstable GFP fusion
proteins to facilitate the investigation of protein stability in the progeny. Ultimately, this approach will
reveal whether or not the Clp protease complex is directly involved in the N-end rule degradation
pathway in chloroplast.
Les mitochondries et les chloroplastes sont des organites de cellules eucaryotes issus d’événements endosymbiotiques impliquant une bactérie et une cellule hôte il y a plus d’un milliard d’années. Aujourd’hui la très grande majorité des protéines présentes dans ces organites sont codées dans le noyau. Le ciblage des protéines cytosoliques vers les mitochondries et les chloroplastes pourrait dériver d’un mécanisme de résistance bactérienne aux attaques de peptides antimicrobiens, ces acteurs majeurs de l’immunité innée, présents dans tous les domaines du vivant. Cette hypothèse se base sur les similarités frappantes entre ces deux mécanismes. Au cours de mon doctorat, j’ai mis à l’épreuve cette hypothèse. Dans une première partie, j’ai pu montrer qu’un sous-ensemble de peptides antimicrobiens se structurant en hélice ↵-amphipathique et les peptides d’adressage possédaient des propriétés physico-chimiques communes, qui sont distinctes de celles partagées entre les peptides signaux de sécrétion bactériens et eucaryotes dont l’origine évolutive commune est bien établie. De plus, ils peuvent se complémenter fonctionnellement in vivo, confortant l’hypothèse de leur origine commune (Garrido et al. 2020). La transition moléculaire nécessaire pour passer d’un peptide antimicrobien à un peptide d’adressage comporte trois étapes cruciales : i) le remplacement des lysines par des arginines qui permet de diminuer l’activité microbienne et de favoriser l’activité d’adressage, ii) l’acquisition d’un site de clivage au sein des peptides d’adressage, iii) l’acquisition d’un domaine N-terminal peu structuré afin d’orienter l’adressage vers le chloroplaste et non vers la mitochondrie au sein des eucaryotes photosynthétiques (Caspari, Garrido et al., article soumis). Dans une deuxième partie, j’ai établi le catalogue exhaustif des familles d’homologues des peptidases impliquées dans la dégradation des peptides d’adressage dans l’arbre du vivant. J’ai pu démontrer que chacune de ces peptidases a été acquise via un évènement de transfert horizontal depuis une bactérie. En accord avec notre hypothèse, on retrouve de nombreux homologues de bactéries résistantes aux peptides antimicrobiens proches des peptidases des organites (Garrido et al., article soumis).
Protein homeostasis (proteostasis) is a dynamic balance of protein synthesis and degradation. Because of the endosymbiotic origin of the chloroplasts and the massive transfer of their genetic information to the nucleus of the host cell, many protein complexes in the chloroplasts are constituted by subunits encoded by both genomes. Hence, the proper function of chloroplasts relies on the coordinated expression of chloroplast- and nucleus-encoded genes. The biogenesis and maintenance of chloroplast proteostasis are dependent on the synthesis of chloroplast-encoded proteins, import of nucleus-encoded chloroplast proteins from the cytosol, and clearance of damaged or otherwise undesired “old” proteins. This review focuses on the regulation of chloroplast proteostasis, the interaction with the proteostasis of the cytosol and its retrograde control to the nuclear gene expression. We also discuss the significant issues and perspectives for the future studies, and the potential applications to improve the photosynthetic performance and stress tolerance of crops.
Plants encode > 100 metalloproteases representing > 19 different protein families. Tools to study this large and diverse class of proteases have not yet been introduced into plant research.
We describe the use of hydroxamate‐based photoaffinity probes to explore plant proteomes for metalloproteases.
We detected labelling of 23 metalloproteases in leaf extracts of the model plant Arabidopsis thaliana that belong to nine different metalloprotease families and localize to different subcellular compartments. The probes identified several chloroplastic FtsH proteases, vacuolar aspartyl aminopeptidase DAP1, peroxisomal metalloprotease PMX16, extracellular matrix metalloproteases and many cytosolic metalloproteases. We also identified nonproteolytic metallohydrolases involved in the release of auxin and in the urea cycle. Studies on tobacco plants (Nicotiana benthamiana) infected with the bacterial plant pathogen Pseudomonas syringae uncovered the induced labelling of PRp27, a secreted protein with implicated metalloprotease activity. PRp27 overexpression increases resistance, and PRp27 mutants lacking metal binding site are no longer labelled, but still show increased immunity.
Collectively, these studies reveal the power of broad‐range metalloprotease profiling in plants using hydroxamate‐based probes.
We have isolated a soluble import-competent 15 kDa N-terminal fragment of the overexpressed Nicotiana plumbaginifolia F(1)beta precursor of the ATP synthase (N(15)pF(1)beta). The isolation was achieved after chemical cleavage, with CNBr, of the insoluble precursor collected in inclusion bodies, followed by purification of the fragment using ion-exchange chromatography. The purity of the final product was estimated to be more than 99%. N(15)pF(1)beta contained a presequence of 54 amino acid residues (except for the N-terminal methionine residue) and 82 N-terminal residues of the mature protein. N(15)pF(1)beta was shown to be imported into isolated potato tuber mitochondria and to be processed by the isolated mitochondrial processing peptidase (MPP) integrated into the cytochrome bc(1) complex of the respiratory chain. Addition of N(16)pF(1)beta at micromolar concentrations resulted in the inhibition of import of F(1)beta precursor and alternative oxidase precursor, synthesized in vitro, into isolated mitochondria as well as the processing of these precursors catalysed by the isolated MPP-bc(1) complex. N(15)pF(1)beta conjugated via a biotin link to avidin blocked import sites even after the reisolation of mitochondria and inhibited the import of the mitochondrial precursors, indicating that it can be used as a substrate for the generation of a stable translocation intermediate. Our results present a novel procedure for the production of an N-terminal fragment of the F(1)beta precursor that contains all information necessary for mitochondrial targeting and processing and that can be used for structural and functional studies of the mitochondrial protein import system. This procedure has a general value because it can be used for the production of chemical quantities of any mitochondrial import substrate and presequence peptide.
Here we show, using the green fluorescent protein (GFP) fusion system, that an Arabidopsis thaliana zinc-metalloprotease (At Zn-MP) is targeted to both mitochondria and chloroplasts. A deletion mutant lacking the amino-terminal 28 residues, with translation initiation at the second methionine residue, was imported into chloroplasts only. However, a mutated form of the full-length targeting peptide, in which the second methionine residue is changed to leucine, was imported to both organelles. No GFP fluorescence was detected when a frame-shift mutation was introduced between the first and second ATG codons of the Zn-MP–GFP construct, suggesting no alternative translational initiation. Our results show that the dual targeting of the Zn-MP is due to an ambiguous targeting peptide. Furthermore, we show that the recombinant At Zn-MP degrades mitochondrial and chloroplastic targeting peptides, indicating its function as a signal peptide degrading protease in both mitochondria and chloroplasts.
Several precursors transported from the cytoplasm to the intermembrane space of yeast mitochondria are first cleaved by the MAS-encoded protease in the matrix space and then by additional proteases that have not been characterized. We have now developed a specific assay for one of these other proteases. The enzyme is an integral protein of the inner membrane; it requires divalent cations and acidic phospholipid for activity, and is defective in yeast mutant pet ts2858 which accumulates an incompletely processed cytochrome b2 precursor. The protease contains a 21.4 kd subunit whose C-terminal part is exposed on the outer face of the inner membrane. An antibody against this polypeptide inhibits the activity of the protease. As overproduction of the polypeptide does not increase the activity of the protease in mitochondria, the enzyme may be a hetero-oligomer. This 'inner membrane protease I' shares several key features with the leader peptidase of Escherichia coli and the signal peptidase of the endoplasmic reticulum.
The transport of proteins across the thylakoid membrane in higher plant chloroplasts is usually mediated by an amino-terminal peptide extension which is subsequently removed by a specific thylakoidal processing peptidase. We have previously shown that the reaction specificity of this enzyme is very similar to those of signal peptidases located in the endoplasmic reticulum and bacterial plasma membrane. In the present report, the reaction mechanism of the thylakoidal peptidase has been investigated by substituting a variety of amino acids for the alanine residues at the -3 and -1 positions of a thylakoid lumen protein precursor. Small neutral side chains are known to be essential at these positions for cleavage by signal peptidases, and we find that these residues likewise play a critical role in defining the thylakoidal processing peptidase cleavage site. However, the requirements of the thylakoidal enzyme at these sites are significantly more restrictive than those of the bacterial or endoplasmic reticulum peptidases. Whereas leucine at the -3 position in the substrate is tolerated by the latter two enzymes, cleavage by the thylakoidal peptidase is almost completely inhibited. At the -1 position the presence of alanine appears to be critical; substitution of this residue by glycine, serine, threonine, leucine, lysine, or glutamate leads to either substantial or complete inhibition of cleavage at this site. Substitutions at either -3 or -1 which blocked cleavage at the correct site led to cleavage taking place at an alternative site, probably after the -21 residue.
Several precursors transported from the cytoplasm to the intermembrane space of yeast mitochondria are first cleaved by the MAS‐encoded protease in the matrix space and then by additional proteases that have not been characterized. We have now developed a specific assay for one of these other proteases. The enzyme is an integral protein of the inner membrane; it requires divalent cations and acidic phospholipid for activity, and is defective in yeast mutant pet ts2858 which accumulates an incompletely processed cytochrome b2 precursor. The protease contains a 21.4 kd subunit whose C‐terminal part is exposed on the outer face of the inner membrane. An antibody against this polypeptide inhibits the activity of the protease. As overproduction of the polypeptide does not increase the activity of the protease in mitochondria, the enzyme may be a hetero‐oligomer. This ‘inner membrane protease I’ shares several key features with the leader peptidase of Escherichia coli and the signal peptidase of the endoplasmic reticulum.
Arabidopsis thaliana is, perhaps, the most important model species in modern plant biology. However, the isolation of organelles from leaves of this plant has been difficult. Here, we present two different protocols for the isolation of mitochondria, yielding either highly functional crude mitochondria or highly purified mitochondria. The crude mitochondria were well coupled with the substrates tested (malate + glutamate, glycine and NADH), exhibiting respiratory control ratios of 2.1–3.9. Purified mitochondria with very low levels of chlorophyll contamination were obtained by Percoll gradient centrifugation, yielding 1.2 mg of mitochondrial protein from 50 g of leaves.
Sample purity is the key for a successful in-depth analysis of any given subcellular proteome. The suitability of free-flow electrophoresis to assist conventional, centrifugation-based techniques in the preparation of plant mitochondria from green and non-green tissue was assessed by various means, including functional assays, immunoblots, electron microscopy and differential gel electrophoresis. Results indicated a significant increase in purity of the mitochondrial samples, highlighted specific contaminants previously reported as mitochondrial proteins, and also pointed to new means for separating plastids and peroxisomes from mitochondria in plant organellar extracts by exploiting differences in surface charge. This approach has the potential to allow a deeper and more comprehensive investigation of the Arabidopsis organellar proteomes, by providing a second dimension of separation based on surface charge in addition to conventional centrifugation purification protocols relying on size and density.
: Genetic information in plants is localized in three compartments: the nucleus, pastids and mitochondria. Despite both plastids
and mitochondria containing their own DNA, most of the proteins of these organelles are nuclear encoded. The biogenesis of
a functional organelle requires coordinate expression of both nuclear and organellar genomes and specific intracellular protein
trafficking, processing and assembly machinery. Most nuclear-encoded organellar proteins are synthesized in the cytosol as
precursor proteins with N-terminal signal peptides, required for sorting and targeting the protein to the correct organelle.
Both plastid and plant mitochondrial signal peptides show a remarkable similarity in amino acid composition. They are rich
in hydrophobic, hydroxylated and positively charged amino acid residues and deficient in acidic amino acids. However, despite
great similarities of signal peptides, the plastid and mitochondrial protein targeting is specific. Yet, there is a group
of proteins that are dually targeted to both organelles. Molecular chaperones and other cytosolic factors are involved in
the import process: maintaining precursors in an import-competent conformation, guiding them to the organelle and assisting
in transport through the membrane. Precursor proteins are transported into plastids through the translocons of the outer and
the inner envelope membrane, TOC and TIC complexes, and into mitochondria through the translocases of the outer and the inner
mitochondrial membrane, TOM and TIM complexes. Mitochondrial molecular chaperone Hsp70 functions as a molecular motor pulling
precursor proteins into the mitochondria. The signal peptides are proteolytically cleaved off inside the organelles by highly
specific organellar processing peptidases, the mitochondrial processing peptidases, that in plant mitochondria are an integral
part of the cytochrome bc1 complex of the respiratory chain, and the stromal processing peptidase, SPP, in plastids. The mature
proteins, after intraorganellar sorting, are assembled by molecular chaperones into functional oligomeric protein complexes,
whereas the cleaved targeting peptides, potentially destructive for biological membranes, are degraded inside the organelles
by a newly identified signal peptide degrading zinc metalloprotease, both in plastids and in mitochondria.
Measurements of the quantum yields of chlorophyll fluorescence and CO2 assimilation for a number of plant species exposed to changing light intensity and atmospheric CO2 concentrations and during induction of photosynthesis are used to examine the relationship between fluorescence quenching parameters and the quantum yield of non-cyclic electron transport. Over a wide range of physiological conditions the quantum yield of non-cyclic electron transport was found to be directly proportional to the product of the photochemical fluorescence quenching (qQ) and the efficiency of excitation capture by open Photosystem II (PS II) reaction centres (Fv/Fm). A simple fluorescence parameter, ΔφF/φFm, which is defined by the difference in fluorescence yield at maximal φFm, and steady-state φFs, divided by φFm, can be used routinely to estimate changes in the quantum yield of non-cyclic electron transport. It is demonstrated that both the concentration of open PS II reaction centres and the efficiency of excitation capture by these centres will determine the quantum yield of non-cyclic electron transport in vivo and that deactivation of excitation within PS II complexes by non-photochemical processes must influence the quantum yield of non-cyclic electron transport.
Characterisation of the amount of protein import of the alternative oxidase (AOX) and the F(A)d precursor proteins (previously shown to use different import pathways) into mitochondria from developing soybean tissues indicated that they displayed different patterns. Import of the AOX declined in both cotyledon and root mitochondria with increasing age, whereas the import of the F(A)d into cotyledon mitochondria remained high throughout the same period. Using primary leaf mitochondria, it was evident that import of AOX remained high while it declined in cotyledon and root mitochondria. The amount of import of the AOX into mitochondria from different tissues closely matched the amount of the Tom 20 receptor.