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The Curious Status of the Golgi Apparatus
... Variety of essential cellular mechanisms occur through formation of lipid vesicles by budding and tubulation. Such important biochemical processes are for example the signal transport in neutral synapses [13], transport in the endoplasmic reticulum and Golgi apparatus [14], the formation of mitochondrial structure [15], etc. Their occurrence is influenced by the emergence of local instabilities which alter the shape of the tubules. ...
... Equations (14) are valid only for |α| > 1/2, i.e., shapes mimicking the distorted tubular vesicles under perling can only be observed for pressures |p/k c | > 81c 3 0 /128. One also notices that the spontaneous curvature entering in the above equations serves only as a scale factor, determining the dimensions of the tube. ...
... The main result is that for the force acting between a 3D object (say a colloid particle) B ≡ {(x, y, z), (x, y, z) ∈ B} of general shape S (x, y) = z and a flat surface bounded by the (x, y)−plane of a Cartesian coordinate system, one has Here L designates the minimal distance. From (14) follows that at its thickest the radius of the tube is R ≡ |ρ[(2n + 1)t * ]|, while the one of the "joining segment" between neighboring pearls is r ≡ |ρ(2nt * )|, with n being a nonnegative integer. The dashed line spanned between the points characterized by t 1 and t 2 = 2t * − t 1 , depicts the side view of a cross section "facing" towards the plate, and is situated at some distance x p from it. ...
In the current article we study the behaviour of the van der Waals force between a planar substrate and an axisymmetric bilayer lipid membrane undergoing pearling instability, caused by uniform hydrostatic pressure difference. To do so, the recently suggested " surface integration approach " is used, which can be considered a generalization of the well known and widely used Derjaguin approximation. The static equilibrium shape after the occurrence of the instability is described in the framework of Helfrich's spontaneous curvature model. Some specific classes of exact analytical solutions to the corresponding shape equation are considered, and the components of the respective position vectors given in terms of elliptic integrals and Jacobi elliptic functions. The mutual orientation between the interacting objects is chosen such that the axis of revolution of the distorted cylinder be parallel to the plane bounding the substrate. Based on the discussed models and approaches we made some estimations for the studied force in real experimentally realizable systems, thus showing the possibility of pearling as an useful technique for reduction of the adhesion in variety of industrial processes using lipid membranes as carriers.
... Thus, our results imply that the pressure to produce the target glycan code for a given cell type with high fidelity places strong constraints on the cisternal number and enzyme specificity (Sengupta and Linstedt, 2011). Taken together, our quantitative analysis of the trade-offs has deep implications for the non-equilibrium self-assembly of Golgi cisternae and suggests that the control of cisternal number must involve a coupling of non-equilibrium self-assembly of cisternae with enzymatic chemical reaction kinetics (Glick and Malhotra, 1998). This combined dynamics of chemical processing with non-equilibrium membrane dynamics involving fission, fusion, and transport (Sachdeva et al., 2016;Sens and Rao, 2013) opens up a new direction for future research. ...
... The reaction rates close to the local minima are sloppy directions, and moving along these directions does not change the glycan profile much. v. Taken together, our quantitative analysis of the trade-offs has deep implications for nonequilibrium self-assembly of the Golgi cisternae, and suggests that the non-equilibrium control of cisternal number must involve a coupling of non-equilibrium self-assembly of cisternae with enzymatic chemical reaction kinetics (Glick and Malhotra, 1998). ...
Many proteins that undergo sequential enzymatic modification in the Golgi cisternae are displayed at the plasma membrane as cell identity markers. The modified proteins, called glycans, represent a molecular code. The fidelity of this glycan code is measured by how accurately the glycan synthesis machinery realises the desired target glycan distribution for a particular cell type and niche. In this paper, we construct a simplified chemical synthesis model to quantitatively analyse the tradeoffs between the number of cisternae, and the number and specificity of enzymes, required to synthesize a prescribed target glycan distribution of a certain complexity to within a given fidelity. We find that to synthesize complex distributions, such as those observed in real cells, one needs to have multiple cisternae and precise enzyme partitioning in the Golgi. Additionally, for fixed number of enzymes and cisternae, there is an optimal level of specificity (promiscuity) of enzymes that achieves the target distribution with high fidelity. The geometry of the fidelity landscape in the multidimensional space of the number and specificity of enzymes, inter-cisternal transfer rates, and number of cisternae, provides a measure for robustness and identifies stiff and sloppy directions. Our results show how the complexity of the target glycan distribution and number of glycosylation enzymes places functional constraints on the Golgi cisternal number and enzyme specificity.
... Cisternal maturation is thought to be driven by COPI vesiclemediated intra-Golgi recycling of resident transmembrane proteins (Schnepf, 1993;Glick and Malhotra, 1998;Pelham, 1998;Rabouille and Klumperman, 2005). COPI vesicles also mediate retrograde traffic from the Golgi (and mammalian ERGIC) to the ER (Szul and Sztul, 2011;Barlowe and Miller, 2013), and some researchers have divided COPI vesicles into two categories: COPIa vesicles that mediate recycling to the ER, and COPIb vesicles that mediate intra-Golgi traffic (Donohoe et al., 2007). ...
... A consequence of the scheme shown in Figure 1A is that between the times of COPIa vesicle budding and COPIb vesicle budding, a cisterna may experience an interval when traffic is minimal. This interval would allow for glycosylation and lipid metabolism, and it might be prolonged in cell types that produce elaborate carbohydrate structures (Glick and Malhotra, 1998). We emphasize that while the model illustrated in Figure 1A is appealing, the COPIb vesicle pathway is still incompletely characterized, and other membrane flow patterns could support intra-Golgi recycling (Rabouille and Klumperman, 2005;Day et al., 2013). ...
A long-standing assumption is that the cisternae of the Golgi apparatus can be grouped into functionally distinct compartments, yet the molecular identities of those compartments have not been clearly described. The concept of a compartmentalized Golgi is challenged by the cisternal maturation model, which postulates that cisternae form de novo and then undergo progressive biochemical changes. Cisternal maturation can potentially be reconciled with Golgi compartmentation by defining compartments as discrete kinetic stages in the maturation process. These kinetic stages are distinguished by the traffic pathways that are operating. For example, a major transition occurs when a cisterna stops producing COPI vesicles and begins producing clathrin-coated vesicles. This transition separates one kinetic stage, the “early Golgi,” from a subsequent kinetic stage, the “late Golgi” or “trans–Golgi network (TGN).” But multiple traffic pathways drive Golgi maturation, and the periods of operation for different traffic pathways can partially overlap, so there is no simple way to define a full set of Golgi compartments in terms of kinetic stages. Instead, we propose that the focus should be on the series of transitions experienced by a Golgi cisterna as various traffic pathways are switched on and off. These traffic pathways drive changes in resident transmembrane protein composition. Transitions in traffic pathways seem to be the fundamental, conserved determinants of Golgi organization. According to this view, the initial goal is to identify the relevant traffic pathways and place them on the kinetic map of Golgi maturation, and the ultimate goal is to elucidate the logic circuit that switches individual traffic pathways on and off as a cisterna matures.
... Many reviews have addressed the details of these different models [2][3][4]. The most popular models include cisternal maturation [5], vesicular transport between stable compartments [6] and the rapid partitioning model [7]. Each of these models possesses strengths and weaknesses, and on their own does not fully accommodate all of the data present in the literature. ...
... For several years, the field has been predominately arguing between two models of cargo progression through the Golgi: cisternal maturation versus vesicular transport, which have been extensively described in the past [3,5,[17][18][19] (Box 1). Briefly, the cisternal maturation model views cisternae as transient carriers born out of the ER and maturing over time into a TGN cisterna in a sort of conveyor belt fashion. ...
... Properly folded cargoes are delivered to the Golgi apparatus, which operates as the main sorting station along the biosynthetic pathway. The Golgi complex possesses a unique architecture as it is composed of highly ordered stacks of membrane cisternae that are frequently interconnected into a ribbon-like structure (Glick and Malhotra, 1998;Mellman and Simons, 1992;Polishchuk and Mironov, 2004;Rambourg and Clermont, 1990). During the transition through the Golgi complex cargoes receive post-translation modifications, including phosphorylation, glycosylation and proteolytic cleavage (Glick and Malhotra, 1998;Mellman and Simons, 1992;Polishchuk and Mironov, 2004;Rambourg and Clermont, 1990). ...
... The Golgi complex possesses a unique architecture as it is composed of highly ordered stacks of membrane cisternae that are frequently interconnected into a ribbon-like structure (Glick and Malhotra, 1998;Mellman and Simons, 1992;Polishchuk and Mironov, 2004;Rambourg and Clermont, 1990). During the transition through the Golgi complex cargoes receive post-translation modifications, including phosphorylation, glycosylation and proteolytic cleavage (Glick and Malhotra, 1998;Mellman and Simons, 1992;Polishchuk and Mironov, 2004;Rambourg and Clermont, 1990). At the trans side of the Golgi the modified proteins and lipids undergo sorting and packaging into forming transport intermediates, which, following fission from the trans-Golgi network (TGN), deliver cargoes to their final destinations (De Matteis and Luini, 2008;Luini et al., 2008;Mellman and Nelson, 2008;Rodriguez-Boulan et al., 2005). ...
It is widely accepted that the Golgi complex operates as a main sorting station in the biosynthetic pathway. On the other hand, the Golgi complex harbors numerous signaling molecules that generate the platform for the coordination of the transduction of specific signals and of membrane transport events. A part of these processes, which require the complex integration of transport-, cytoskeleton- and polarity-associated mechanisms, is tightly regulated by molecular machineries comprising guanine nucleotide exchange factors (GEF) and their down-stream effectors, such as the small GTPase Cdc42. Dysfunction of several Cdc42-specific GEFs has been shown to cause a number of human diseases, which are associated with impaired intracellular trafficking at the level of the Golgi complex as well as in other compartments. Here we briefly overview how mutations in Cdc42-specific GEFs have an impact on the organization of intracellular trafficking fluxes and how such trafficking aberrations could be associated with a number of human disorders.
... Until recently however, this theory has received little attention except in the context of membrane synthesis and lipid flow between organelles (Voelker, 1991). Once thought of as a series of stable compartments, we are now increasingly forced to accept that membrane trafficking through the secretory/endosomal pathways occurs, to a large extent, via organelle maturation, and a biochemical continuum of hybrid organelles (Glick, 2002;Glick and Malhotra, 1998;Glick and Nakano, 2009;Lippincott-Schwartz and Zaal, 2000;Lowe and Barr, 2007). ...
... In this model physical maturation of the cisternae drives cargo processing, with the cisternae existing transiently in various states of development, selectively retaining cargo while acquiring and losing distinct populations of resident Golgi proteins over time. Cargo processing in stacked Golgi, such as those found in mammals, would occur simultaneously with an outward, cis-to medial-to trans-flow of the maturing cisternae and concurrent retrograde trafficking of resident proteins through vesicular trafficking or cisternae-cisternae membrane contacts ( Fig. 1) (Glick and Malhotra, 1998;Matsuura-Tokita et al., 2006;Mironov et al., 1997b;Moelleken et al., 2007;Mollenhauer and Morre, 1991;Pelham, 2001;Pelham and Rothman, 2000). ...
The Golgi complex constitutes a central way station of the eukaryotic endomembrane system, an intricate network of organelles engaged in control of membrane trafficking and the processing of various cellular components. Previous ideas of compartmental stability within this network are gradually being reshaped by concepts describing a biochemical continuum of hybrid organelles whose constitution is regulated by compartmental maturation. Membrane lipid composition and lipid signaling processes make fundamental contributions to compartmentalization strategies that are themselves critical for organizing cellular architecture and biochemical activities. Phosphatidylinositol transfer proteins (PITPs) are increasingly recognized as key regulators of membrane trafficking through the secretory pathway. They do so by coordinating lipid metabolism with lipid signaling, translating this information to core protein components of the membrane trafficking machinery. In this capacity, PITPs can be viewed as regulators of an essential lipid-protein interface of cisternal maturation. It is also now becoming appreciated, for the first time, that such an interface plays important roles in larger systems processes that link secretory pathway function with cell proliferation.
... One famous and memorable debate regarding trafficking in the Golgi dealt with the question of how cargo molecules are conveyed within the Golgi (Mellman and Simons, 1992;Glick and Malhotra, 1998;Pelham and Rothman, 2000). Live imaging of the budding yeast provided strong support for the cisternal maturation model (Losev et al., 2006;Matsuura-Tokita et al., 2006;Glick and Nakano, 2009;Nakano and Luini, 2009;Glick and Luini, 2011), but many unanswered questions remain. ...
The Golgi apparatus represents a central compartment of membrane traffic. Its apparent architecture, however, differs considerably among species, from unstacked and scattered cisternae in the budding yeast Saccharomyces cerevisiae to beautiful ministacks in plants and further to gigantic ribbon structures typically seen in mammals. Considering the well-conserved functions of the Golgi, its fundamental structure must have been optimized despite seemingly different architectures. In addition to the core layers of cisternae, the Golgi is usually accompanied by next-door compartments on its cis and trans sides. The trans-Golgi network (TGN) can be now considered as a compartment independent from the Golgi stack. On the cis side, the intermediate compartment between the ER and the Golgi (ERGIC) has been known in mammalian cells, and its functional equivalent is now suggested for yeast and plant cells. High-resolution live imaging is extremely powerful for elucidating the dynamics of these compartments and has revealed amazing similarities in their behaviors, indicating common mechanisms conserved along the long course of evolution. From these new findings, I would like to propose reconsideration of compartments and suggest a new concept to describe their roles comprehensively around the Golgi and in the post-Golgi trafficking.
... As a central meeting point for endocytic and exocytotic systems, Golgi apparatus harbors its best-known role in protein secretion and modification in eukaryotic cells [69]. Similar to ER, the important functions of the Golgi system in sMECs primarily include the synthesis and secretion of the major milk constituents (milk proteins/lipids and lactose), along with the maintenance of calcium homeostasis. ...
The mammary gland undergoes a spectacular series of changes during its development and maintains a remarkable capacity to remodel and regenerate during progression through the lactation cycle. This flexibility of the mammary gland requires coordination of multiple processes including cell proliferation, differentiation, regeneration, stress response, immune activity, and metabolic changes under the control of diverse cellular and hormonal signaling pathways. The lactating mammary epithelium orchestrates synthesis and apical secretion of macromolecules including milk lipids, milk proteins, and lactose as well as other minor nutrients that constitute milk. Knowledge about the subcellular compartmentalization of these metabolic and signaling events, as they relate to milk production and secretion during lactation, is expanding. Here we review how major organelles (endoplasmic reticulum, Golgi apparatus, mitochondrion, lysosome, and exosome) within mammary epithelial cells collaborate to initiate, mediate, and maintain lactation, and how study of these organelles provides insight into options to maintain mammary/breast health.
... Cargoes are not packed into COPI vesicles during maturation, but are instead carried forward while remaining in the maturing cisterna, which effectively acts as the transport carrier. Enzymes and other Golgi-resident proteins are transported in the retrograde direction by COPI vesicles, hence maintaining the observed polarized distribution of these proteins along the stack [46,47] (Fig. 1B). The main weakness of this initial formulation of the model was that it could not account for the different intra-Golgi transport and export kinetic rates of different cargoes. ...
The Golgi complex is the membrane-bound organelle that lies at the center of the secretory pathway. Its main functions are to maintain cellular lipid homeostasis, to orchestrate protein processing and maturation, and to mediate protein sorting and export. These functions are not independent of one another, and they all require that the membranes of the Golgi complex have a well-defined biochemical composition. Importantly, a finely-regulated spatiotemporal organization of the Golgi membrane components is essential for the correct performance of the organelle. In here, we review our current mechanistic and molecular understanding of how Golgi membranes are spatially organized in the lateral and axial directions to fulfill their functions. In particular, we highlight the current evidence and proposed models of intra-Golgi transport, as well as the known mechanisms for the retention of Golgi residents and for the sorting and export of transmembrane cargo proteins. Despite the controversies, conflicting evidence, clashes between models, and technical limitations, the field has moved forward and we have gained extensive knowledge in this fascinating topic. However, there are still many important questions that remain to be completely answered. We hope that this review will help boost future investigations on these issues.
... Les cargos sont transportés à travers l'appareil de Golgi par la progression des citernes. Le maintient des protéines résidentes de Golgi est entraînée par le transport rétrograde des vésicules COPI au sein de l'appareil de Golgi, et peut également impliquer le recyclage de clathrine d'un compartiment maturé au TGN(Glick and Malhotra, 1998)(Pelham, 1998)(Glick and Nakano, 2009) (Figure 23). Le modèle de progression / maturation des citernes avec le transport tubulaire hétérotypique. ...
Les septines sont une famille de protéines GTPases qui peuvent former des structures d'ordre supérieur, comme les filaments et les anneaux, et capables de se lier avec les membranes cellulaires par leur interaction avec les phosphoinositides (PIs) via un domaine polybasique en N-terminal de leur domaine de liaison au GTP. Nous avons montré par une analyse transcriptomique réalisée en utilisant les données GSE14323 que la septine 9 est significativement surexprimée dans la cirrhose induite par le virus de l'hepatite C (VHC). Nos résultats montrent, ainsi, que la septine 9 induit l’augmentation en taille des gouttelettes lipidiques (GLs) par un mécanisme dépendant le phosphatidylinositol-5-phosphate et des microtubules. Nous avons montré, également, que cette voie de régulation des GLs est exploité par le VHC. De plus, nous avons montré que la septine 9 est impliquée dans la régulation de la morphologie de l’appareil Golgi et la mise en place de la polarité cellulaire par son interaction avec les phosphoinositides via deux domaines polybasiques. Ces résultats apportent une nouvelle compréhension du mécanisme moléculaire de l’interaction des septines avec les phosphoinositides et montrent pour la première fois l’importance de cette interaction dans des fonctions cellulaires de la septine 9.
... Curiously, plant cells contain stacked Golgi cisternae, yet they do not express any GRASP or GRASP-related proteins. And some nonvertebrate organisms with stacked Golgi cisternae express just one GRASP-related protein, while the Golgi cisternae are not stacked in other nonvertebrate organisms (e.g., yeast) that express a single GRASP (Glick and Malhotra, 1998). Apparently, the presence or number of GRASP proteins expressed does not correlate with stacked cisternae. ...
Cisternae of the Golgi apparatus adhere to each other to form stacks, which are aligned side by side to form the Golgi ribbon. Two proteins, GRASP65 and GRASP55, previously implicated in stacking of cisternae, are shown to be required for the formation of the Golgi ribbon.
... The adaptor protein (AP-2) is a multimeric protein that acts on the cell membrane to lead the transmembrane proteins to endocytosis. Clathrin and transmembrane proteins are turned into clathrin-coated vesicles (CCVs) and are transported to early endosomes (Fig. 4) (27,28). ...
... Molecular mechanisms of anterograde cargo transport through the Golgi remain a major question in the field of membrane trafficking (Malhotra et al., 1989;Glick and Malhotra, 1998;Glick and Luini, 2011;Beznoussenko et al., 2014;Mironov and Beznoussenko, 2019). In this context, there is still a big debate in the field about how anterograde cargo, such as MMPs that arrive in the cis-Golgi, are segregated from retrograde cargo trafficking to the ER (Glick and Nakano, 2009;Mironov and Beznoussenko, 2019;Kurokawa et al., 2019). ...
Matrix metalloproteinases (MMPs) degrade several ECM components and are crucial modulators of cell invasion and tissue organization. Although much has been reported about their function in remodeling ECM in health and disease, their trafficking across the Golgi apparatus remains poorly understood. Here we report that the cis-Golgi protein nucleobindin-1 (NUCB1) is critical for MMP2 and MT1-MMP trafficking along the Golgi apparatus. This process is Ca2+-dependent and is required for invasive MDA-MB-231 cell migration as well as for gelatin degradation in primary human macrophages. Our findings emphasize the importance of NUCB1 as an essential component of MMP transport and its overall impact on ECM remodeling.
... Сформированные вновь транс цистерны превращаются в мембранные переносчики секретируемых продуктов. Однако, не привлекая везикулярный транспорт, трудно объяснить сохранение полярности АГ и постоянного ферментного состава определённых цистерн, находящихся в состоянии динамического равновесия(Farquhar, Palade, 1981;Glick, Malhotra, 1998). Согласно везикулярноматурационной модели, белки, синтезированные в ER, упаковываются в везикулы, окаймленные COPII в сайтах tER (переходного компартмента между АГ и ERGIC (ER -Гольджи промежуточный компартмент)). ...
The dissertation summarizes my work on cytology, life cycles, taxonomy and phylogeny of microsporidia belonging to various systematic groups. As it concerns cell biology of microsporidia, it is particularly focused on the structural and functional organization of the secretory system and Golgi complex. In addition, is covers some aspects of host-parasite interactions, namely suppression of host cell apoptosis by microsporidia parasites.
... From exo-to endocytosis and pathogen intrusion to immune response, the lipid bilayer plays an important role in signal transduction. 1 For instance, lipid−protein interactions influence crucial cellular processes such as neuro-synaptic signaling, 2 membrane fusion, 3 Golgi vesicle genesis, 4 and lipidassociated enzymatic reactions. 5 During kinase-based phosphorylation, a lipid microdomain (lipid raft) induces a protein recruitment process which leads to a cascade of signaling pathway. ...
From pathogen intrusion to immune response, cell membrane plays an important role in signal transduction. Such signals are important for cellular proliferation and survival. However, measurement of these subtle signals through the lipid membrane scaffold is challenging. We present a chromatic model membrane vesicle system engineered to covalently bind with lysine residues of protein molecules for investigation of cellular interactions and signaling. We discovered that different protein molecules induced differential spectroscopic signals, which is based on the chemical and physical properties of protein interacting at the vesicle surface. The observed chromatic response (CR) for bound protein molecules with higher molecular weight was much larger (~5-15X) than those for low molecular weight proteins. Through mass spectrometry (MS), we found that only six out of sixty (10%) lysine groups present in bovine serum albumin (BSA) were accessible to membrane of the vesicles. Finally, a “sphere-shell” model representing protein-vesicle complex was used for evaluating contribution of the van der Waals interactions between proteins and vesicle. Our analysis points toward contributions from van der Waals, hydrophobic, and electrostatic interactions toward observed CR signals resulting from molecular interactions at the vesicle membrane surface. Overall, this study provided a convenient, chromatic, semi-quantitative way of detecting biomolecules and their interactions with model membranes at sub-nanomolar concentration.
... Translocation of MICs, ROPs and GRAs via ER mediated by their N-terminal signal sequences is followed by their transfer into ER-to-Golgi transport vesicles. In the Golgi apparatus all T. gondii secretory proteins translocate via the same pathway from the cis to the trans face of cisternae (Glick and Malhotra, 1998); however, the following route from the trans-Golgi to the parasite cell surface differs between MICs, ROPs, GRAs (Ngô et al., 2000;Venugopal et al., 2017;Dogga et al., 2017;Mercier and Cesbron-Delauw, 2015;Venugopal and Marion, 2018;Hammoudi et al., 2018) (Fig. 3). While GRAs are sorted by protein aggregation, and fusion with plasma membrane is mediated by the NSF/SNAP/SNARE/Rabs machinery, MICs and ROPs are sorted by tyrosine motif/adaptin/clathrin pathways (Fig. 3). ...
T. gondii is a major opportunistic pathogen chronically infecting nearly one third of the world's population. Due to the high infection and mortality rates in immunocompromised patients and newborns, the extent or magnitude of T. gondii pathogenesis is determined mainly by host-pathogen interactions. T. gondii utilizes specialized secretory proteins to modify host cellular factors and facilitate invasion and replication. This review provides update on the recent progress in this field of research with particular emphasis on the T. gondii secretory proteins and their role in invasion and pathogenesis.
... In WT cells, lateral buds emerging from the Golgi cisternae were clearly observed (Figure 6a). Based on current models of Golgi trafficking, the lateral buds are likely to be COPI-coated vesicles predicted to mediate the retrograde transport of resident Golgi proteins (Glick and Malhotra, 1998;Love et al., 1998;Pimpl et al., 2000;Donohoe et al., 2007;Glick and Nakano, 2009). The COPI vesicles are usually of diameter 50-90 nm (Bonfanti et al., 1998;Pimpl et al., 2000;Donohoe et al., 2007;Figure 5. Mis-sorting of vacuolar protein AALP in tno1 is reduced by overexpression of SYP41 or SYP61. ...
The trans‐Golgi network (TGN) is a major site for sorting of cargo to either the vacuole orapoplast. The TGN‐localized coiled‐coil protein TNO1 is a putative tethering factor that interacts with the TGN t‐SNARE SYP41 and is required for correct localization of the SYP61 t‐SNARE. An Arabidopsis thalianatno1 mutant is hypersensitive to salt stress and partially mislocalizes vacuolar proteins to the apoplast, indicating a role in vacuolar trafficking. Here, we show that overexpression of SYP41 or SYP61 significantly increases SYP41‐SYP61 complex formation in a tno1 mutant and rescues the salt sensitivity and defective vacuolar trafficking of the tno1 mutant. The TGN is disrupted and vesicle budding from Golgi cisternae is reduced in the tno1 mutant and these defects arealsorescued by overexpression of SYP41 or SYP61. Our results suggest that the trafficking and Golgi morphology defects caused by loss of TNO1 can be rescued by increasing SYP41‐SYP61 t‐SNARE complex formation, implicating TNO1 as a tethering factor mediating efficient vesicle fusion at the TGN.
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... The two favoured hypotheses are the vesicular transport and the cisternal maturation model. The vesicular transport model describes the Golgi as a stable organelle with cisternae separated from each other in cis to trans direction, with membrane bound vesicles that shuttle cargo between different cisternae (Glick and Malhotra, 1998;Glick and Luini, 2011). In the cisternal maturation model, COPII vesicles from the ERES fuse and form the first cis-cisterna of the Golgi stack, which maturates and finally forms secretory vesicles (Glick, 2000;Glick and Luini, 2011). ...
African trypanosomes are the causative agents of fatal diseases in humans and livestock. Trypanosomes show a complex lifecycle and shuttle between the transmitting vector, the tsetse (Glossina spec.), and the mammalian host. As a result of this the parasite undergoes tremendous changes in morphology and metabolism to adapt to the different living environments. The two best-studied lifecycle stages are the procyclic forms (PCF) that live in the tsetse fly and the proliferative bloodstream form (BSF) that resides in the mammalian blood. The most conspicuous weapon that trypanosomes use to evade the host immune attack is a dense layer of a single protein type, the variant surface glycoprotein (VSG), which shields the entire cell surface. Immune evasion required high rates of surface membrane turnover and surface coat recycling. Trypanosomes show highly polarised cell architecture with all major eukaryotic organelles (endoplasmic reticulum, Golgi apparatus, endosomal apparatus, lysosome, mitochondrion and peroxisome-like glycosomes) generally present in single copy. Furthermore, trypanosomes possess a single flagellum, which is important not only for cellular motility but also for cell division. How the duplication of all these cellular components is coordinated in order to progresss through the cell division cycle is poorly understood. We used trypanosomes as a model organism due to the relative simplicity and the polarised nature of their cell architecture and determined the duplication of all their compartments. This was only possible due to a new synchronisation approach developed during this project. In the first part of the thesis a precise temporal map of the cell division cycle of the BSF T. brucei cell division cycle was generated. By the use of well-described morphological markers (K/N status, new flagellum outgrowth and DNA synthesis) the position of individual cells was determined with high temporal resolution; this allowed us for the first time to synchronise a cell population in silico without affecting the naturally asynchronous growth. In the second part of the thesis we used this tool to follow duplication events of the Major organelles during progression through the cell division cycle. We precisely determined the time points of organelle duplication and found that it is ordered in trypanosomes. Furthermore we found that BSF T. brucei cells do not grow continuously, cell size start to increase rapidly, during a short period of time, late in the cell division cycle. We speculate that the initiation of cell volume increase is temporally separated from the formation of all secretory organelles in order to ensure maintenance of the protective coat, which must remain intact at all times in order for BSF trypanosomes to be able to evade the host immune response.
... The proteins in the ER lumen are subsequently packaged into transport vesicles that fuse with the cis-Golgi vesicles. Cis-Golgi vesicles then move toward the plasma membrane and change to trans-Golgi cisternae (Glick and Malhotra, 1988). Some secretory factors such as hormones are stored in secretory vesicles and are only released upon triggers of hormonal or neural signals. ...
... Given their abundance and importance, an understanding of the mechanism by which collagens are sorted, packaged and exported across the secretory pathway, is of fundamental importance. A challenge arises from the fact that procollagens contain rigid, rod-like triple-helical domains that can reach 450 nm in length (Burgeson et al., 1985) and are too large to be exported from the ER by conventional COPII (Coat Protein complex II)-coated vesicles of 60-90 nm diameter (Glick and Malhotra, 1998;Miller and Schekman, 2013;Saito and Katada, 2015). Vesicles generated by a COPII-dependent mechanism have been characterised extensively (Barlowe et al., 1994;Lee et al., 2004) and it is clear that they are too small for procollagen export from the ER. ...
Previously we showed that membrane fusion is required for TANGO1-dependent export of procollagen VII from the endoplasmic reticulum (ER) (Nogueira, et al., 2014). Along with the t-SNARE Syntaxin 18, we now reveal the complete complement of SNAREs required in this process, t-SNAREs BNIP1 and USE1, and v-SNARE YKT6. TANGO1 recruits YKT6-containing ER Golgi Intermediate Compartment (ERGIC) membranes to procollagen VII-enriched patches on the ER. Moreover residues 1214-1396, that include the first coiled coil of TANGO1, specifically recruit ERGIC membranes even when targeted to mitochondria. TANGO1 is thus pivotal in concentrating procollagen VII in the lumen and recruiting ERGIC membranes on the cytoplasmic surface of the ER. Our data reveal that growth of a mega transport carrier for collagen export from the ER is not by acquisition of a larger patch of ER membrane, but instead by addition of ERGIC membranes to procollagen-enriched domains of the ER by a TANGO1-mediated process.
... The unstacked cisternae expose a larger surface area that becomes more accessible to recruit the components required for vesicle budding [78]. During interphase, budding and fusion of COPI transport vesicles at the Golgi are delicately balanced to maintain its function and morphology [79]. Upon entry into mitosis, phosphorylation of GM130 by Cdk1 prevents the vesicle tethering factor p115 from binding and thus blocks vesicle docking [80]. ...
The Golgi apparatus is tightly integrated into the cellular system where it plays essential roles required for a variety of cellular processes. Its vital functions include not only processing and sorting of proteins and lipids, but also serving as a signaling hub and a microtubule-organizing center. Golgi stacks in mammalian cells are interconnected into a compact ribbon in the perinuclear region. However, the ribbon can undergo distinct disassembly processes that reflect the cellular state or environmental demands and stress. For instance, its most dramatic change takes place in mitosis when the ribbon is efficiently disassembled into vesicles through a combination of ribbon unlinking, cisternal unstacking and vesiculation. Furthermore, the ribbon can also be detached and positioned at specific cellular locations to gain additional functionalities during differentiation, or fragmented to different degrees along disease progression or upon cell death. Here, we describe the major morphological alterations of Golgi ribbon disassembly under physiological and pathological conditions and discuss the underlying mechanisms that drive these changes.
... A typical P. pastoris cell contains two to six discrete tER sites, each of which is adjacent to a Golgi stack (Rossanese et al., 1999). According to the cisternal maturation model for Golgi function, new Golgi cisternae form next to tER sites by the homotypic fusion of COPII vesicles (Bannykh and Balch, 1997;Mironov et al., 1997;Pelham, 1998;Glick and Malhotra, 1998). In S. cerevisiae, the entire ER network seems to function as tER, and therefore new Golgi cisternae form throughout the cytoplasm (Rossanese et al., 1999). ...
Pichia pastoris has discrete transitional ER sites and coherent Golgi stacks, making this yeast an ideal system for studying the organization of the early secretory pathway. To provide molecular tools for this endeavour, we isolated P. pastoris homologues of the SEC12, SEC13, SEC17, SEC18 and SAR1 genes. The P. pastorisSEC12, SEC13, SEC17 and SEC18 genes were shown to complement the corresponding S. cerevisiae mutants. The SEC17 and SAR1 genes contain introns at the same relative positions in both P. pastoris and S. cerevisiae, whereas the SEC13 gene contains an intron in P. pastoris but not in S. cerevisiae. Intron structure is similar in the two yeasts, although the favoured 5′ splice sequence appears to be GTAAGT in P. pastoris vs. GTATGT in S. cerevisiae. The predicted amino acid sequences of Sec13p, Sec17p, Sec18p and Sar1p show strong conservation in the two yeasts. By contrast, the predicted lumenal domain of Sec12p is much larger in P. pastoris, suggesting that this domain may help localize Sec12p to transitional ER sites. A comparison of the SEC12 loci in various budding yeasts indicates that the SEC12-related gene SED4 is probably unique to the Saccharomyces lineage. GenBank Accession Nos are: SEC12, AF216960; SEC13, AF242186; SEC17, AF216957; SEC18, AF216958; SAR1, AF216959; ACT1, AF216956. Copyright
... in the Golgi, to arrive at general conditions for compartment 40 biogenesis. Such an exercise would be useful in addressing the fundamental issue of whether 41 the Golgi organelle is constructed from a pre-existing template or generated de novo, a result 42 of self-organization [4, 6, 13, 14]. In the process, it may lead us to revisit and make precise, the 43 notion of an organelle. ...
A central issue in cell biology is the physico-chemical basis of organelle biogenesis in intracellular trafficking pathways, its most impressive manifestation being the biogenesis of Golgi cisternae. At a basic level, such morphologically and chemically distinct compartments should arise from an interplay between the molecular transport and chemical maturation. Here, we formulate analytically tractable, minimalist models, that incorporate this interplay between transport and chemical progression in physical space, and explore the conditions for de novo biogenesis of distinct cisternae. We propose new quantitative measures that can discriminate between the various models of transport in a qualitative manner–this includes measures of the dynamics in steady state and the dynamical response to perturbations of the kind amenable to live-cell imaging.
... Secretory and transmembrane proteins make up 30–50% of all cellular proteins, and are trafficked through the endoplasmic reticulum (ER) to the Golgi for folding and modifications before delivery to their final destination. Secretory cargo molecules are thought to travel through the Golgi complex mostly inside flat cisternae that are constantly maturing in a cis-to-trans (anterograde) fashion via the so called cisternal maturation mechanism (Glick and Malhotra, 1998). However, resident Golgi proteins and Soluble NSF Attachment protein Receptors (SNAREs) are constantly recycled back in vesicular 50 | carriers to replenish the content of newly formed ciscisternae . ...
This eBook contains 13 reviews which address the molecular mechanisms of Golgi pathology in Parkinson and Alzheimer disease, amyotrophic lateral sclerosis (ALS) and spinal muscular atrophies, and discuss their potential relevance to the pathological loss of neuronal cell bodies, axons and synapses.
... This scheme has dominated the field for the last few decades, and has provided an elegant framework by which to rationalize a wealth of molecular and genetic data (Rothman and Wieland, 1996;Schekman and Orci, 1996); however, it has never been validated in vivo, and in recent years it has come under increasing criticism. The second is the progression-maturation scheme, by which cargo remains confined within the lumen of cisternae while cisternae move through the stack by gradually maturing from cis into trans compartments (Bannykh and Balch, 1997;Mironov et al., 1997;Glick and Malhotra, 1998). The third model (flow through continuities) posits that cargo flows along permanent or transient continuities, connecting successive compartments (Weidman, 1995;Mironov et al., 1997Mironov et al., , 1998. ...
... COG1-4) and lobe B (COG5-8) (Fotso et al., 2005;Ungar et al., 2005). Secretory andGlick and Malhotra, 1998). However, resident 105Willett et al., 2013b). ...
The Conserved Oligomeric Golgi (COG) complex is an evolutionarily conserved hetero-octameric protein complex that has been proposed to organize vesicle tethering at the Golgi apparatus. Defects in seven of the eight COG subunits are linked to Congenital Disorders of Glycosylation (CDG)-type II, a family of rare diseases involving misregulation of protein glycosylation, alterations in Golgi structure, variations in retrograde trafficking through the Golgi and system-wide clinical pathologies. A troublesome aspect of these diseases are the neurological pathologies such as low IQ, microcephaly, and cerebellar atrophy. The essential function of the COG complex is dependent upon interactions with other components of trafficking machinery, such as Rab-GTPases and SNAREs. COG-interacting Rabs and SNAREs have been implicated in neurodegenerative diseases like Alzheimer's disease and Parkinson's disease. Defects in Golgi maintenance disrupts trafficking and processing of essential proteins, frequently associated with and contributing to compromised neuron function and human disease. Despite the recent advances in molecular neuroscience, the subcellular bases for most neurodegenerative diseases are poorly understood. This article gives an overview of the potential contributions of the COG complex and its Rab and SNARE partners in the pathogenesis of different neurodegenerative disorders.
... The Golgi apparatus is responsible for glycolipid biosynthesis and has a critical function in the protein secretory pathway involved in glycosylation, sorting and secretion of newly synthesised proteins received from the endoplasmic reticulum (ER) (Glick and Malhotra, 1998;Glick, 2000;Maccioni et al, 2011). The Golgi comprises a series of cisternae organised in a cis-medialtrans manner. ...
Background:
The aetiology of Barrett's oesophagus (BO) and oesophageal cancer is poorly understood. We previously demonstrated that Golgi structure and function is altered in oesophageal cancer cells. A Golgi-associated protein, GOLPH2, was previously established as a tissue biomarker for BO. Cellular functions for GOLPH2 are currently unknown, therefore in this study we sought to investigate functional roles for this Golgi-associated protein in oesophageal disease.
Methods:
Expression, intracellular localisation and secretion of GOLPH2 were identified by immunofluorescence, immunohistochemistry and western blot. GOLPH2 expression constructs and siRNA were used to identify cellular functions for GOLPH2.
Results:
We demonstrate that the structure of the Golgi is fragmented and the intracellular localisation of GOLPH2 is altered in BO and oesophageal adenocarcinoma tissue. GOLPH2 is secreted by oesophageal cancer cells and GOLPH2 expression, cleavage and secretion facilitate cell migration and invasion. Furthermore, exposure of cells to DCA, a bile acid component of gastric refluxate and known tumour promoter for oesophageal cancer, causes disassembly of the Golgi structure into ministacks, resulting in cleavage and secretion of GOLPH2.
Conclusions:
This study demonstrates that GOLPH2 may be a useful tissue biomarker for oesophageal disease. We provide a novel mechanistic insight into the aetiology of oesophageal cancer and reveal novel functions for GOLPH2 in regulating tumour cell migration and invasion, important functions for the metastatic process in oesophageal cancer.British Journal of Cancer advance online publication, 13 October 2015; doi:10.1038/bjc.2015.350 www.bjcancer.com.
... Generation of a mega carrier containing PC VII at the ER. general (Glick & Malhotra 1998). This model has been challenged. ...
COPII vesicles mediate export of secretory cargo from the endoplasmic reticulum (ER). However, a standard COPII vesicle with a diameter of 60- 90 nm is too small to export collagens that are composed of rigid triple helices of up to 400 nm in length. How do cells pack and secrete such bulky molecules? This issue is fundamentally important, as collagens constitute approximately 25% of our dry body weight and are essential for almost all cell-cell interactions. Recently, a potential mechanism for the biogenesis of mega-transport carriers was identified, involving packing collagens and increasing the size of COPII coats. Packing is mediated by TANGO1, which binds procollagen VII in the lumen and interacts with the COPII proteins Sec23/Sec24 on the cytoplasmic side of the ER. Cullin3, an E3 ligase, and its specific adaptor protein, KLHL12, ubiquitinate Sec31, which could increase the size of COPII coats. Recruitment of these proteins and their specific interactors into COPII-mediated vesicle biogenesis may be all that is needed for the export of bulky collagens from the ER. Nonetheless, we present an alternative pathway in which TANGO1 and COPII cooperate to export collagens without generating a mega-transport carrier. Expected final online publication date for the Annual Review of Cell and Developmental Biology Volume 31 is October 06, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
... Protein aggregates have been shown to traffic through the Golgi en route to functional activity elsewhere inside or outside the cell. A well-characterized example is the transport of procollagen aggregates in mammalian cells, which helped prove the validity of the cisternal maturation model (56)(57)(58). The aggregates in our tomograms were more similar in appearance to the developing scales of the green alga Scherffelia dubia (17,59,60), which also traverse the Golgi by cisternal maturation before they are secreted to the cell wall and flagella (61). ...
Significance
To our knowledge, this is the first detailed study of Golgi ultrastructure within unperturbed cells. Three intracisternal structures were identified, with implications for Golgi architecture and trafficking: ( i ) Bundles of filaments show how cargoes may oligomerize to increase their local concentration at trans-Golgi buds. ( ii ) Granular aggregates provide evidence for cisternal maturation, as they are likely too large to transit the Golgi via vesicles. ( iii ) Protein arrays link the membranes of the central trans-Golgi cisternae, simultaneously maintaining the narrow luminal spacing while promoting cargo exit from the Golgi periphery by excluding material from the center. The asymmetry of the array structure indicates that the apposing membranes of a single cisterna have distinct compositions. The assembly of arrays may also enhance glycosyltransferase kinetics.
... Protein transport through the Golgi apparatus may occur according to the vesicular transport model (stable Golgi cisternae) or the cisternal maturation model (Glick and Malhotra, 1998 ) . Many lines of evidence now support this later model in which the Golgi cisternae themselves move through the Golgi stack. ...
The mammary gland acts as a bio-factory to produce in large amount few proteins by transcribing temporally and spatially regulated genes and translating their mRNA. The aim of this chapter is to summarize briefly our knowledge on the structure of the milk protein genes and to put into context the rapid growth of information on the regulatory elements involved in controlling the expression of these genes. It also describes the amino acid supply to the mammary gland and the intracellular transport and sorting of milk proteins in the secretory pathway of mammary cells.
... Delivery of secretory proteins to the cis-side of the Golgi apparatus is followed by their passage across the Golgi stack of cisternae, which contains enzymes involved in sequential processing of oligosaccharide chains on glycoproteins, and in proteolytic cleavage (Mellman et al., 2000). The favored model of anterograde intra-Golgi transport is the cisternal maturation and progression (Glick et al., 1998). It assumes that new cisternae form at the cis-Golgi, progress through the stack carrying anterograde cargo, and disassemble at the trans-Golgi. ...
Several members of the Src family of non-receptor protein tyrosine kinases (e.g. Yes), as well as Leishmania hydrophilic acylated surface protein B (HASPB) harbor a short N-terminal motif called the Src homology 4 (SH4) domain, which undergoes tandem modification with the saturated acyl chains myristate and palmitate. SH4 domains are responsible for stable anchoring of these otherwise soluble proteins to the cytoplasmic leaflet of cellular membranes, mediate their targeting to the plasma membrane, and moreover, confer affinity for cholesterol- and sphingolipid-enriched membrane microdomains, that is lipid rafts. N-terminal myristoylation occurs in the cytosol, concurrently with translation of the protein, and is a prerequisite for subsequent palmitoylation. The latter is thought to occur at perinuclear (most probably Golgi) membranes. It has been hypothesized that doubly acylated SH4 domain proteins partition into lipid rafts soon after being palmitoylated at the Golgi, and that these microdomains are necessary for the transport of SH4 proteins to the plasma membrane, by playing a role in their sorting and/or formation of transport carriers at the trans-Golgi network.
The aim of the first part of this study was to characterize membrane (both lipid and protein) environment of diacylated SH4 domain proteins residing in lipid rafts. The approach was to analyze lipid and protein components of immunoaffinity-purified detergent-resistant membranes (DRMs) containing SH4 domain reporter fusion proteins. DRMs immunoisolated using a HASPB SH4 domain fusion protein as bait differed in lipid composition from the total DRM pool, being enriched in sphingomyelin, and depleted in phosphatidylcholine and phosphatidylethanolamine, and therefore seem to be a subset of total DRMs. This may suggest that in vivo, the protein associates with a specific subset of lipid rafts, thus indicating that the heterogeneity of lipid rafts can be appreciated also using the detergent method. Our immunoaffinity purification approach seemed to enrich for bona fide raft proteins, as suggested by an increase compared to total DRMs in the proportion of plasma membrane and lipid-anchored proteins, as well as proteins whose association with DRMs is sensitive to cholesterol depletion. We estimated relative amounts of proteins in immunoisolated SH4 DRMs and total DRMs, with the use of label-free mass spectrometry-based quantification, which was validated for a subset of proteins by quantitative Western blotting. We could observe a lack of enrichment of endogenous Yes kinase in immunoisolated SH4 DRMs, which may indicate that the identity of lipid rafts into which SH4 domain proteins partition, is determined not only by the dual fatty acylation with myristate and palmitate, but also by interactions conferred by domains distal from the SH4 domain.
In the second part of this study, we intended to investigate the previously reported role of the COPI coatomer complex, as well as of the secretory pathway in general, in the plasma membrane transport of diacylated SH4 domain proteins. Conditions that disrupted the structure and function of the Golgi apparatus, i.e. siRNA-mediated knockdown of the β subunit of the COPI complex, expression of constitutively active mutant (Q71L) of the small GTPase Arf1, as well as brefeldin A treatment, resulted in increased intracellular accumulation of a HASPB SH4 fluorescent fusion protein, pointing out to the role of the Golgi in its transport to the plasma membrane. None of the above treatments, however, blocked the appearance of the SH4 reporter protein at the plasma membrane, suggesting the existence of an alternative trafficking pathway that does not require the Golgi complex.
... Following post-transcriptional processing and quality control, they are packed into cargo vesicles coated with coat protein complex II (COPII) PROTEIN HOMEOSTASIS at specialized membrane domains, called ER exit sites (mammals) or tER sites (yeast or drosophila melanogaster) and directed towards the Golgiapparatus [132] . The transport vesicles fuse to form new cis-Golgi vesicles, which then move towards the trans-face of the Golgi stack [133] . After passing the Golgi-apparatus, where they are modified, processed and sorted, cargo proteins are directed into vesicles and transported towards the cell membrane. ...
Transcription and translation are precisely-regulated processes that enable a cell to react to environmental stimuli by the production of appropriate sets of proteins. In order to analyze gene expression regulation, two novel methods have been developed that facilitate affinity purification approaches in combination with quantitative mass spectrometry.
First, the concepts underlying transcriptional control are investigated by identifying proteins binding to cis-regulatory modules (CRMs) in a sequence-specific manner. The method facilitated the determination of proteins specifically binding to DNA sequences with a length of 500 base pairs. Quantitative comparison of the binding pattern of three CRMs active in Drosophila melanogaster muscle development revealed 72 candidates potentially regulating the activity of these CRMs among thousands of proteins that interact non-specifically. In vivo validation of biological activity of several candidates is in progress and will probably reveal new circuits of Drosophila melanogaster muscle development.
Second, a novel approach to specifically enrich and quantify newly synthesized proteins by combining click-chemistry and pulsed SILAC labeling was developed. This method was introduced as a useful tool to study protein synthesis and the sensitive detection of rapid response to cellular stimulation.
In addition, the method was adapted to the selective and precise quantification of secreted proteins. This important subset of mammalian proteins is currently understudied because of technical limitations in the detection of low-abundant proteins against a background of serum. In-depth and differential secretome analysis of various cell lines and primary cells revealed, e.g., profound effects of serum starvation on secretome composition. Moreover, a unique application studying the kinetics of protein secretion was introduced.
The approach will have broad implications in studying the responsiveness of cells grown under optimal conditions.
Finally, in combination with RNA and protein abundance measurements, the developed approaches were used to investigate regulatory mechanism establishing response programs in lipopolysaccharides stimulated mouse macrophages with temporal resolution. These data for the first time provide a comprehensive view on the kinetics of macrophage activation. Transcriptional, translational and localization regulation was distinguished and starting points for further investigation of these mechanisms were proposed.
... COPI vesicles retrieve chaperones which escaped the lumen of the ER and recycle SNARE proteins (Letourneur et al., 1994;Pelham et al., 1988). Furthermore, COPI-coated vesicles retrieve cisand medial-Golgi-resident proteins which reach later Golgi compartments during cisternal maturation of the Golgi apparatus (reviewed by Glick and Malhotra, 1998;Pelham, 2001). Yet, there is also evidence for a role of COPI in late secretory trafficking Gu et al., 1997;Robinson et al., 2006;. ...
Vesicular transport is an essential process allowing communication between different organelles in an eukaryotic cell. The small GTPase Arf1p regulates the generation of coated vesicles from donor organelles at many different levels of the secretory pathway. Arf1p cooperates with GTPase-activating proteins (GAPs) to hydrolyze the bound GTP, which subsequently induces the shedding of the proteinacious vesicle coat. Only then the vesicle is capable to undergo SNARE-mediated (soluble NSF attachment protein receptor) fusion with a target organelle to deliver its content. Previously, our lab showed that ArfGAPs can catalytically induce an altered conformation in vesicle SNARE proteins (v-SNAREs). The SNARE in the altered conformation is able to interact with Arf1p. Thus, the uptake of v-SNAREs in budding vesicles is facilitated.
The current study extends the previous results to target membrane SNAREs (t-SNAREs) and shows that the ArfGAP-induced conformation enhances the formation of SNARE complexes. SNARE complex formation is an essential step during membrane fusion. Thus, the altered SNARE conformation is not only important during vesicle generation, but also for the consumption of transport vesicles. This let us speculate that ArfGAP proteins might act in a chaperone-like function on SNARE proteins. We were also able to show that SNARE proteins in the ArfGAP-induced SNARE conformation can interact with Sec17p and Sec18p, the yeast homologs of alpha-SNAP (soluble NSF attachment protein) and AAA-ATPase NSF (N-ethylmaleimide-sensitive factor). Both factors play a key role in membrane fusion by resolving cis-SNARE complexes. Data from this study indicate that the ATPase Sec18p has a second function during vesicle fusion as it can displace Arf1p from SNAREs. This process requires neither ATP-hydrolysis nor Sec18p´s co-factor Sec17p. Residual coat on the vesicle could still be required during an initial contact between the two fusing membranes. However, it would likely obstruct the final fusion event. Thus, Sec18p might be able to remove residual coat from a vesicle and allow final fusion to take place.
After one round of vesicle fusion, v-SNAREs are recycled back to the donor compartment. Data presented in this study show that Snc1p and Snc2p, which are v-SNAREs involved in yeast exo- and endocytosis, interact physically and genetically with the ArfGAP Gcs1p, and that this interaction is essential for recycling of SNAREs via the trans-Golgi-Network (TGN) and endosomes. Furthermore, we were able to show that the COPI vesicle coat, which has been implicated in retrograde trafficking from the Golgi apparatus to the endoplasmic reticulum (ER) as well as in retrograde traffic within the Golgi apparatus, plays also an important role in the recycling of the post-Golgi SNAREs Snc1p and Snc2p.
Besides Arf1p´s role in the formation of retrograde directed vesicles, Arf1p also participates in anterograde trafficking from the TGN, which is thought to be the main sorting station for anterograde cargo in an eukaryotic cell. In an attempt to identify new interactors of the small GTPase Arf1p, we found a novel fungi-specific protein family: the ChAPs (for Chs5p and Arf1p-interacting proteins). The ChAP family of proteins consists out of four members: Bch1p, Bch2p, Chs6p and Bud7p. These factors at least partially localize to the TGN, dependent on their interaction with Chs5p. Chs5p has been previously reported to be essential for the delivery of chitin-synthase III (Chs3p) to the yeast bud neck-region; a process Arf1p is also implicated in. We are able to show that Arf1p, Chs5p and individual members of the ChAP family interact genetically and physically. Arf1p, Chs5p and the ChAP proteins form high molecular complexes that contain the potential cargo Chs3p. Based on our findings, we suggest that the ChAP proteins are required for the transport of certain cargo in specialized transport vesicles. The ChAPs might function as cargo receptors, coat adaptors or even as novel coat.
Altogether, this work has highlighted new interactors for the small GTPase Arf1p and new modes of function for its regulatory GAP proteins.
... PKD was found to bind to the trans-Golgi network (TGN) via DAG. The TGN represents the outermost cisternae of the Golgi stack from which vesicles are released by fission destined to the cell surface, lysosomes and the secretory pathway (Glick and Malhotra, 1998). The interaction of PKD with DAG at the TGN is mediated via its CRD1 domain (Prestle et al., 1996;Maeda et al., 2001; Baron and Malhotra, 2002). ...
The pancreas ductal adenocarcinoma (PDAC) is one of the most common types of cancer, accounting for a large number of cancer related deaths. Rapid tumour metastasis is a major problem in pancreatic cancer, and little is known on the molecular events governing this process, however, features like changes in cell shape, modulation of cell-to-cell adhesion, enhanced cell motility and matrix degrading potential seem to be important. In the literature the Protein kinase D (PKD) family of serine/threonine kinases, which consists of 3 structurally related isoforms, PKD1/PKCμ, PKD2 and PKD3/PKCnu has been implicated in the regulation of some of these processes, yet the molecular mechanisms involved largely remained unclear. That's why this study, using PDAC cell lines, focused on the function of PKD in the metastatic progression of cancer cells. In this work, PKD was found to be strongly expressed and also active in Panc89 cells. Focusing on this cell line, PKD was shown to colocalise with F-Actin and respective markers, indicating active Actin remodeling. For example, Arp3, a member of the Actin-related Arp2/3 protein complex, which is responsible for de novo Actin nucleation and dendritic branching of Actin filaments, colocalised with PKD. Further, PKD colocalised with a subcellular pool of Vinculin at the edge of membrane protrusions, again indicating active Actin turnover, possibly at nascent focal complexes, however PKD was not localised to Vinculin-positive mature focal adhesions to the substratum. In addition, PKD localised with Cortactin, which is enriched within lamellipodia and membrane ruffles. It is implicated in the stabilisation of F-Actin branch points and therefore exhibits important functions at the cortical F-Actin cytoskeleton, which are directly linked to cell migration. Surprisingly, PKD was also found to directly bind to F-Actin in vitro. The binding domain was mapped to 46 amino acids in the N-terminal region of PKD. An alignment of the respective PKD sequence indicated highly conserved motifs, both amongst PKD isoforms and also between human, mouse, and Drosophila species, pointing to a general feature of PKDs. Apart from the binding to F-Actin, PKD and Cortactin also interacted biochemically and PKD was shown to phosphorylate Cortactin in vitro at S298, as well as at additional unspecified sites. Unfortunately an in vivo phosphorylation of Cortactin could not be demonstrated up to now. Employing stable Panc89 cell lines, PKD1 impaired 3D cell migration, while for PKD1KD expressing cells, migration was enhanced. These effects can either be explained by a model implicating Cortactin phosphorylation by PKD in the regulation of F-Actin turnover and rigidity, or by the phosphorylation of yet unknown PKD substrates at the respective F-Actin-rich structures, negatively regulating cell migration. Since mutation of the potential Cortactin phosphorylation site Ser298 to an alanine residue also increased cell migration in stable Panc89 cells, Cortactin might be a potential target of PKD in the regulation of F-Actin dynamics.
PKD has also been implicated in the regulation of Ca2+-dependent cell-to-cell adhesion. PKD2KD and, to lesser extent, PKD1KD, strongly enhanced cell-to-cell adhesion of stable Panc89 cells, whereas E-Cadherin expression in the respective parental cell lines was reduced. At least in the case of PKD2KD cells, aggregation was only partially dependent on E-Cadherin, pointing to the expression of additional Cadherin isoforms. In the PKD2 expressing cell line, aggregation was very weak, in some assays even resembling the vector control. This phenotype could be correlated with processed E-Cadherin fragments in the supernatant of PKD2 expressing cells, which have been implicated in the literature with the inhibition of cell-to-cell adhesion of cancer cell lines, thereby also increasing their invasive potential. Initial results demonstrated that the E-Cadherin fragments from the supernatant of stable PKD2 expressing Panc89 cells were processed by a serine-protease, possibly Plasmin or cationic Trypsin. How the PKD2 isoform is implicated in this process remains to be investigated further. Yet, these findings are in line with data obtained from expression profiling experiments with the respective stable Panc89 cells, indicating that the urokinase-like Plasminogen-activator-receptor (PLAUR) is strongly up-regulated in PKD2 expressing cells, triggering the activation of the uPA-Plasminogen-Plasmin-cascade, which has been demonstrated to be involved in the processing of E-Cadherin.
During the course of this work novel aspects concerning the role of PKD in cell migration and cellular adhesion were revealed. The localisation of PKD at the F-Actin cytoskeleton and its in vitro binding to F-Actin implicate possible functions for the PKD protein family in the regulation of F-Actin dynamics, which might influence cell adhesion and motility.
Stimulator of interferon genes (STING) is a sensor of cyclic dinucleotides including cyclic GMP-AMP (cGAMP), which is produced by cGAMP synthase (cGAS) in response to cytosolic DNA. The cGAS-STING signaling pathway regulates both innate and adaptive immune responses, as well as fundamental cellular functions such as autophagy, senescence, and apoptosis. Mutations leading to constitutive activation of STING cause devastating human diseases. Thus, the cGAS-STING pathway is of great interest because of its role in diverse cellular processes and because of the potential therapeutic implications of targeting cGAS and STING. Here, we review molecular and cellular mechanisms of STING signaling, and we propose a framework for understanding the immunological and other cellular functions of STING in the context of disease.
As the foremost cause of cancer‐related death, metastasis consists of three steps: invasion, circulation, and colonization. Only targeting one single phase of the metastasis cascade may be insufficient since there are many alternative routes for tumor cells to disseminate. Here, to target the whole cascade of metastasis, hybrid erythrocyte and tumor cell membrane‐coated nanoparticle (Hyb‐NP) is designed with dual functions of increasing circulation time and recognizing primary, circulating, and colonized tumors. After loading with monensin, a recently reported metastasis inhibitor, the delivery system profoundly reduces spontaneous metastasis in an orthotopic breast cancer model. Underlying mechanism studies reveal that Hyb‐NP can deliver monensin to its action site in the Golgi apparatus, and in return, monensin can block the exocytosis of Hyb‐NP from the Golgi apparatus, forming a reservoir‐like subcellular structure. Notably, the Golgi apparatus reservoir displays three vital functions for suppressing metastasis initialization, including enhanced subcellular drug retention, metastasis‐related cytokine release inhibition, and directional migration inhibition. Collectively, based on metastasis cascade targeting at the tissue level, further formation of the Golgi apparatus drug reservoir at the subcellular level provides a potential therapeutic strategy for cancer metastasis suppression.
Many newly synthesized proteins pass through the Golgi apparatus. This passage is driven by the maturation of Golgi cisternae, which are disk-shaped membrane compartments that contain the newly synthesized proteins together with resident Golgi processing enzymes. Golgi cisternae continually form, transform, and then dissolve into transport carriers. Meanwhile, resident Golgi proteins recycle by pathways that involve either intra-Golgi recycling or transport to another organelle and back. These processes are regulated by multiple GTPases. The different kinetics of the various recycling pathways generate polarized distributions of resident Golgi proteins. An ongoing challenge is to elucidate the mechanisms that control Golgi dynamics.
This thesis provides in vivo information about two proteins - p23 and calnexin - which play major roles in the early secretory pathway of mammalian cells. The aim was to gain further understanding about the function of these proteins by generating mice deficient in the expression of p23 and calnexin. p23 is a member of the integral p24 protein family. These proteins are highly abundant in the endoplasmic reticulum and Golgi apparatus and are thought to play a role in protein transport and vesicle formation. Disruption of both p23 alleles results in early embryonic lethality. Inactivation of one allele leads not only to reduced levels of p23 itself but also of other family members. The reduction in steady state protein levels also leads to an altered subcellular distribution of p23 as well as p26 (another family member) in p23 heterozygous cells. In addition, structural changes in the Golgi apparatus, in particular dilated saccules, were observed. These changes in p23 heterozygous mice have functional consequences, resulting in specific defects in the secretion of some plasma proteins. Calnexin is a molecular chaperone molecule which is involved in the correct folding of newly synthesised polypeptide chains in the ER. 40% of the calnexin homozygous deficient mice died shortly after birth, the remaining 60% developed a severe neuronal phenotype and had to be sacrificed within 2 months. They were about one third smaller than their littermates and displayed motor disorders which included shaking of the body and a wobbly unsteady gait. Histological examination of brain, cerebellum, spinal cord, neuromuscular junctions and muscle did not reveal any abnormalities in the calnexin-deficient mice. Although these mice showed clear neurodegenerative symptoms, the molecular basis for the phenotype has not yet been characterised.
This chapter characterizes the most important cellular organellesOrganelles such as the endoplasmatic reticulumEndoplasmatic Reticulum (ER) and the biological and physical functions based on the Golgi apparatusGolgi apparatus and relates the properties of these organelles to the overall mechanical propertiesMechanical properties of the cell. More specific, the connections between the endoplasmatic reticulumEndoplasmatic Reticulum (ER) and the Golgi apparatusGolgi apparatus are presented. All cell organellesOrganelles consist of highly specialized subcompartments fulfilling distinct tasks, and hence, they provide on this discrete level a higher organization of the overall cell. What we understand about the function of organellesOrganelles is mainly based on microscopic observations and less on the effects of stress on the structure and function of organelles, which is also addressed and discussed.
The sections in this article are
The Problem
The Models
The Data
The Solution?
The Implications
The sections in this article are
Introduction
Golgi Organization in Budding Yeasts
G olgi Apparatus Organization in Fission Yeasts
Comparison of Yeast and Plant G olgi Structures
Acknowledgements
Combined harvester: Supramolecular co‐assembly of lower‐rim dodecyl‐modified sulfonatocalix[4]arene (SC4AD) and naphthyl‐1,8‐diphenyl pyridinium derivative (NPS) gives a light‐harvesting platform showing significant aggregation induced emission enhancement (AIEE). By using these near‐infrared (NIR) emissive nanoparticles imaging in the Golgi apparatus is possible.
Abstract
Light‐harvesting systems are an important way for capturing, transferring and utilizing light energy. It remains a key challenge to develop highly efficient artificial light‐harvesting systems. Herein, we report a supramolecular co‐assembly based on lower‐rim dodecyl‐modified sulfonatocalix[4]arene (SC4AD) and naphthyl‐1,8‐diphenyl pyridinium derivative (NPS) as a light‐harvesting platform. NPS as a donor shows significant aggregation induced emission enhancement (AIEE) after assembling with SC4AD. Upon introduction of Nile blue (NiB) as an acceptor into the NPS‐SC4AD co‐assembly, the light‐harvesting system becomes near‐infrared (NIR) emissive (675 nm). Importantly, the NIR emitting NPS‐SC4AD‐NiB system exhibits an ultrahigh antenna effect (33.1) at a high donor/acceptor ratio (250:1). By co‐staining PC‐3 cells with a Golgi staining reagent, NBD C6‐ceramide, NIR imaging in the Golgi apparatus has been demonstrated using these NIR emissive nanoparticles.
Light‐harvesting systems are an important way for capturing, transferring and utilizing light energy. It remains a key challenge to develop highly efficient artificial light‐harvesting systems. Herein, we report a supramolecular co‐assembly based on lower‐rim dodecyl‐modified sulfonatocalix[4]arene (SC4AD) and naphthyl‐1,8‐diphenyl pyridinium derivative (NPS) as a light‐harvesting platform. NPS as a donor shows significant aggregation induced emission enhancement (AIEE) after assembling with SC4AD. Upon introduction of Nile blue (NiB) as an acceptor into the NPS‐SC4AD co‐assembly, the light‐harvesting system becomes near‐infrared (NIR) emissive (675 nm). Importantly, the NIR emitting NPS‐SC4AD‐NiB system exhibits an ultrahigh antenna effect (33.1) at a high donor/acceptor ratio (250:1). By co‐staining PC‐3 cells with a Golgi staining reagent, NBD C6‐ceramide, NIR imaging in the Golgi apparatus has been demonstrated using these NIR emissive nanoparticles.
Phosphatidylinositol (PtdIns) transfer proteins (PITPs) stimulate PtdIns-4-P synthesis and signaling in eukaryotic cells, but to what biological outcomes such signaling circuits are coupled remains unclear. Herein, we show that two highly related StART-like PITPs, PITPNA and PITPNB, act in a redundant fashion to support development of the embryonic mammalian neocortex. PITPNA/PITPNB do so by driving PtdIns-4-P-dependent recruitment of GOLPH3, and likely ceramide transfer protein (CERT), to Golgi membranes with GOLPH3 recruitment serving to promote MYO18A- and F-actin-directed loading of the Golgi network to apical processes of neural stem cells (NSCs). We propose the primary role for PITP/PtdIns-4-P/GOLPH3/CERT signaling in NSC Golgi is not in regulating bulk membrane trafficking but in optimizing apically directed membrane trafficking and/or apical membrane signaling during neurogenesis. Xie et al. describe a cell-autonomous phosphatidylinositol-4-phosphate-dependent pathway essential for neocortex development that regulates neural stem cell function by loading the Golgi network into the cellular apical compartment. The circuit involves two lipid transfer proteins (PITPNA/PITPNB) that potentiate phosphatidylinositol-4-phosphate synthesis, and GOLPH3 and CERT as effectors of phosphatidylinositol-4-phosphate signaling.
Extracellular vesicles (EVs) released by eukaryotes, archaea, and bacteria contain proteins, lipids, polysaccharides, and other molecules. The cargo analysis of EVs shows that they contain virulence factors suggesting a role in the pathogenesis of infection. The proteome, lipidome, RNA content, and carbohydrate composition of EVs from Paracoccidioides brasiliensis and Paracoccidioides lutzii were characterized. However, the effects of P. brasiliensis EVs on the host immune system have not yet been investigated. Herein, we verified that EVs from P. brasiliensis induce the production of proinflammatory mediators by murine macrophages in a dose-dependent manner. Addition of EV to macrophages also promoted transcription of the M1-polarization marker iNOs and diminish that of the M2 markers Arginase-1, Ym-1, and FIZZ-1. Furthermore, the augmented expression of M2-polarization markers, stimulated by IL-4 plus IL-10, was reverted toward an M1 phenotype in response to secondary stimulation with EVs from P. brasiliensis. The ability of EVs from P. brasiliensis to promote M1 polarization macrophages favoring an enhanced fungicidal activity, demonstrated by the decreased CFU recovery of internalized yeasts, with comparable phagocytic efficacy. Our results suggest that EVs from P. brasiliensis can modulate the innate immune response and affect the relationship between P. brasiliensis and host immune cells.
The Golgi apparatus plays a pivotal role in membrane traffic of eukaryotic cells. Cargo proteins synthesized in the endoplasmic reticulum (ER) are transported to the cis-face of the Golgi, processed and glycosylated, and then delivered from the trans-face of the Golgi for their final destinations. Debates exist, though, on the mechanism how proteins are transported through the Golgi apparatus. Here, we review current understanding of protein transport through the Golgi apparatus, and describe our models based on the observation of their dynamics by the 3D live imaging microscopy we developed.
A central issue in cell biology is the physico-chemical basis of organelle biogenesis in intracellular trafficking pathways, its most impressive manifestation being the biogenesis of Golgi cisternae. At a basic level, such morphologically and chemically distinct compartments should arise from an interplay between the molecular transport and chemical maturation. Here, we formulate analytically tractable, minimalist models, that incorporate this interplay between transport and chemical progression in physical space, and explore the conditions for de novo biogenesis of distinct cisternae. We propose new quantitative measures that can discriminate between the various models of transport in a qualitative manner- this includes measures of the dynamics in steady state and the dynamical response to perturbations of the kind amenable to live-cell imaging.
This chapter outlines the secretory pathway. The secretory pathway has two main functions. First, it transports newly synthesized proteins and lipids from the endoplasmic reticulum (ER) to the Golgi apparatus and then on to their final destinations. Second, it modifies many of these proteins and lipids during the transport process. The organelles of the secretory and endocytic pathways collectively form the endomembrane system, which has the distinctive property that all of its compartments communicate with one another and with the extracellular environment by means of transport vesicles. This chapter also depicts the relationship between tER sites, ERGIC elements, and the Golgi in vertebrate cells. The mechanism by which secretory cargo molecules move through the Golgi has been debated for over 40 years. A new cisterna would form at the cis face of the stack by the homotypic fusion of ERGIC elements. Equally mysterious are the mechanisms that control Golgi organization. Some of the proteins that are initially translocated into or across the ER membrane are not destined for secretion or for delivery to the endosomal/lysosomal/vacuolar system, but instead reside permanently in the ER. Most resident Golgi enzymes are type II membrane proteins with short N-terminal cytosolic tails, and the localization signals for these proteins are contained within the transmembrane domains and flanking regions. Recently, several groups have proposed that blocking the microtubule-dependent transport of ERGIC elements to the Golgi ribbon leads to the formation of new Golgi stacks next to tER sites, with the concomitant shrinking of the preexisting Golgi ribbon.
Technological breakthroughs of the last decade have advanced our understanding of vesicular trafficking from a largely descriptive approach to a molecular science. An impressive conservation of the molecular transport machinery across phyla has been revealed (30). Although the current consensus is that intracellular protein transport is mediated by transport vesicles, evidence suggestive of nonvesicular transport mechanisms continues to accumulate. As a consequence, two formerly rejected models, namely transport by cisternal progression (13) and traffic via membranous tubules (24, 37), are attracting renewed interest. Here, we provide updated versions of these models, review both new and old evidence relevant to their application to transport pathways, and discuss the implications as well as the difficulties posed by these alternative mechanisms.
During mitosis, the ribbon of the Golgi apparatus is transformed into dispersed tubulo-vesicular membranes, proposed to facilitate stochastic inheritance of this low copy number organelle at cytokinesis. Here, we have analyzed the mitotic disassembly of the Golgi apparatus in living cells and provide evidence that inheritance is accomplished through an ordered partitioning mechanism. Using a Sar1p dominant inhibitor of cargo exit from the endoplasmic reticulum (ER), we found that the disassembly of the Golgi observed during mitosis or microtubule disruption did not appear to involve retrograde transport of Golgi residents to the ER and subsequent reorganization of Golgi membrane fragments at ER exit sites, as has been suggested. Instead, direct visualization of a green fluorescent protein (GFP)-tagged Golgi resident through mitosis showed that the Golgi ribbon slowly reorganized into 1–3-μm fragments during G2/early prophase. A second stage of fragmentation occurred coincident with nuclear envelope breakdown and was accompanied by the bulk of mitotic Golgi redistribution. By metaphase, mitotic Golgi dynamics appeared to cease. Surprisingly, the disassembly of mitotic Golgi fragments was not a random event, but involved the reorganization of mitotic Golgi by microtubules, suggesting that analogous to chromosomes, the Golgi apparatus uses the mitotic spindle to ensure more accurate partitioning during cytokinesis.
The distribution of beta 1,2 N-acetylglucosaminyltransferase I (NAGT I), alpha 1,3-1,6 mannosidase II (Mann II), beta 1,4 galactosyltransferase (GalT), alpha 2,6 sialyltransferase (SialylT) was determined by immuno-labelling of cryo-sections from HeLa cell lines. Antibody labelling in the HeLa cell line was made possible by stable expression of epitope-tagged forms of these proteins or forms from species to which specific antibodies were available. NAGT I and Mann II had the same distribution occupying the medial and trans cisternae of the stack. GalT and SialylT also had the same distribution but they occupied the trans cisterna and the trans-Golgi network (TGN). These results generalise our earlier observations on the overlapping distribution of Golgi enzymes and show that each of the trans compartments of the Golgi apparatus in HeLa cells contains unique mixtures of those Golgi enzymes involved in the construction of complex, N-linked oligosaccharides.
The CHO cell temperature-sensitive mutant ldlF exhibits two defects in membrane traffic at the nonpermissive temperature (39.5 degrees C): rapid degradation of LDL receptors, possibly caused by endocytic missorting, and disruption of ER-through-Golgi transport. Here, we show that at 39.5 degrees C, the Golgi in ldlF cells dissociated into vesicles and tubules. This dissociation was inhibited by AlF4-, suggesting trimeric G proteins are involved in the dissociation mechanism. This resembled the effects of brefeldin A on wild-type cells. We isolated a hamster cDNA that specifically corrected the ts defects of ldlF cells, but not those of other similar ts mutants (ldlE, ldlG, ldlH, and End4). Its predicted protein sequence is conserved in humans, rice, Arabidopsis, and Caenorhabditis elegans, and is virtually identical to that of bovine epsilon-COP, a component of the coatomer complex implicated in membrane transport. This provides the first genetic evidence that coatomers in animal cells can play a role both in maintaining Golgi structure and in mediating ER-through-Golgi transport, and can influence normal endocytic recycling of LDL receptors. Thus, along with biochemical and yeast genetics methods, mammalian somatic cell mutants can provide powerful tools for the elucidation of the mechanisms underlying intracellular membrane traffic.
alpha-mannosidases I and II (Man I and II) are resident enzymes of the Golgi complex involved in oligosaccharide processing during N-linked glycoprotein biosynthesis that are widely considered to be markers of the cis- and medial-Golgi compartments, respectively. We have investigated the distribution of these enzymes in several cell types by immunofluorescence and immunoelectron microscopy. Man II was most commonly found in medial- and/or trans- cisternae but showed cell type-dependent variations in intra-Golgi distribution. It was variously localized to either medial (NRK and CHO cells), both medial and trans (pancreatic acinar cells, enterocytes), or trans- (goblet cells) cisternae, or distributed across the entire Golgi stack (hepatocytes and some enterocytes). The distribution of Man I largely coincided with that of Man II in that it was detected primarily in medial- and trans-cisternae. It also showed cell type dependent variations in its intra-Golgi distribution. Man I and Man II were also detected within secretory granules and at the cell surface of some cell types (enterocytes, pancreatic acinar cells, goblet cells). In the case of Man II, cell surface staining was shown not to be due to antibody cross-reactivity with oligosaccharide epitopes. These results indicate that the distribution of Man I and Man II within the Golgi stack of a given cell type overlaps considerably, and their distribution from one cell type to another is more variable and less compartmentalized than previously assumed.
To analyze the mechanism of integral membrane protein localization in the early Golgi apparatus of Saccharomyces cerevisiae, we have used Och1p, a cis-Golgi mannosyltransferase. A series of influenza virus hemagglutinin (HA) epitope-tagged fusion proteins was constructed in which invertase is appended to the Golgi-luminal carboxy terminus of full-length Och1p. Several constructs included a Kex2p cleavage site between the Och1p and invertase moieties to monitor transit to the Kex2p-containing TGN. Cells expressing an Och1p-invertase fusion do not secrete invertase, but those expressing an Och1p-Kex2p site-invertase fusion protein secrete high levels of invertase in a Kex2p-dependent manner. The Och1p-Kex2p site-invertase fusion protein is cleaved with a half-time of 5 min, and the process proceeds to completion. Before cleavage the protein receives glycosyl modifications indicative of passage through the medial- and trans-Golgi, therefore cleavage occurs after ordered anterograde transport through the Golgi to the TGN. Transit to distal compartments is not induced by the invertase moiety, since noninvertase fusion constructs encounter the same glycosyltransferases and Kex2p as well. The Och1p-HA moiety, irrespective of whether it is generated by cleavage of the fusion protein in the TGN or synthesized de novo, is degraded with a half-time of about 60 min. Thus, the half-time of degradation is 12-fold longer than the time required to reach the TGN. At steady state, de novo-synthesized and TGN-generated HA epitope-tagged Och1p reside in a compartment with a buoyant density identical to that of wild-type Och1p and distinct from that of the vacuole or the TGN. Finally, och1 null cells that express an Ochlp fusion construct known to rapidly encounter the TGN glycosylate invertase to the same extent as wild-type cells, indicating that they have phenotypically wild-type Och1p activity. These results lead us to propose a model for Och1p-HA localization that involves movement to distal compartments, at least as far as the TGN, followed by retrieval to the cis compartment, presumably by vesicular transport.
The mechanism by which Golgi membrane proteins are retained within the Golgi complex in the midst of a continuous flow of
protein and lipid is not yet understood. The diffusional mobilities of mammalian Golgi membrane proteins fused with green
fluorescent protein from Aequorea victoria were measured in living HeLa cells with the fluorescence photobleaching recovery technique. The diffusion coefficients ranged
from 3 × 10−9 square centimeters per second to 5 × 10−9 square centimeters per second, with greater than 90 percent of the chimeric proteins mobile. Extensive lateral diffusion
of the chimeric proteins occurred between Golgi stacks. Thus, the chimeras diffuse rapidly and freely in Golgi membranes,
which suggests that Golgi targeting and retention of these molecules does not depend on protein immobilization.
COP I-coated vesicles were analyzed for their content of resident Golgi enzymes (N-acetylgalactosaminyltransferase; N-acetylglucosaminyltransferase I; mannosidase II; galactosyltransferase), cargo (rat serum albumin; polyimmunoglobulin receptor), and recycling proteins (-KDEL receptor; ERGIC-53/p58) using biochemical and morphological techniques. The levels of these proteins were similar when the vesicles were prepared under interphase or mitotic conditions showing that sorting was unaffected. The average density relative to starting membranes for resident enzymes (14-30%), cargo (16-23%), and recycling proteins (81-125%) provides clues to the function of COP I vesicles in transport through the Golgi apparatus.
All cells secrete a diversity of macromolecules to modify their environment or to protect themselves. Eukaryotic cells have evolved a complex secretory pathway consisting of several membrane-bound compartments which contain specific sets of proteins. Experimental work on the secretory pathway has focused mainly on mammalian cell lines or on yeasts. Now, some general principles of the secretory pathway have become clear, and most components of the secretory pathway are conserved between yeast cells and mammalian cells. However, the structure and function of the secretory system in protists have been less extensively studied. In this review, we summarize the current knowledge about the secretory pathway of five different groups of protists: Giardia lamblia, one of the earliest lines of eukaryotic evolution, kinetoplastids, the slime mold Dictyostelium discoideum, and two lineages within the "crown" of eukaryotic cell evolution, the alveolates (ciliates and Plasmodium species) and the green algae. Comparison of these systems with the mammalian and yeast system shows that most elements of the secretory pathway were presumably present in the earliest eukaryotic organisms. However, one element of the secretory pathway shows considerable variation: the presence of a Golgi stack and the number of cisternae within a stack. We suggest that the functional separation of the plasma membrane from the nucleus-endoplasmic reticulum system during evolution required a sorting compartment, which became the Golgi apparatus. Once a Golgi apparatus was established, it was adapted to the various needs of the different organisms.
It is thought that residents of the Golgi stack are localized by a retention mechanism that prevents their forward progress. Nevertheless, some early Golgi proteins acquire late Golgi modifications. Herein, we describe GPP130 (Golgi phosphoprotein of 130 kDa), a 130-kDa phosphorylated and glycosylated integral membrane protein localized to the cis/medial Golgi. GPP130 appears to be the human counterpart of rat Golgi integral membrane protein, cis (GIMPc), a previously identified early Golgi antigen that acquires late Golgi carbohydrate modifications. The sequence of cDNAs encoding GPP130 indicate that it is a type II membrane protein with a predicted molecular weight of 81,880 and an unusually acidic lumenal domain. On the basis of the alignment with several rod-shaped proteins and the presence of multiple predicted coiled-coil regions, GPP130 may form a flexible rod in the Golgi lumen. In contrast to the behavior of previously studied type II Golgi proteins, overexpression of GPP130 led to a pronounced accumulation in endocytotic vesicles, and endogenous GPP130 reversibly redistributed to endocytotic vesicles after chloroquine treatment. Thus, localization of GPP130 to the early Golgi involves steps that are saturable and sensitive to lumenal pH, and GPP130 contains targeting information that specifies its return to the Golgi after chloroquine washout. Given that GIMPc acquires late Golgi modifications in untreated cells, it seems likely that GPP130/GIMPc continuously cycles between the early Golgi and distal compartments and that an unidentified retrieval mechanism is important for its targeting.
Transport between Golgi dsternae in a cell-free system is blocked hy cither nonhydrolyzable analogue ol'GTP (GTPyS) or by the sulfhydryl reagenl N-cIhylmaleimide (NEM). With GTPyS, vesicles accumulate encased in a coat which is fro/en in place because GTP cannot he hydrnlyzed. With NEM, uncoated vesicles accumulate ducked to their target cisterna. but tail to fuse; NEM inactivates a protein required tor fusion, termed NSF, for NEM Sensitive Fusion protein. The coal is composed uf many copies of a protein termed coatomcr (short for coat protomer), an assembly of seven distinct subunils. termed COP proteins. The coated vesicles also contain many copies of ;i GTPase, ARF. Cytosohc ARF binds GTP, binds to the Golgi, and then recruits cytosolic coatomer lo join the assembling coat. Later, after budding, ARF hydrolyzes its bound GTP, releasing coatomer; this explains why non-hydrolyzable GTP blocks transport and accumulates coated transport vesicles. Similar or identical COPI-coated vesicles mediate antcrograde and retrograde transport both in isolated Golgi stacks ;md in whole cells.
An important post-translational modification on most cellular proteins and lipids is represented by the acquisition of oligosaccharide side chains. The Golgi apparatus (GA) of yeast, invertebrate and vertebrate cells and of plant cells fulfills major functions in the complex process of glycosylation. Studies on the enzymes involved in glycosylation have revealed the complexity of functional subcompartmentation of the GA.
After successfully preparing hCG crystals, X-ray crystallographic analysis was finally performed. The fine 3D structure of deglycosylated hCG was determined and disulfide bridges, charge interactions, hydrophobic interactions, and hydrogen bonding of the αβ dimer were all investigated. It is clear that X-ray crystallographic methods will probably never reveal the actual structure of the real hormone; glycosylated hCG with sugars greatly affecting structure; hCG-H, the invasion-promoting autocrine; pituitary hCG, the sulfated form of hCG; and hyperglycosylated free β-subunit as produced by cancer cells. They are four separate molecules. They bind three or four different receptors and have separate functions, but they all share the same deglycosylated structure. This chapter has a misleading title, because "The Three-Dimensional Structure of hCG" is actually based on pure speculation. This chapter subjectively predicts what hCG, hCG-H, pituitary hCG, and free β-subunit might really look like. Nonetheless, we will carefully examine the X-ray crystallography of a hypothetical deglycosylated root molecule. X-ray crystallography has shown us sequences common with the evolution of TGF β and unique-to-TGFβ -like cystine knots. Each of these will be discussed in this chapter.
The Golgi apparatus (GA) is an extensive reticulum consisting of small stacks of cisternae connected to each other by tubules. At the onset of mitosis, this reticulum fragments extensively into clusters of vesicles and tubules visible throughout the cytoplasm. At the end of mitosis, small stacks reassemble and migrate towards the nucleus to re-form the interphase reticulum. We have mimicked in vitro this disassembly/reassembly cycle and this has allowed us to identify proteins involved in these transitions, and so understand some of the rules which govern both the fragmentation and the rebuilding of this complex organelle. Specifically, we have found that fragmentation involves two pathways, one of which involves the continued budding of COP I vesicles. We have also discovered that the rebuilding of single cisternae involves two fusion machineries, one of them being NSF/SNAPs/p115, the other p97.
The structure of the Golgi apparatus (GA) of 15 mammalian cell types has been examined by three-dimensional electron microscopy. In these cells, the GA appears as a single organelle consisting of a branching and anastomosing ribbon forming a juxta-or perinuclear network. Along the Golgi ribbon, compact regions made up of stacked saccules alternate with non-compact, highly fenestrated or tubular regions that bridge the saccules either of the same or of several adjacent compact zones. On its cis-trans axis, the compact region of the Golgi ribbon may be subdivided into three main compartments. The cis-compartment, made up exclusively of anastomosed membranous tubules, forms a network referred to as the cis-tubular network (CTN). The mid-compartment comprises poorly fenestrated, more or less dilated saccules depending on the presence of secretory material within their lumen. The trans-compartment is composed of three or more sacculo-tubular elements which are not continuous along the Golgi ribbon but display instead a ‘peeling off’ configuration. In cells not engaged in the production of secretion granules, the tubular portions of the trans-elements are well developed and form extensive trans-tubular polygonal networks referred to as TTN. In contrast, in glandular cells, where the formation of secretion granules mobilizes most of the membrane of the trans-Golgi elements, these tubular portions or TTN are small or even absent. The structural changes of the GA under some physiological stimulatory and inhibitory conditions in glandular cells are also described.
alpha-mannosidases I and II (Man I and II) are resident enzymes of the Golgi complex involved in oligosaccharide processing during N-linked glycoprotein biosynthesis that are widely considered to be markers of the cis- and medial-Golgi compartments, respectively. We have investigated the distribution of these enzymes in several cell types by immunofluorescence and immunoelectron microscopy. Man II was most commonly found in medial- and/or trans- cisternae but showed cell type-dependent variations in intra-Golgi distribution. It was variously localized to either medial (NRK and CHO cells), both medial and trans (pancreatic acinar cells, enterocytes), or trans- (goblet cells) cisternae, or distributed across the entire Golgi stack (hepatocytes and some enterocytes). The distribution of Man I largely coincided with that of Man II in that it was detected primarily in medial- and trans-cisternae. It also showed cell type dependent variations in its intra-Golgi distribution. Man I and Man II were also detected within secretory granules and at the cell surface of some cell types (enterocytes, pancreatic acinar cells, goblet cells). In the case of Man II, cell surface staining was shown not to be due to antibody cross-reactivity with oligosaccharide epitopes. These results indicate that the distribution of Man I and Man II within the Golgi stack of a given cell type overlaps considerably, and their distribution from one cell type to another is more variable and less compartmentalized than previously assumed.
The CHO cell temperature-sensitive mutant ldlF exhibits two defects in membrane traffic at the nonpermissive temperature (39.5 degrees C): rapid degradation of LDL receptors, possibly caused by endocytic missorting, and disruption of ER-through-Golgi transport. Here, we show that at 39.5 degrees C, the Golgi in ldlF cells dissociated into vesicles and tubules. This dissociation was inhibited by AlF4-, suggesting trimeric G proteins are involved in the dissociation mechanism. This resembled the effects of brefeldin A on wild-type cells. We isolated a hamster cDNA that specifically corrected the ts defects of ldlF cells, but not those of other similar ts mutants (ldlE, ldlG, ldlH, and End4). Its predicted protein sequence is conserved in humans, rice, Arabidopsis, and Caenorhabditis elegans, and is virtually identical to that of bovine epsilon-COP, a component of the coatomer complex implicated in membrane transport. This provides the first genetic evidence that coatomers in animal cells can play a role both in maintaining Golgi structure and in mediating ER-through-Golgi transport, and can influence normal endocytic recycling of LDL receptors. Thus, along with biochemical and yeast genetics methods, mammalian somatic cell mutants can provide powerful tools for the elucidation of the mechanisms underlying intracellular membrane traffic.
The function of the Golgi apparatus in the secretion of plant slimes is reviewed. It is shown how the research on slime secretion has increased the knowledge on the structure and dynamics of dictyosomes. Current models on intradictyosomal membrane traffic-anterograde progression of complete cisternae or anterograde movement of lateral vesicles with stationary cisternae-are discussed in the light of old and new results on slime secreting plant cells.
Coat complexes facilitate the formation of transport vesicles which are essential for proper trafficking of protein and lipids through the secretory pathway. Since its initial identification in the mid-1980s, the COPI coat complex has been credited with mediating multiple distinct transport events and intracellular processes in the exocytic pathway. Not surprisingly, the diversity of these functions has led to significant debate concerning the primary function of COPI. Specifically, within the ER/Golgi and intra-Golgi systems, does COPI mediate anterograde protein transport, retrograde protein transport, or both? This review will focus on the in vivo roles of COPI, primarily examining data from studies of yeast COPI mutants but also including evidence from mammalian systems as appropriate. Some of the current controversies surrounding whether COPI acts directly or indirectly in anterograde and retrograde transport will also be addressed. Because recruitment of COPI to membranes requires the small GTP-binding protein ARF, we will also discuss ARF and proteins that regulate ARF function, and how these proteins might modulate both COPI-driven events and overall membrane composition. Finally, we will point out some of the links still missing from our understanding of COPI-driven events and discuss possible future directions for studies of COPI function.
The Golgi apparatus (GA) is a key organelle in biosynthetic membrane transport. It is a morphologically complex compartment composed of a central domain of stacked flattened cisternae, flanked at its entry and exit sites by tubular networks with sorting functions, thecis- andtrans-Golgi networks (CGN and TGN), respectively. Interactions with the cytoskeleton appear to play important roles in both function and morphology of these membranes. Microtubules and their associated motors are involved in determining the position of the GA which is usually associated with the juxtanuclear microtubule organizing center (MTOC). They are also involved in directing incoming and outgoing membrane traffic. Because the GA is the junction at which transport to and from the endoplasmic reticulum (ER) and to the cell surface, as well as endocytic transport, meet, it can be considered a central station of membrane traffic. The role of microfilaments in Golgi function is less well understood. The presence of members of the protein families of spectrin and ankyrin on Golgi membranes, however, indicates that these cytoskeletal structures may be involved in the distinct morphological structure of the GA. The association of myosins with Golgi membranes may furthermore suggest a role of the microfilament system in aspects of membrane transport of this organelle. The roles of the different cytoskeletal structures in Golgi function will be summarized in this review, and their relevance to the organization of membrane traffic discussed.
This chapter discusses the transport of protein between endoplasmic reticulum and golgi compartments in semiintact cells. Preparation of cells that actively transport VSV G protein are most thoroughly characterized using CHO cells and CHO cytosol. However, there is considerable variability in transport using different cell lines or conditions. Where indicated in the procedures, measurement of transport using different marker proteins, or the use of different cell lines or cytosol preparations, may need to be optimized to obtain maximal efficiencies of transport. Extensive fragmentation and lysis of the ER (particularly in the case of strongly adherent cells) generally result in reduced transport. In particular, perforation conditions can lead to the release of soluble marker proteins from the ER during preparation of semiintact cells. In contrast, poor perforation leads to a transport that is efficient, but cytosol independent. In the latter case, cytosol dependence can be enhanced by gently homogenizing the semiintact cells with a loose-fitting pestle in a glass Dounce (10-20 strokes) prior to washing.
The membrane compartments responsible for Golgi functions in wild-type Saccharomyces cerevisiae were identified and characterized by immunoelectron microscopy. Using improved fixation methods, Golgi compartments were identified by labeling with antibodies specific for alpha 1-6 mannose linkages, the Sec7 protein, or the Ypt1 protein. The compartments labeled by each of these antibodies appear as disk-like structures that are apparently surrounded by small vesicles. Yeast Golgi typically are seen as single, isolated cisternae, generally not arranged into parallel stacks. The location of the Golgi structures was monitored by immunoelectron microscopy through the yeast cell cycle. Several Golgi compartments, apparently randomly distributed, were always observed in mother cells. During the initiation of new daughter cells, additional Golgi structures cluster just below the site of bud emergence. These Golgi enter daughter cells at an early stage, raising the possibility that much of the bud's growth might be due to secretory vesicles formed as well as consumed entirely within the daughter. During cytokinesis, the Golgi compartments are concentrated near the site of cell wall synthesis. Clustering of Golgi both at the site of bud formation and at the cell septum suggests that these organelles might be directed toward sites of rapid cell surface growth.
The recent identification of an endoplasmic reticulum-Golgi intermediate compartment has added to the complexity of the structural and functional organization of the early secretory pathway. Protein sorting along the endoplasmic reticulum-Golgi pathway depends on different signals and mechanisms, some of which guarantee recycling from various levels of the Golgi apparatus to biosynthetically earlier compartments.
Protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus may be monitored in vitro with gently lysed semiintact cells or with crude homogenates fractionated by differential centrifugation. A complete resolution of this reaction requires the purification of donor ER and acceptor Golgi membranes. Toward this end there is a development of an in vitro assay that utilizes an ER-enriched membrane fraction to reconstitute ER-to-Golgi transport. This new assay allows a detailed analysis of the specific contributions of both membrane and soluble components to the transport process not possible in semiintact cells and crude homogenates. The development of an enriched ER in vitro assay represents a methodological advance in defining the molecular requirements for ER-to- Golgi transport. The enriched ER transport assay offers more flexibility than assays using semiintact cells in that membrane fractions from different sources can readily be mixed. The development of the enriched ER transport assay represents an advance toward the reconstitution of ER-to-Golgi transport in a fully resolved system. The use of this assay in analyzing the biochemical requirements of transport in vitro will aid in defining the molecular details of transport in vivo.
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In 1898, Camillio Golgi reported a new cellular constituent with the form of an extensive intracellular network (the apparato reticolare interno ), which now bears his name. However, the history of Golgi's apparatus is replete with controversy regarding its reality, what components of the cell should be included under its aegis, and what terminology should be used when referring to it. Electron microscopy has resolved many of these controversies and it is appropriate that this volume emphasize that aspect of Golgi apparatus discovery.
The principal structural component of the Golgi apparatus is the stack of cisternae, or dictyosome. As determined both biochemically and at the level of electron microscopy, the dictyosome is a highly ordered and polarized structure. The maintenance of order within the stack is thought to result from either intercisternal bonding constituents, or filamentous structures (or both) that bridge the space between adjacent cisternae.
Mechanisms proposed for movement of membrane and product into and out of the dictyosome (i.e., the Golgi apparatus stack) include a serial mode which functions exclusively by the formation, displacement, and loss of cisternae from the stack, and a parallel mode which functions exclusively by the movement of membrane, product, or precursor molecules directly into the peripheral edges of the cisternae. In the parallel mode, all cisternae can be accessed either singly or simultaneously, at least in theory, at any position within the stack. It is probable that both the serial and the parallel modes function concomitantly and need not be mutually exclusive.
Finally, the peripheral tubules of the cisternae represent a major membranous constituent of the cell with potentially unique functions. These tubules interconnect cisternae of adjacent stacks and may represent the major site of receptors for the shuttle (i.e., parallel) type of transfer among cisternae. Peripheral tubules as extensions of the cisternal lumina into the cytoplasm presumably have other functions, but these, like the tubules themselves, have only rarely been accommodated into functional models of Golgi apparatus dynamics in secretion or membrane flow.
The production of type I collagen by fibroblasts, odontoblasts, and osteoblasts is reviewed on the basis of results obtained by electron microscopy, 3H‐proline radioautography, and immunostaining for type I procollagen.
In the three cell types, the percursors of type I collagen are processed along the rough endocplasmic reticulum (rER)‐Golgic‐secretory granule pathway in the same manner as secretory proteins, but the available evidence suggests a few special features: (1) From the rER site of synthesis, the initial collagen procursors, known as pro‐alpha chains, are transported to the Golgi apparatus within tubular structures, referred to as intermediate tubules, rather than within vesicles. (2) The pro‐alpha chains coil into a triple helix within spherical distensions present along the saccules on the cis side of Golgi stacks. (3) The resulting procollagens are fairly raigid and form bundles that cause spherical distensions to lengthen into cylindrical ones, whereas by an unknown mechanism these distensions become part of the saccules on the trans‐side of Golgi stacks. (4) The procollagen‐containing cylindrical distensions are resleased from trans‐saccules to become secretory granules, and some procollagen material finds its way into lysosomes. (5) The secretory granules release their procollagen content by exocytosis at the cell surface. (6) The released procollagen is transformed into collagen before or, more probably, after associating with the surface of a collagen fibril.
Le processus des N-oligosides dans l'appareil de Golgi se fait par etapes successivement dans les citernes cis, centrales et trans. La mise en evidence de ce processus provient de 4 types d'experience: la separation des marqueurs enzymatiques P par fractionnement des membranes de Golgi, la localisation in situ d'enzymes marqueurs aux citernes par microscopie electronique; la localisation in situ des glycoproteines modifiees par ces enzymes et une carte fonctionnelle des compartiments observes par les glycoproteines transportes
This chapter discusses the structure and function of the Golgi apparatus. The basic ultrastructure of the Golgi apparatus is relatively consistent throughout both the animal and plant kingdoms. The Golgi apparatus is readily observed in the living cell under the phase contrast microscope, where it generally appears comparable to that seen following the metallic impregnation methods. Metallic impregnation shows the Golgi apparatus as a dense, somewhat pleomorphic and ill-defined morphological structure. Early studies revealed its form to vary greatly in different types of cells and in the same cells depending on their state of activity. It may exist as a compact mass or be dispersed through the cytoplasm as isolated fragments. It has been described as a filamentous or platelike reticulum, a network of varying degrees of complexity, an arrangement of clear canals, vacuoles of varying size and crescent, cup or diskshaped bodies (dictyosomes) composed of an outer rim of osmiophilic substance (“Golgi externum”) and an inner central portion of osmiophobic substance (“Golgi internum”). Electron microscope studies served to confirm and strengthen the classical concept of the Golgi apparatus and to clearly show its complex nature. Golgi complex might be involved in the synthesis of this cell product or to what extent it might be dealing with the secretory product in other ways. In some cells, it appears that the Golgi complex may collaborate with other cell organelles in the production of complex or heterogeneous secretory product. Other complex functions are also discussed in the chapter.
When an organism has only one or two mitochondria or chloroplasts per cell, it is probable that their partitioning is always stringently controlled so that each daughter cell always receives half the organelles in the parent cell. When there are more copies of an organelle, the available data suggest that partitioning is stochastic but far from random, with a strong tendency toward equality. The molecular mechanisms that promote equal partitioning are not known in any case, but the great variety of organelle behavior suggests that many different mechanisms are involved in different organisms. As Wilson (1925) pointed out, the precision of partitioning of cytoplasmic organelles rarely if ever equals that of mitosis, but it is still an expression of selection for mechanisms that will ensure the hereditary continuity of the organelles. How cells compensate for unequal partitioning by controlling organelle replication is known for only one case. But when one considers that Tetrahymena and Paramecium use different methods to compensate for unequal partitioning of macronuclear DNA, it would not be surprising if organisms use a variety of different compensating replication modes for organelles as well. What is surprising is that so little attention has been paid to these problems. Nothing could be simpler than counting organelles in dividing cells, but this has been done on a large scale in only two systems. Quantitative techniques in cell biology have been developed to the point where such studies could be done even on cells that have too many organelles for direct counting. Molecular mechanisms of partitioning have scarcely been touched on. Much more has been done on the role of the cytoskeleton in determining cell shape, and some observations have been made on its role in positioning organelles in interphase cells, but these kinds of studies have not been extended to dividing cells. Some experiments and observations have been made on the role of microtubules and microfilaments in moving cytoplasmic organelles around the cell during interphase, but again nothing has been done on their possible role in partitioning organelles at cytokinesis. The major lesson of this article is how little has been done, and how much can be done. The partitioning of cytoplasmic organelles at cell division is a wide-open field for future research, and one of great importance for both genetics and cell biology.
Membrane biogenesis, the synthesis of specific molecular components and their assembly into cellular membranes, is a critical, major activity in which cells engage throughout their existence. This chapter provides an overview of the membrane biogenesis. The main molecular components of cellular membranes are: (a) polar lipids, such as phospholipids, glycolipids, and cholesterol, organized into a continuous bilayer, fluid at the temperature of the immediate ambient; and (b) proteins, many of them glycoproteins—interacting in different ways with the lipid bilayer. The physical state and molecular architecture of cellular membranes is revealed by the formulation of fluid mosaic model. On account of their hydrophobic lipid bilayer, cell membranes function essentially as diffusion barriers, primarily for electrolytes and hydrophilic micro- and macromolecular solutes. Membrane fluidity is also a prerequisite for membrane fusion-fission, an event that occurs at the last step in cell division and at the first step in zygote formation. The chapter concludes that membrane fluidity is a sine qua noncondition for cellular existence. Cells could not exist without it.
Despite controversies and debates, some fundamental properties of endosomes become apparent when comparing results from in vivo and in vitro strategies used to study endosomal membrane traffic. In addition, recent studies are starting to unravel the complex organization of early endosomes, in particular along the route followed by recycling receptors.
The highlight of the past year was the demonstration that retrieval of endoplasmic reticulum membrane proteins containing the di-lysine motif involves COPI coat proteins. Other findings contributed to the debate about the nature of the 'intermediate compartment' between the endoplasmic reticulum and the Golgi apparatus, and the mechanism by which transported proteins are concentrated at this step.
Recent advances have uncovered the general protein apparatus used by all eukaryotes for intracellular transport, including secretion and endocytosis, and for triggered exocytosis of hormones and neurotransmitters. Membranes are shaped into vesicles by cytoplasmic coats which then dissociate upon GTP hydrolysis. Both vesicles and their acceptor membranes carry targeting proteins which interact specifically to initiate docking. A general apparatus then assembles at the docking site and fuses the vesicle with its target.
Dilysine motifs in cytoplasmic domains of transmembrane proteins are signals for their continuous retrieval from the Golgi back to the endoplasmic reticulum (ER). We describe a system to assess retrieval to the ER in yeast cells making use of a dilysine-tagged Ste2 protein. Whereas retrieval was unaffected in most sec mutants tested (sec7, sec12, sec13, sec16, sec17, sec18, sec19, sec22, and sec23), a defect in retrieval was observed in previously characterized coatomer mutants (sec21-1, sec27-1), as well as in newly isolated retrieval mutants (sec21-2, ret1-1). RET1 was cloned by complementation and found to encode the alpha subunit of coatomer. While temperature-sensitive for growth, the newly isolated coatomer mutants exhibited a very modest defect in secretion at the nonpermissive temperature. Coatomer from beta'-COP (sec27-1) and alpha-COP (ret1-1) mutants, but not from gamma-COP (sec21) mutants, had lost the ability to bind dilysine motifs in vitro. Together, these results suggest that coatomer plays an essential role in retrograde Golgi-to-ER transport and retrieval of dilysine-tagged proteins back to the ER.
Lactating mammary glands fixed by perfusion with 5% glutaraldehyde subsequently were postfixed with potassium ferrocyanide reduced osmium or were treated with tannic acid. Stained thin sections were examined with the electron microscope and stereopairs were prepared. The distribution of casein submicelles was analyzed in the various components of the Golgi apparatus. The Golgi stacks were composed of five or six elements, all of which contained casein submicelles 20 nm in diameter. The cis-tubular network or cis-element, as well as the underlying three or four midsaccules, showed these casein submicelles either attached to their membrane or free in the lumen. The trans-most element of the stacks formed distended prosecretory granules in which both isolated or clustered casein submicelles were suspended in an electron-lucent fluid. These micellar aggregates increased in size and became progressively more compact to form spherical dense bodies or casein micelles, in which the individual 20 nm particles could easily be resolved. Casein micelles were seen in secretory granules in addition to a wispy material of low density. The numerous small spherical vesicles (80 nm or larger) seen on the cis, lateral, or trans aspects of the stacks did not appear to contain free casein submicelles. This raises questions regarding the role of these vesicles in the transport of casein macromolecules through the Golgi stacks. It was noticeable that in this Golgi apparatus a trans-Golgi network was limited to a few small residual tubules free from casein submicelles. It thus appears that the greater part of the trans-most Golgi element gives rise to the large prosecretory granules. After leaving the Golgi region and prior to exocytosis, the secretory granules often fuse to form larger granules before exocytosis.
We have identified a novel natural metabolite, ilimaquinone (IQ), from sea sponges that causes Golgi membranes to break down completely in vivo into small vesicular structures (called vesiculated Golgi membranes [VGMs]). Under these conditions, transport of newly synthesized proteins from endoplasmic reticulum (ER) to the cis-Golgi-derived VGMs is unaffected; however, further transport along the secretory pathway is blocked. Upon removal of the drug, VGMs reassemble rapidly into a Golgi complex, and protein transport is restored. By employing a cell-free system that reconstitutes vesicular transport between successive Golgi cisternae, we provide evidence that the inhibition of protein transport by IQ is specifically due to an inhibition of transport vesicle formation. In addition, like brefeldin A (BFA), IQ treatment prevents the association of beta-COP and ADP-ribosylation factor to the Golgi membranes; however, unlike BFA treatment, there is no retrograde transport of Golgi enzymes into ER.
Eukaryotic life depends on the spatial and temporal organization of cellular membrane systems. Recent advances in understanding
the machinery of vesicle transport have established general principles that underlie a broad variety of physiological processes,
including cell surface growth, the biogenesis of distinct intracellular organelles, endocytosis, and the controlled release
of hormones and neurotransmitters.
Microtubule disruption has dramatic effects on the normal centrosomal localization of the Golgi complex, with Golgi elements remaining as competent functional units but undergoing a reversible "fragmentation" and dispersal throughout the cytoplasm. In this study we have analyzed this process using digital fluorescence image processing microscopy combined with biochemical and ultrastructural approaches. After microtubule depolymerization, Golgi membrane components were found to redistribute to a distinct number of peripheral sites that were not randomly distributed, but corresponded to sites of protein exit from the ER. Whereas Golgi enzymes redistributed gradually over several hours to these peripheral sites, ERGIC-53 (a protein which constitutively cycles between the ER and Golgi) redistributed rapidly (within 15 minutes) to these sites after first moving through the ER. Prior to this redistribution, Golgi enzyme processing of proteins exported from the ER was inhibited and only returned to normal levels after Golgi enzymes redistributed to peripheral ER exit sites where Golgi stacks were regenerated. Experiments examining the effects of microtubule disruption on the membrane pathways connecting the ER and Golgi suggested their potential role in the dispersal process. Whereas clustering of peripheral pre-Golgi elements into the centrosomal region failed to occur after microtubule disruption, Golgi-to-ER membrane recycling was only slightly inhibited. Moreover, conditions that impeded Golgi-to-ER recycling completely blocked Golgi fragmentation. Based on these findings we propose that a slow but constitutive flux of Golgi resident proteins through the same ER/Golgi cycling pathways as ERGIC-53 underlies Golgi Dispersal upon microtubule depolymerization. Both ERGIC-53 and Golgi proteins would accumulate at peripheral ER exit sites due to failure of membranes at these sites to cluster into the centrosomal region. Regeneration of Golgi stacks at these peripheral sites would re-establish secretory flow from the ER into the Golgi complex and result in Golgi dispersal.
Electron microscope immunocytochemistry reveals that both anterograde-directed (proinsulin and VSV G protein) and retrograde-directed (the KDEL receptor) cargo are present in COPI-coated vesicles budding from every level of the Golgi stack in whole cells; however, they comprise two distinct populations that together can account for at least 80% of the vesicles budding from Golgi cisternae. Segregation of anterograde- from retrograde-directed cargo into distinct sets of COPI-coated vesicles is faithfully reproduced in the cell-free Golgi transport system, in which VSV G protein and KDEL receptor are packaged into separable vesicles, even when budding is driven by highly purified coatomer and a recombinant ARF protein.