Stochastic Model of Maturation and Vesicular Exchange in Cellular Organelles

The dynamical organization of membrane-bound organelles along intracellular transport pathways relies on vesicular exchange between organelles and on the maturation of the organelle’s composition by enzymatic reactions or exchange with the cytoplasm. The relative importance of each mechanism in controlling organelle dynamics remains controversial, in particular for transport through the Golgi apparatus. Using a stochastic model, we identify two classes of dynamical behavior that can lead to full maturation of membrane-bound compartments. In the first class, maturation corresponds to the stochastic escape from a steady state in which export is dominated by vesicular exchange, and is very unlikely for large compartments. In the second class, it occurs in a quasi-deterministic fashion and is almost size independent. Whether a system belongs to the first or second class is largely controlled by homotypic fusion.     

IntraGolgi distribution of the Conserved Oligomeric Golgi (COG) complex

The Conserved Oligomeric Golgi (COG) complex is an eight-subunit (Cog1-8) peripheral Golgi protein involved in membrane trafficking and glycoconjugate synthesis. COG appears to participate in retrograde vesicular transport and is required to maintain normal Golgi structure and function. COG mutations interfere with normal transport, distribution, and/or stability of Golgi proteins associated with glycoconjugate synthesis and trafficking, and lead to failure of spermatogenesis in Drosophila melanogaster, misdirected migration of gonadal distal tip cells in Caenorhabditis elegans, and type II congenital disorders of glycosylation in humans. The mechanism by which COG influences Golgi structure and function is unclear. Immunogold electron microscopy was used to visualize the intraGolgi distribution of a functional, hemagglutinin epitope-labeled COG subunit, Cog1-HA, that complements the Cog1-deficiency in Cog1-null Chinese hamster ovary cells. COG was found to be localized primarily on or in close proximity to the tips and rims of the Golgi's cisternae and their associated vesicles and on vesicles and vesiculo-tubular structures seen on both the cis and trans-Golgi Network faces of the cisternal stacks, in some cases on COPI containing vesicles. These findings support the proposal that COG is directly involved in controlling vesicular retrograde transport of Golgi resident proteins throughout the Golgi apparatus.     

Molecular basis for Golgi maintenance and biogenesis

The Golgi apparatus contains thousands of different types of integral and peripheral membrane proteins, perhaps more than any other intracellular organelle. To understand these proteins' roles in Golgi function and in broader cellular processes, it is useful to categorize them according to their contribution to Golgi creation and maintenance. This is because all of the Golgi's functions derive from its ability to maintain steady-state pools of particular proteins and lipids, which in turn relies on the Golgi's dynamic character - that is, its ongoing state of transformation and outgrowth from the endoplasmic reticulum. Here, we categorize the expanding list of Golgi-associated proteins on the basis of their role in Golgi reformation after the Golgi has been disassembled. Information gained on how different proteins participate in this process can provide important insights for understanding the Golgi's global functions within cells.     

The Golgi puppet master: COG complex at center stage of membrane trafficking interactions

The central organelle within the secretory pathway is the Golgi apparatus, a collection of flattened membranes organized into stacks. The cisternal maturation model of intra-Golgi transport depicts Golgi cisternae that mature from cis to medial to trans by receiving resident proteins, such as glycosylation enzymes via retrograde vesicle-mediated recycling. The conserved oligomeric Golgi (COG) complex, a multi-subunit tethering complex of the complexes associated with tethering containing helical rods family, organizes vesicle targeting during intra-Golgi retrograde transport. The COG complex, both physically and functionally, interacts with all classes of molecules maintaining intra-Golgi trafficking, namely SNAREs, SNARE-interacting proteins, Rabs, coiled-coil tethers, vesicular coats, and molecular motors. In this report, we will review the current state of the COG interactome and analyze possible scenarios for the molecular mechanism of the COG orchestrated vesicle targeting, which plays a central role in maintaining glycosylation homeostasis in all eukaryotic cells.     

Models for Golgi traffic: a critical assessment

A variety of secretory cargoes move through the Golgi, but the pathways and mechanisms of this traffic are still being debated. Here, we evaluate the strengths and weaknesses of five current models for Golgi traffic: (1) anterograde vesicular transport between stable compartments, (2) cisternal progression/maturation, (3) cisternal progression/maturation with heterotypic tubular transport, (4) rapid partitioning in a mixed Golgi, and (5) stable compartments as cisternal progenitors. Each model is assessed for its ability to explain a set of key observations encompassing multiple cell types. No single model can easily explain all of the observations from diverse organisms. However, we propose that cisternal progression/maturation is the best candidate for a conserved core mechanism of Golgi traffic, and that some cells elaborate this core mechanism by means of heterotypic tubular transport between cisternae.     

Sequential transport of protein between the endoplasmic reticulum and successive Golgi compartments in semi-intact cells.

The vectorial transport of vesicular stomatitis virus (VSV) G protein between the ER and the cis and medial Golgi compartments has been reconstituted using semi-intact (perforated) cells. The transport of VSV-G protein between successive compartments is measured by the sequential processing of the two N-linked oligosaccharide chains present on VSV-G protein to the endoglycosidase (endo) H-resistant structures which have unique electrophoretic mobilities during sodium dodecyl sulfate-gel electrophoresis. The appearance of a form of VSV-G which contains only one endo H-resistant oligosaccharide chain (GH1) is kinetically and biochemically indistinguishable from the appearance of the Man5, endo D-sensitive form (GD), the latter being a processing reaction diagnostic of transport from the ER to the cis Golgi compartment. These results provide evidence that the cis Golgi compartment may contain in addition to alpha-1,2-mannosidase I, both N-acetylglueosamine transferase I and alpha-1,2-mannosidase II. VSV-G protein is subsequently processed to the form which contains two endo H-resistant oligosaccharides (GH2) after a second wave of vesicular transport. Processing of GH1 to GH2 in vitro occurs only after a lag period following the appearance of GH1; processing is sensitive to N-ethylmaleimide, guanosine-5'-O-(3-thiotriphosphate), and a synthetic peptide homologous to the rab1 protein effector domain, and processing is inhibited in the absence of free Ca2+ (in the presence of EGTA), reagents which potently inhibit ER to cis Golgi transport. These results suggest that VSV-G protein proceeds through at least two rounds of vesicular transport from the ER to the medial Golgi compartment for processing to the GH2 form, providing a model system to study the regulation of the vectorial membrane fission and fusion events involved in vesicular trafficking and organelle dynamics in the early stages of the secretory pathway.     

Intracellular Salmonella enterica redirect exocytic transport processes in a Salmonella pathogenicity island 2-dependent manner

During intracellular life, Salmonella enterica proliferate within a specialized membrane compartment, the Salmonella-containing vacuole (SCV), and interfere with the microtubule cytoskeleton and cellular transport. To characterize the interaction of intracellular Salmonella with host cell transport processes, we utilized various model systems to follow microtubule-dependent transport. The vesicular stomatitis virus glycoprotein (VSVG) is a commonly used marker to follow protein transport from the Golgi to the plasma membrane. Using a VSVG-GFP fusion protein, we observed that virulent intracellular Salmonella alter exocytotic transport and recruit exocytotic transport vesicles to the SCV. This virulence function was dependent on the function of the type III secretion system encoded by Salmonella Pathogenicity Island 2 (SPI2) and more specifically on a subset of SPI2 effector proteins. Furthermore, the Golgi to plasma membrane traffic of the shingolipid C(5)-ceramide was redirected to the SCV by virulent Salmonella. We propose that Salmonella modulates the biogenesis of the SCV by deviating this compartment from the default endocytic pathway to an organelle that interacts with the exocytic pathway. This observation might reveal a novel element of the intracellular survival and replication strategy of Salmonella.     

Golgi and Related Vesicle Proteomics: Simplify to Identify

Despite more than six decades of successful Golgi research, the fundamental question as to how biosynthetic material is transported through the secretory pathway remains unanswered. New technologies such as live cell imaging and correlative microscopy have highlighted the plastic nature of the Golgi, one that is sensitive to perturbation yet highly efficient in regaining both structure and function. Single molecule-microscopy and super resolution-microscopy further adds to this picture. Various models for protein transport have been put forward, each with its own merits and pitfalls but we are far from resolving whether one is more correct than the other. As such, our laboratory considers multiple mechanisms of Golgi transport until proven otherwise. This includes the two classical modes of transport, vesicular transport and cisternal progression/maturation as well as more recent models such as tubular inter- and intra-cisternal connections (long lasting or transient) and inter-Golgi stack transport. In this article, we focus on an emerging inductive technology, mass spectrometry-based proteomics that has already enabled insight into the relative composition of compartments and subcompartments of the secretory pathway including mechanistic aspects of protein transport. We note that proteomics, as with any other technology, is not a stand-alone technology but one that works best alongside complementary approaches.     

Cell cycle maintenance and biogenesis of the Golgi complex

How organelle identity is established and maintained, and how organelles divide and partition between daughter cells, are central questions of organelle biology. For the membrane-bound organelles of the secretory and endocytic pathways [including the endoplasmic reticulum (ER), Golgi complex, lysosomes, and endosomes], answering these questions has proved difficult because these organelles undergo continuous exchange of material. As a result, many "resident" proteins are not localized to a single site, organelle boundaries overlap, and when interorganellar membrane flow is interrupted, organelle structure is altered. The existence and identity of these organelles, therefore, appears to be a product of the dynamic processes of membrane trafficking and sorting. This is particularly true for the Golgi complex, which resides and functions at the crossroads of the secretory pathway. The Golgi receives newly synthesized proteins from the ER, covalently modifies them, and then distributes them to various final destinations within the cell. In addition, the Golgi recycles selected components back to the ER. These activities result from the Golgi's distinctive membranes, which are organized as polarized stacks (cis to trans) of flattened cisternae surrounded by tubules and vesicles. Golgi membranes are highly dynamic despite their characteristic organization and morphology, undergoing rapid disassembly and reassembly during mitosis and in response to perturbations in membrane trafficking pathways. How Golgi membranes fragment and disperse under these conditions is only beginning to be clarified, but is central to understanding the mechanism(s) underlying Golgi identity and biogenesis. Recent work, discussed in this review, suggests that membrane recycling pathways operating between the Golgi and ER play an indispensable role in Golgi maintenance and biogenesis, with the Golgi dispersing and reforming through the intermediary of the ER both in mitosis and in interphase when membrane cycling pathways are disrupted.     

Traffic through the Golgi apparatus.

The role of vesicles in cargo transport through the Golgi apparatus has been controversial. Large forms of cargo such as protein aggregates are thought to progress through the Golgi stack by a process of cisternal maturation, balanced by a return flow of Golgi resident proteins in COPI-coated vesicles. However, whether this is the primary role of vesicles, or whether they also serve to transport small cargo molecules in a forward direction has been debated. Two papers (Martínez-Menárguez et al., 2001; Mironov et al., 2001, this issue) use sophisticated light and electron microscopy to provide evidence that the vesicular stomatitis virus membrane glycoprotein (VSV G)* is largely excluded from vesicles in vivo, and does not move between cisternae, whereas resident Golgi enzymes freely enter vesicles as predicted by the cisternal maturation model. Both papers conclude that vesicles are likely to play only a minor role in the anterograde transport of cargo through the Golgi apparatus in mammalian tissue culture cells.     

Gastric mucosal cell homeostatic physiome. Critical role of ER-initiated membranes restitution in the fidelity of cell function renewal.

Homeostatic cell physiology is preserved through the fidelity of the cell membranes restitution. The task is accomplished through the assembly of the precisely duplicated segments of the cell membranes, and transport to the site of their function. Here we examined the mechanism that initiates and directs the restitution of the intra- and extracellular membranes of gastric mucosal cell. The homeostatic restitution of gastrointestinal epithelial cell membrane components was investigated by studying the lipidomic processes in endoplasmic reticulum (ER) and Golgi. The biomembrane lipid synthesis during the formation of transport vesicles in the systems containing isolated organelle and the cell-specific cytosol (Cyt) from rat gastric mucosal epithelial cells was assessed. The results revealed that lipids of ER transport vesicle and the transmembrane and intravesicular cargo are delivered en bloc to the point of destination. En bloc delivery of proteins, incorporated into predetermined in ER lipid environment, ensures fidelity of the membrane modification in Golgi and the restitution of the lipid and protein elements that are consistent with the organelle and the cell function. The mechanism that maintains apical membrane restitution is mediated through the synthesis of membrane segments containing ceramide (Cer). The Cer-containing membranes and protein cargo are further specialized in Golgi. The portion of the vesicles destined for apical membrane renewal contains glycosphingolipids and phosphatidylinositol 3-phosphate. The vesicles containing phosphatidylinositol 4-phosphate are directed to endosomes. Our findings revealed that the preservation of the physiological equilibrium in cell structure and function is attributed to (1) a complete membrane segment synthesis in ER, (2) its transport in the form of ER-transport vesicle to Golgi, (3) the membrane components-defined maturation of lipids and proteins in Golgi, and (4) en bloc transfer of the new segment of the membrane to the cell apical membrane or intracellular organelle.     

The Yeast Golgi Apparatus

The Golgi apparatus is an organelle that has been extensively studied in the model eukaryote, yeast. Its morphology varies among yeast species; the Golgi exists as a system of dispersed cisternae in the case of the budding yeast Saccharomyces cerevisiae, whereas the Golgi cisternae in Pichia pastoris and Schizosaccharomyces pombe are organized into stacks. In spite of the different organization, the mechanism of trafficking through the Golgi apparatus is believed to be similar, involving cisternal maturation, in which the resident Golgi proteins are transported backwards while secretory cargo proteins can stay in the cisternae. Questions remain regarding the organization of the yeast Golgi, the regulatory mechanisms that underlie cisternal maturation of the Golgi and transport machinery of cargo proteins through this organelle. Studies using different yeast species have provided hints to these mechanisms.      

Protein transport in plant cells: in and out of the Golgi

In plant cells, the Golgi apparatus is the key organelle for polysaccharide and glycolipid synthesis, protein glycosylation and protein sorting towards various cellular compartments. Protein import from the endoplasmic reticulum (ER) is a highly dynamic process, and new data suggest that transport, at least of soluble proteins, occurs via bulk flow. In this Botanical Briefing, we review the latest data on ER/Golgi inter-relations and the models for transport between the two organelles. Whether vesicles are involved in this transport event or if direct ER-Golgi connections exist are questions that are open to discussion. Whereas the majority of proteins pass through the Golgi on their way to other cell destinations, either by vesicular shuttles or through maturation of cisternae from the cis- to the trans-face, a number of membrane proteins reside in the different Golgi cisternae. Experimental evidence suggests that the length of the transmembrane domain is of crucial importance for the retention of proteins within the Golgi. In non-dividing cells, protein transport out of the Golgi is either directed towards the plasma membrane/cell wall (secretion) or to the vacuolar system. The latter comprises the lytic vacuole and protein storage vacuoles. In general, transport to either of these from the Golgi depends on different sorting signals and receptors and is mediated by clathrin-coated and dense vesicles, respectively. Being at the heart of the secretory pathway, the Golgi (transiently) accommodates regulatory proteins of secretion (e.g. SNAREs and small GTPases), of which many have been cloned in plants over the last decade. In this context, we present a list of regulatory proteins, along with structural and processing proteins, that have been located to the Golgi and the 'trans-Golgi network' by microscopy.     

PKCδ and ε regulate the morphological integrity of the ER-Golgi intermediate compartment (ERGIC) but not the anterograde and retrograde transports via the Golgi apparatus

The ER-Golgi intermediate compartment (ERGIC) is an organelle through which cargo proteins pass and are being transferred by either anterograde or retrograde transport between the endoplasmic reticulum (ER) and the Golgi apparatus. We examined the effect of 80 different kinase inhibitors on ERGIC morphology and found that rottlerin, a PKCδ inhibitor, induced the dispersion of the perinuclear ERGIC into punctate structures. Rottlerin also delayed anterograde transport of vesicular stomatitis virus G protein (VSVG) from the ER to the Golgi and retrograde transport of cholera toxin from cell surface to the ER via the Golgi. RNA interference revealed that knockdown of PKCδ or ε resulted in the dispersion of the ERGIC, but unexpectedly did not inhibit VSVG and cholera toxin transport. We also found that rottlerin depolarized the mitochondrial membrane potential, as does carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), an uncoupler, and demonstrated that a decrease in the intracellular adenosine triphosphate (ATP) levels by rottlerin might underlie the block in transports. These results suggest that PKCδ and ε specifically regulate the morphology of the ERGIC and that the maintenance of ERGIC structure is not necessarily required for anterograde and retrograde transports.     

How Camillo Golgi became “the Golgi”

On April 1898 Camillo Golgi communicated to the Medical-Surgical Society of Pavia, the discovery of the “internal reticular apparatus”, a novel intracellular organelle which he observed in nerve cells with the silver impregnation he had introduced for the staining of the nervous system. Soon after the discovery it became evident that this cellular component, which was also named the “Golgi apparatus”, was a ubiquitous structure in eukaryotic cells. However the reality of the organelle was questioned for years and many cytologists considered the internal reticular apparatus as an artefact due to the fixation and/or metallic impregnation procedure. The controversy was finally solved in the mid-1950s by electron microscopy when the Golgi apparatus definitely acquired its dignity of being a genuine cell organelle. The designation of “Golgi complex” entered officially in the literature in 1956. Both the terms Golgi apparatus and Golgi complex are currently interchangeable. However a quick “the Golgi” and the introduction of Golgi in adjectival form are now prevalent in the blooming scientific literature on the organelle. Thus Camillo Golgi underwent his final transformation and, becoming the eponym of the organelle he had discovered, he found a way to immortality.     

The internal reticular apparatus of Camillo Golgi: a complex, heterogeneous organelle, enriched in acid, neutral, and alkaline phosphatases, and involved in glycosylation, secretion, membrane flow, lysosome formation, and intracellular digestion

Its topography is one of the most characteristic features of the Golgi apparatus and the reticular nature of this organelle is evident in Golgi's first drawings, in light microscopic enzyme cytochemical preparations, and in high voltage electron micrographs of thick sections. Although individual components of the Golgi apparatus may differ in staining characteristics, morphology, contents, and enzymatic activities, they are integrated into a dynamic topographical and functional unit that is closely associated with the endoplasmic reticulum. Modulation of enzymatic activities and morphological and enzymatic heterogeneity are not surprising in an organelle that is the site of both synthetic and digestive events, including glycosylation, sulfation, formation of secretory granules and lysosomes, and the degradation of endocytized material.     

Exclusion of golgi residents from transport vesicles budding from Golgi cisternae in intact cells

A central feature of cisternal progression/maturation models for anterograde transport across the Golgi stack is the requirement that the entire population of steady-state residents of this organelle be continuously transported backward to earlier cisternae to avoid loss of these residents as the membrane of the oldest (trans-most) cisterna departs the stack. For this to occur, resident proteins must be packaged into retrograde-directed transport vesicles, and to occur at the rate of anterograde transport, resident proteins must be present in vesicles at a higher concentration than in cisternal membranes. We have tested this prediction by localizing two steady-state residents of medial Golgi cisternae (mannosidase II and N-acetylglucosaminyl transferase I) at the electron microscopic level in intact cells. In both cases, these abundant cisternal constituents were strongly excluded from buds and vesicles. This result suggests that cisternal progression takes place substantially more slowly than most protein transport and therefore is unlikely to be the predominant mechanism of anterograde movement.     

The yeast Golgi apparatus: Insights and mysteries

The Golgi apparatus is known to modify and sort newly synthesized secretory proteins. However, fundamental mysteries remain about the structure, operation, and dynamics of this organelle. Important insights have emerged from studying the Golgi in yeasts. For example, yeasts have provided direct evidence for Golgi cisternal maturation, a mechanism that is likely to be broadly conserved. Here, we highlight features of the yeast Golgi as well as challenges that lie ahead.     

COPI-mediated retrograde trafficking from the Golgi to the ER regulates EGFR nuclear transport

Emerging evidence indicates that cell surface receptors, such as the entire epidermal growth factor receptor (EGFR) family, have been shown to localize in the nucleus. A retrograde route from the Golgi to the endoplasmic reticulum (ER) is postulated to be involved in the EGFR trafficking to the nucleus; however, the molecular mechanism in this proposed model remains unexplored. Here, we demonstrate that membrane-embedded vesicular trafficking is involved in the nuclear transport of EGFR. Confocal immunofluorescence reveals that in response to EGF, a portion of EGFR redistributes to the Golgi and the ER, where its NH₂-terminus resides within the lumen of Golgi/ER and COOH-terminus is exposed to the cytoplasm. Blockage of the Golgi-to-ER retrograde trafficking by brefeldin A or dominant mutants of the small GTPase ADP-ribosylation factor, which both resulted in the disassembly of the coat protein complex I (COPI) coat to the Golgi, inhibit EGFR transport to the ER and the nucleus. We further find that EGF-dependent nuclear transport of EGFR is regulated by retrograde trafficking from the Golgi to the ER involving an association of EGFR with γ-COP, one of the subunits of the COPI coatomer. Our findings experimentally provide a comprehensive pathway that nuclear transport of EGFR is regulated by COPI-mediated vesicular trafficking from the Golgi to the ER, and may serve as a general mechanism in regulating the nuclear transport of other cell surface receptors.     

Secretory vesicular transport from the Golgi is altered during ATP-binding cassette protein A1 (ABCA1)-mediated cholesterol efflux

Apolipoprotein AI (apoAI)-mediated cholesterol efflux is a process by which cells export excess cellular cholesterol to apoAI to form high density lipoprotein. ATP-binding cassette protein A1 (ABCA1) has recently been identified as the key regulator of this process. The pathways of intracellular cholesterol transport during efflux are largely unknown nor is the molecular mechanism by which ABCA1 governs cholesterol efflux well understood. Here, we report that, in both macrophages and fibroblasts, the secretory vesicular transport changes in response to apoAI-mediated cholesterol efflux. Vesicular transport from the Golgi to the plasma membrane increased 2-fold during efflux. This increase in vesicular transport during efflux was observed in both raft-poor and raft-rich vesicle populations originated from the Golgi. Importantly, enhanced vesicular transport in response to apoAI is absent in Tangier fibroblasts, a cell type with deficient cholesterol efflux due to functional ABCA1 mutations. These findings are consistent with an efflux model whereby cholesterol is transported from the storage site to the plasma membrane via the Golgi. ABCA1 may influence cholesterol efflux in part by enhancing vesicular trafficking from the Golgi to the plasma membrane.