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The endoplasmic reticulum (ER)-Golgi system has been studied using biochemical, genetic, electron and light microscopic techniques. We now understand many aspects of trafficking from the ER to the Golgi apparatus, including some of the signals and mechanisms for selective retention and retrieval of ER resident proteins and export of cargo proteins. Proteins that leave the ER emerge in ‘export complexes’ or ER ‘exit sites’ and accumulate in pleiomorphic transport carriers referred to sometimes as VTCs or intermediate compartments. These structures then transit from the ER to the Golgi apparatus along microtubules using the dynein/dynactin motor and fuse with the cis cisterna of the Golgi apparatus. Many proteins (including vSNAREs, ERGIC53/p58 and the KDEL receptor) must cycle back to the ER from pre-Golgi intermediates or the Golgi. We will discuss both the currently favored model that this cycling occurs via 50-nm COPI-coated vesicles and in vivo evidence that suggests retrograde trafficking may occur via tubular structures. [ABSTRACT FROM AUTHOR]

The mammalian Golgi apparatus is a highly dynamic organelle, which is normally localized in the juxtanuclear space and plays an essential role in the regulation of cellular homeostasis. While posttranslational modification of cargo is mediated by the resident enzymes (glycosyltransferases, glycosidases, and kinases), the ribbon structure of Golgi and its cisternal stacking mostly rely on the cooperation of coiled-coil matrix golgins. Among them, giantin, GM130, and GRASPs are unique, because they form a tripartite complex and serve as Golgi docking sites for cargo delivered from the endoplasmic reticulum (ER). Golgi undergoes significant disorganization in many pathologies associated with a block of the ER-to-Golgi or intra-Golgi transport, including cancer, different neurological diseases, alcoholic liver damage, ischemic stress, viral infections, etc. In addition, Golgi fragments during apoptosis and mitosis. Here, we summarize and analyze clinically relevant observations indicating that Golgi fragmentation is associated with the selective loss of Golgi residency for some enzymes and, conversely, with the relocation of some cytoplasmic proteins to the Golgi. The central concept is that ER and Golgi stresses impair giantin docking site but have no impact on the GM130-GRASP65 complex, thus inducing mislocalization of giantin-sensitive enzymes only. This cardinally changes the processing of proteins by eliminating the pathways controlled by the missing enzymes and by activating the processes now driven by the GM130-GRASP65-dependent proteins. This type of Golgi disorganization is different from the one induced by the cytoskeleton alteration, which despite Golgi de-centralization, neither impairs function of golgins nor alters trafficking. [ABSTRACT FROM AUTHOR]

Despite its discovery more than three decades ago and well-established role in protein sorting and trafficking in the early secretory pathway, the intermediate compartment (IC) has remained enigmatic. The prevailing view is that the IC evolved as a specialized organelle to mediate long-distance endoplasmic reticulum (ER)-Golgi communication in metazoan cells, but is lacking in other eukaryotes, such as plants and fungi. However, this distinction is difficult to reconcile with the high conservation of the core machineries that regulate early secretory trafficking from yeast to man. Also, it has remained unclear whether the pleiomorphic IC components—vacuoles, tubules and vesicles—represent transient transport carriers or building blocks of a permanent pre-Golgi organelle. Interestingly, recent studies have revealed that the IC maintains its compositional, structural and spatial properties throughout the cell cycle, supporting a model that combines the dynamic and stable aspects of the organelle. Moreover, the IC has been assigned novel functions, such as cell signaling, Golgi-independent trafficking and autophagy. The emerging permanent nature of the IC and its connections with the centrosome and the endocytic recycling system encourage reconsideration of its relationship with the Golgi ribbon, role in Golgi biogenesis and ubiquitous presence in eukaryotic cells. [ABSTRACT FROM AUTHOR]

This review summarizes the data describing the role of cellular microtubules in transportation of membrane vesicles - transport containers for secreted proteins or lipids. Most events of early vesicular transport in animal cells (from the endoplasmic reticulum to the Golgi apparatus and in the opposite recycling direction) are mediated by microtubules and microtubule motor proteins. Data on the role of dynein and kinesin in early vesicle transport remain controversial, probably because of the differentiated role of these proteins in the movements of vesicles or membrane tubules with various cargos and at different stages of secretion and retrograde transport. Microtubules and dynein motor protein are essential for maintaining a compact structure of the Golgi apparatus; moreover, there is a set of proteins that are essential for Golgi compactness. Dispersion of ribbon-like Golgi often occurs under physiological conditions in interphase cells. Golgi is localized in the leading part of crawling cultured fibroblasts, which also depends on microtubules and dynein. The Golgi apparatus creates its own system of microtubules by attracting γ-tubulin and some microtubule-associated proteins to membranes. Molecular mechanisms of binding microtubule-associated and motor proteins to membranes are very diverse, suggesting the possibility of regulation of Golgi interaction with microtubules during cell differentiation. To illustrate some statements, we present our own data showing that the cluster of vesicles induced by expression of constitutively active GTPase Sar1a[H79G] in cells is dispersed throughout the cell after microtubule disruption. Movement of vesicles in cells containing the intermediate compartment protein ERGIC53/LMANI was inhibited by inhibiting dynein. Inhibiting protein kinase LOSK/SLK prevented orientation of Golgi to the leading part of crawling cells, but the activity of dynein was not inhibited according to data on the movement of ERGIC53/LMANI-marked vesicles. [ABSTRACT FROM AUTHOR]

Notch signaling has a pivotal role in numerous cell-fate decisions, and its aberrant activity leads to developmental disorders and cancer. To identify molecules that influence Notch signaling, we screened nearly 17,000 compounds using automated microscopy to monitor the trafficking and processing of a ligand-independent Notch-enhanced GFP (eGFP) reporter. Characterization of hits in vitro by biochemical and cellular assays and in vivo using zebrafish led to five validated compounds, four of which induced accumulation of the reporter at the plasma membrane by inhibiting γ-secretase. One compound, the dihydropyridine FLI-06, disrupted the Golgi apparatus in a manner distinct from that of brefeldin A and golgicide A. FLI-06 inhibited general secretion at a step before exit from the endoplasmic reticulum (ER), which was accompanied by a tubule-to-sheet morphological transition of the ER, rendering FLI-06 the first small molecule acting at such an early stage in secretory traffic. These data highlight the power of phenotypic screening to enable investigations of central cellular signaling pathways. [ABSTRACT FROM AUTHOR]

Coat protein complex I (COPI) and COPII are required for bidirectional membrane trafficking between the endoplasmic reticulum (ER) and the Golgi. While these core coat machineries and other transport factors are highly conserved across species, high-resolution imaging studies indicate that the organization of the ER-Golgi interface is varied in eukaryotic cells. Regulation of COPII assembly, in some cases to manage distinct cellular cargo, is emerging as one important component in determining this structure. Comparison of the ER-Golgi interface across different systems, particularly mammalian and plant cells, reveals fundamental elements and distinct organization of this interface. A better understanding of how these interfaces are regulated to meet varying cellular secretory demands should provide key insights into the mechanisms that control efficient trafficking of proteins and lipids through the secretory pathway. [ABSTRACT FROM AUTHOR]

Secretory proteins are transported to the Golgi complex in vesicles that bud from the endoplasmic reticulum. The cytoplasmic coat protein complex II (COPII) is responsible for cargo sorting and vesicle morphogenesis. COPII was first described in Saccharomyces cerevisiae, but its basic function is conserved throughout all eukaryotes. Nevertheless, the COPII coat has adapted to the higher complexity of mammalian physiology, achieving more sophisticated levels of secretory regulation. In this review we cover aspects of mammalian COPII-mediated regulation of secretion, in particular related to the function of COPII paralogues, the spatial organization of cargo export and the role of accessory proteins. [ABSTRACT FROM AUTHOR]

Export of proteins from the endoplasmic reticulum in COPII-coated vesicles occurs at defined sites that contain the scaffolding protein Sec16. We identify TFG-1, a new conserved regulator of protein secretion that interacts directly with SEC-16 and controls the export of cargoes from the endoplasmic reticulum in Caenorhabditis elegans. Hydrodynamic studies indicate that TFG-1 forms hexamers that facilitate the co-assembly of SEC-16 with COPII subunits. Consistent with these findings, TFG-1 depletion leads to a marked decline in both SEC-16 and COPII levels at endoplasmic reticulum exit sites. The sequence encoding the amino terminus of human TFG has been previously identified in chromosome translocation events involving two protein kinases, which created a pair of oncogenes. We propose that fusion of these kinases to TFG relocalizes their activities to endoplasmic reticulum exit sites, where they prematurely phosphorylate substrates during endoplasmic reticulum export. Our findings provide a mechanism by which translocations involving TFG can result in cellular transformation and oncogenesis. [ABSTRACT FROM AUTHOR]

Microtubule array in eukaryotic cells supports directed transport of various cargoes driven by motor proteins. The arrangement of microtubules in cytoplasm is not stochastic; they are organized in a certain way setting a system of coordinates for intracellular transport. Most cultured fibroblast-like cells possess a radial microtubule array with the minus ends of microtubules gathered on the centrosome and plus ends directed towards the periphery of the cell. Mechanisms that regulate the formation of radial microtubule system remain unclear. Usually centrosome works as a microtubule-organizing center; however, the radial system of microtubules can be formed without centrosome participation. At least in some cases microtubule network can be organized by dynein-dynactin complexes associated with membrane vesicles. Membrane vesicles can nucleate microtubules, anchor them and move along them. However, the role of membrane organelles in microtubule organization began to attract attention of researches only recently. It this review we summarize the data indicating that membrane organelles can organize microtubules, providing “tracks” for their subsequent transport. [ABSTRACT FROM AUTHOR]

Reticulons (RTNs) are membrane-spanning proteins sharing a typical domain named reticulon homology domain (RHD). RTN genes have been identified in all eukaryotic organisms examined so far, and the corresponding proteins have been found predominantly associated to the endoplasmic reticulum membranes. In animal and yeast, in which knowledge of the protein family is more advanced, RTNs are involved in numerous cellular processes such as apoptosis, cell division and intracellular trafficking. Up to now, a little attention has been paid to their plant counterparts, i.e., RTNLBs. In this review, we summarize the data available for RTNLB proteins and, using the data obtained with animal and yeast models, several functions for RTNLBs in plant cells are proposed and discussed. [ABSTRACT FROM AUTHOR]

Cholesterol, certain lipids, membrane-bound and soluble proteins, as well as viruses that are synthesized in the endoplasmic reticulum (ER), reach the plasma membrane (PM) via non-classical pathway(s) that remain poorly understood. Typical for this transport is (i) its insensitivity to brefeldin A (BFA), which dissociates selected coat complexes from membranes, resulting in the disassembly of the Golgi apparatus; (ii) its rapid kinetics as compared to the classical secretory pathway; and (iii) its role in the trafficking of lipid raft components. Based on results showing that the intermediate compartment (IC) at the ER-Golgi boundary constitutes a stable tubular network that maintains its dynamics in the presence of BFA, we propose that two bidirectional Golgi-bypass pathways to the PM exist, a direct route from early IC elements, and another, reminescent of the yeast secretory pathway, from late IC elements via the endosomal system. These pathways have implications for the organization of the secretory processes in different cell types. (Part of a Multi-author Review) [ABSTRACT FROM AUTHOR]

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a well-studied glycolytic protein with energy production as its implied occupation. It has established itself lately as a multifunctional protein. Recent studies have found GAPDH to be involved in a variety of nuclear and cytosolic pathways ranging from its role in apoptosis and regulation of gene expression to its involvement in regulation of Ca2+ influx from endoplasmic reticulum. Numerous studies also indicate that GAPDH interacts with microtubules and participates in cell membrane fusion. This review is focused on the cytosolic functions of the protein related to vesicular transport. Suggestions for future directions as well as the model of protein polymer structure and possible post-translational modifications as a basis for its multifunctional activities in the early secretory pathway are given. [ABSTRACT FROM AUTHOR]

Transport of proteins from the endoplasmic reticulum (ER) to the Golgi is mediated by the sequential action of two coat complexes: COPII concentrates cargo for secretion at ER export sites, then COPI is subsequently recruited to nascent carriers and retrieves recycling proteins back to the ER. These carriers then move towards the Golgi along microtubules, driven by the dynein/dynactin complexes. Here we show that the Sec23p component of the COPII complex directly interacts with the dynactin complex through the carboxy-terminal cargo-binding domain of p150Glued. Functional assays, including measurements of the rate of recycling of COPII on the ER membrane and quantitative analyses of secretion, indicate that this interaction underlies functional coupling of ER export to microtubules. Together, our data suggest a mechanism by which membranes of the early secretory pathway can be linked to motors and microtubules for subsequent organization and movement to the Golgi apparatus. [ABSTRACT FROM AUTHOR]

The effects of the fungal metabolite brefeldin A (BFA) on the endocytic routes of internalised wheat germ agglutinin (WGA) were studied in human HepG2 hepatoma cells, drawing particular attention to the application times in relation to the membrane dynamics occurring at the trans Golgi face during endocytosis. As shown in previous studies, transport of internalised WGA into the Golgi apparatus can be classified in three stages being characterised by predominance of vesicular endosomes (stage I), formation of an extended endocytic trans Golgi network (stage II) and uptake of WGA into the stacked Golgi cisternae (stage III). BFA treatment of the cells led to rapid tubular-reticular transformations of the Golgi stacks. Retrograde transport and further destinations of internalised WGA depended on the time of BFA application. When BFA was administered during stages I or II, WGA was localised within the BFA-induced tubules and networks, but never was found within the endoplasmic reticulum. By contrast, BFA treatment during stage III led to a redistribution of internalised WGA into cisternae of the endoplasmic reticulum. These results show that BFA administered according to a precise time schedule can be used as a regulatory agent that allows to control retrograde traffic of internalised molecules into the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

The discovery and development of green fluorescent protein (GFP) from the jellyfish, Aequorea victoria, has revolutionized studies on protein localization and dynamics by allowing direct observation of a protein's life history and pathway in living cells, previously only deduced from genetic, biochemical, or immunolabeling studies. Applied to the secretory membrane system, which regulates delivery of newly synthesized proteins and lipids to the cell surface, GFP-based studies are providing important new insights into the maintenance and biogenesis of organelles, as well as the origin, pathway, and fate of secretory transport intermediates. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

When co-translationally inserted into endoplasmic reticulum (ER) membranes, newly synthesized proteins encounter the lumenal environment of the ER, which contains chaperone proteins that facilitate the folding reactions necessary for protein oligomerization, maturation and export from the ER. Here we show, using a temperature-sensitive variant of vesicular stomatitis virus G protein tagged with green fluorescent protein (VSVG-GFP), and fluorescence recovery after photobleaching (FRAP), the dynamics of association of folded and misfolded VSVG complexes with ER chaperones. We also investigate the potential mechanisms underlying protein retention in the ER. Misfolded VSVG-GFP complexes at 40°C are highly mobile in ER membranes and do not reside in post-ER compartments, indicating that they are not retained in the ER by immobilization or retrieval mechanisms. These complexes are immobilized in ATP-depleted or tunicamycin-treated cells, in which VSVG-chaperone interactions are no longer dynamic. These results provide insight into the mechanisms of protein retention in the ER and the dynamics of protein-folding complexes in native ER membranes. [ABSTRACT FROM AUTHOR]

Summary: Since the advent of green-fluorescent protein (GFP) technology there has been an explosion of interest in applying this molecule to cell biology. This review summarizes new insights in secretory membrane traffic obtained by the use of GFP fusion proteins. Transport steps between the endoplasmic reticulum and the Golgi apparatus, intra-Golgi traffic, and transport from the Golgi to the plasma membrane are discussed. In addition, insights into the dynamics of the Golgi compartment in plant cells and in mitotic mammalian cells have been included. We conclude that membrane traffic in the secretory pathway appears to be much more dynamic and diverse than previously thought and that GFP promises to be a powerful means to unravel these complex processes. [ABSTRACT FROM AUTHOR]