How to get to the right place at the right time: Rab/Ypt small GTPases and vesicle transport

Summary Vesicles often must be transported over long distances in a very crowded cytoplasmic environment encumbered by the cytoskeleton and membranes of different origin that provide an important barrier to their free diffusion. In animal cells with specialised tasks, such as neurons or endothelial cells, vesicles that are directed to the cell periphery are linked to the microtubular cytoskeleton tracks via association with motor proteins that allow their vectorial movement. In lower eukaryotes the actin cytoskeleton plays a prominent role in organising vesicle movement during polarised growth and mating. The Ras-like small GTPases of the Rab/Ypt family play an essential role in vesicle trafficking and due to their diversity and specific localisation have long been implicated in the selective delivery of vesicles. Recent evidence has cast doubt on the classical point of view of how this class of proteins acts in vesicle transport and suggests their involvement also in the events that permit vesicle anchoring to the cytoskeleton. Therefore, after a brief review of what is known about how vesicle movement is achieved in mammalian and yeast systems, and how Rab/Ypt proteins regulate the vesicle predocking events, it is discussed how these proteins might participate in the events that lead to vesicle movement through association with the cytoskeleton machinery.     

SNARE complex proteins, including the cognate pair VAMP-2 and syntaxin-4, are expressed in cultured oligodendrocytes

Myelin membrane synthesis in the CNS by oligodendrocytes (OLs) involves directed intracellular transport and targeting of copious amounts of specialized lipids and proteins over a relatively short time span. As in other plasma membrane-directed fusion, this process is expected to use specific trafficking and vesicle fusion proteins characteristic of the SNARE model. We have investigated the developmental expression of SNARE proteins in highly enriched primary cultures of OLs at discrete stages of differentiation. VAMP-2/synaptobrevin-2, syntaxin-2 and -4, nsec-1/munc-18-1, Rab3a, synaptophysin, and synapsin were expressed. During differentiation, expression of the vesicular SNARE VAMP-2, the small GTP-binding protein Rab3a, and the target SNARE syntaxin-4 were up-regulated. VAMP-2 and Rab3 proteins detected immunocytochemically in cultured OLs were localized within the developing process network; in situ anti-VAMP-2 antibody stained the perikarya of rows of cells with the distribution and appearance of OLs. We discuss the potential involvement of SNARE complex proteins in a plasma membrane-directed transport mechanism targeting nascent myelin vesicles to the forming myelin sheath.     

How to get to the right place at the right time: Rab/Ypt small GTPases and vesicle transport

Vesicles often must be transported over long distances in a very crowded cytoplasmic environment encumbered by the cytoskeleton and membranes of different origin that provide an important barrier to their free diffusion. In animal cells with specialised tasks, such as neurons or endothelial cells, vesicles that are directed to the cell periphery are linked to the microtubular cytoskeleton tracks via association with motor proteins that allow their vectorial movement. In lower eukaryotes the actin cytoskeleton plays a prominent role in organising vesicle movement during polarised growth and mating. The Ras-like small GTPases of the Rab/Ypt family play an essential role in vesicle trafficking and due to their diversity and specific localisation have long been implicated in the selective delivery of vesicles. Recent evidence has cast doubt on the classical point of view of how this class of proteins acts in vesicle transport and suggests their involvement also in the events that permit vesicle anchoring to the cytoskeleton. Therefore, after a brief review of what is known about how vesicle movement is achieved in mammalian and yeast systems, and how Rab/Ypt proteins regulate the vesicle predocking events, it is discussed how these proteins might participate in the events that lead to vesicle movement through association with the cytoskeleton machinery.     

Lipid metabolism and vesicle trafficking: more than just greasing the transport machinery.

The movement of lipids from their sites of synthesis to ultimate intracellular destinations must be coordinated with lipid metabolic pathways to ensure overall lipid homeostasis is maintained. Thus, lipids would be predicted to play regulatory roles in the movement of vesicles within cells. Recent work has highlighted how specific lipid metabolic events can affect distinct vesicle trafficking steps and has resulted in our first glimpses of how alterations in lipid metabolism participate in the regulation of intracellular vesicles. Specifically, (i) alterations in sphingolipid metabolism affect the ability of SNAREs to fuse membranes, (ii) sterols are required for efficient endocytosis, (iii) glycerophospholipids and phosphorylated phosphatidylinositols regulate Golgi-mediated vesicle transport, (iv) lipid acylation is required for efficient vesicle transport mediated membrane fission, and (v) the addition of glycosylphosphatidylinositol lipid anchors to proteins orders them into distinct domains that result in their preferential sorting from other vesicle destined protein components in the endoplasmic reticulum. This review describes the experimental evidence that demonstrates a role for lipid metabolism in the regulation of specific vesicle transport events.     

囊泡转运蛋白SM蛋白家族的研究进展

真核细胞中含有多种不同功能的转运囊泡。虽然转运途径和携带物质各异,但细胞转运的基本分子机制却呈现出高度相似性和保守性。大多数转运途径都需要一种SNARE（Soluble NSF Attachment Protein Receptor）蛋白质复合体介导转运膜泡与靶膜的融合。同时,另一个蛋白家族,Secl／Muncl8蛋白（SM蛋白）也在囊泡运输中发挥重要作用。但是相比于对SNARE蛋白的认识的一致性,在不同的研究中SM蛋白的功能及其与SNARE复合体的相互作用方式却不尽相同。以下综述近年来有关SM蛋白结构和功能的研究进展,并归纳SM蛋白分子的作用机制、功能以及应用。     

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.     

真核细胞内膜泡运输的分子机制

真核细胞内一些蛋白质需靠膜泡进行定向运输,膜泡是在外衣蛋白的作用下形成的,根据外衣蛋白的不同,膜泡分为笼蛋白,COPⅠ和COPⅡ外衣膜泡,这些外衣膜泡分别在细胞内不同供膜（donor membrane)处形成,因为被运输蛋白具有分选信号可与供膜上相应的受体结合,所以能被包裹在特异的膜泡之中,在膜泡形成过程中,外衣蛋白在“芽生”膜泡的细胞质侧组装成笼状外衣,帮助“芽生”膜泡从供膜处脱落,一旦笼状外衣膜泡脱离供膜,笼状外衣蛋白便发生解聚而成为无衣膜泡,无衣膜泡在Rab蛋白的调控下可定向运输蛋白质,而解聚后的外衣蛋白可重新介导新的外衣膜泡形成。     

Vesicle-mediated ER export of proteins and lipids

In eukaryotic cells, the endoplasmic reticulum (ER) is a major site of synthesis of both lipids and proteins, many of which must be transported to other organelles. The COPII coat-comprising Sar1, Sec23/24, Sec13/31-generates transport vesicles that mediate the bulk of protein/lipid export from the ER. The coat exhibits remarkable flexibility in its ability to specifically select and accommodate a large number of cargoes with diverse properties. In this review, we discuss the fundamentals of COPII vesicle production and describe recent advances that further our understanding of just how flexible COPII cargo recruitment and vesicle formation may be. Large or bulky cargo molecules (e.g. collagen rods and lipoprotein particles) exceed the canonical size for COPII vesicles and seem to rely on the additional action of recently identified accessory molecules. Although the bulk of the research has focused on the fate of protein cargo, the mechanisms and regulation of lipid transport are equally critical to cellular survival. From their site of synthesis in the ER, phospholipids, sphingolipids and sterols exit the ER, either accompanying cargo in vesicles or directly across the cytoplasm shielded by lipid-transfer proteins. Finally, we highlight the current challenges to the field in addressing the physiological regulation of COPII vesicle production and the molecular details of how diverse cargoes, both proteins and lipids, are accommodated. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Lipid requirements for endocytosis in yeast

Endocytosis is, besides secretion, the most prominent membrane transport pathway in eukaryotic cells. In membrane transport, defined areas of the donor membranes engulf solutes of the compartment they are bordering and bud off with the aid of coat proteins to form vesicles. These transport vehicles are guided along cytoskeletal paths, often matured and, finally, fuse to the acceptor membrane they are targeted to. Lipids and proteins are equally important components in membrane transport pathways. Not only are they the structural units of membranes and vesicles, but both classes of molecules also participate actively in membrane transport processes. Whereas proteins form the cytoskeleton and vesicle coats, confer signals and constitute attachment points for membrane-membrane interaction, lipids modulate the flexibility of bilayers, carry protein recognition sites and confer signals themselves. Over the last decade it has been realized that all classes of bilayer lipids, glycerophospholipids, sphingolipids and sterols, actively contribute to functional membrane transport, in particular to endocytosis. Thus, abnormal bilayer lipid metabolism leads to endocytic defects of different severity. Interestingly, there seems to be a great deal of interdependence and interaction among lipid classes. It will be a challenge to characterize this plenitude of interactions and find out about their impact on cellular processes.     

Stage-specific assays to study biosynthetic cargo selection and role of SNAREs in export from the endoplasmic reticulum and delivery to the Golgi

To analyze the role of coat protein type II (COPII) coat components and targeting and fusion factors in selective export from the endoplasmic reticulum (ER) and transport to the Golgi, we have developed three novel, stage-specific assays. Cargo selection can be measured using a "stage 1 cargo capture assay," in which ER microsomes are incubated in the presence of glutathione S-transferase (GST)-tagged Sar1 GTPase and purified Sec23/24 components to follow recruitment of biosynthetic cargo to prebudding complexes. This cargo recruitment assay can be followed by two sequential assays that measure separately the budding of COPII-coated vesicles from ER microsomes (stage 2) and, finally, delivery of cargo-containing vesicles to the Golgi (stage 3). We show how these assays provide a means to identify the snap receptor (SNARE) protein rBet1 as an essential component that is not required for vesicle formation, but is required for vesicle targeting and fusion during ER-to-Golgi transport. In general, these assays provide an approach to characterize the biochemical basis for the recruitment of a wide variety of biosynthetic cargo proteins to COPII vesicles and the role of different transport components in the early secretory pathway of mammalian cells.     

Characterization of Yeast Extracellular Vesicles: Evidence for the Participation of Different Pathways of Cellular Traffic in Vesicle Biogenesis

Background

Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown.

Methodology/Principal Findings

We characterized extracellular vesicle production in wild type (WT) and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicle-associated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB) formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100–300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasma membrane; bos1-1, vesicle targeting to the Golgi complex) or MVB functionality (vps23, late endosomal trafficking) revealed a complex and interrelated protein collection. Semi-quantitative analysis of protein abundance revealed that mutations in both MVB- and Golgi-derived pathways affected the composition of yeast extracellular vesicles, but none abrogated vesicle production. Lipid analysis revealed that mutants with defects in Golgi-related components of the secretory pathway had slower vesicle release kinetics, as inferred from intracellular accumulation of sterols and reduced detection of these lipids in vesicle fractions in comparison with WT cells.

Conclusions/Significance

Our results suggest that both conventional and unconventional pathways of secretion are required for biogenesis of extracellular vesicles, which demonstrate the complexity of this process in the biology of yeast cells.     

Proteins affecting thylakoid morphology – the key to understanding vesicle transport in chloroplasts?

We recently showed that a Rab protein, CPRabA5e (CP = chloroplast localized), is located in chloroplasts of Arabidopsis thaliana where it is involved in various processes, such as thylakoid biogenesis and vesicle transport. Using a yeast two-hybrid method, CPRabA5e was shown to interact with a number of chloroplast proteins, including the CURVATURE THYLAKOID 1A (CURT1A) protein and the light-harvesting chlorophyll a/b binding protein (LHCB1.5). CURT1A has recently been shown to modify thylakoid architecture by inducing membrane curvature in grana, whereas LHCB1.5 is a protein of PSII (Photosystem II) facilitating light capture. LHCB1.5 is imported to chloroplasts and transported to thylakoid membranes using the post-translational Signal Recognition Particle (SRP) pathway. With this information as starting point, we here discuss their subsequent protein-protein interactions, given by the literature and Interactome 3D. CURT1A itself and several of the proteins interacting with CURT1A and LHCB1.5 have relations to vesicle transport and thylakoid morphology, which are also characteristics of cprabA5e mutants. This highlights the previous hypothesis of an alternative thylakoid targeting pathway for LHC proteins using vesicles, in addition to the SRP pathway.     

Regulating cytoskeleton-based vesicle motility

During vesicular transport, the assembly of the coat complexes and the selection of cargo proteins must be coordinated with the subsequent translocation of vesicles from the donor to an acceptor compartment. Here, we review recent progress toward uncovering the molecular mechanisms that connect transport vesicles to the protein machinery responsible for cytoskeleton-mediated motility. An emerging theme is that vesicle cargo proteins, either directly or through binding interactions with coat proteins, are able to influence cytoskeletal dynamics and motor protein function. Hence, a vesicle's cargo composition may help direct its intracellular motility and targeting.     

Requirement for Golgi-localized PI(4)P in fusion of COPII vesicles with Golgi compartments

The role of specific membrane lipids in transport between endoplasmic reticulum (ER) and Golgi compartments is poorly understood. Using cell-free assays that measure stages in ER-to-Golgi transport, we screened a variety of enzyme inhibitors, lipid-modifying enzymes, and lipid ligands to investigate requirements in yeast. The pleckstrin homology (PH) domain of human Fapp1, which binds phosphatidylinositol-4-phosphate (PI(4)P) specifically, was a strong and specific inhibitor of anterograde transport. Analysis of wild type and mutant PH domain proteins in addition to recombinant versions of the Sac1p phosphoinositide-phosphatase indicated that PI(4)P was required on Golgi membranes for fusion with coat protein complex II (COPII) vesicles. PI(4)P inhibition did not prevent vesicle tethering but significantly reduced formation of soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes between vesicle and Golgi SNARE proteins. Moreover, semi-intact cell membranes containing elevated levels of the ER-Golgi SNARE proteins and Sly1p were less sensitive to PI(4)P inhibitors. Finally, in vivo analyses of a pik1 mutant strain showed that inhibition of PI(4)P synthesis blocked anterograde transport from the ER to early Golgi compartments. Together, the data presented here indicate that PI(4)P is required for the SNARE-dependent fusion stage of COPII vesicles with the Golgi complex.     

ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat

In eukaryotic cells, secretion is achieved by vesicular transport. Fusion of such vesicles with the correct target compartment relies on SNARE proteins on both vesicle (v-SNARE) and the target membranes (t-SNARE). At present it is not clear how v-SNAREs are incorporated into transport vesicles. Here, we show that binding of ADP-ribosylation factor (ARF)-GTPase-activating protein (GAP) to ER-Golgi v-SNAREs is an essential step for recruitment of Arf1p and coatomer, proteins that together form the COPI coat. ARF-GAP acts catalytically to recruit COPI components. Inclusion of v-SNAREs into COPI vesicles could be mediated by direct interaction with the coat. The mechanisms by which v-SNAREs interact with COPI and COPII coat proteins seem to be different and may play a key role in determining specificity in vesicle budding.     

Association of protein-tyrosine phosphatase MEG2 via its Sec14p homology domain with vesicle-trafficking proteins

The protein-tyrosine phosphatase PTPMEG2 is located on the cytoplasmic face of the enclosing membrane of secretory vesicles, where it regulates vesicle size by promoting homotypic vesicle fusion by dephosphorylating N-ethylmaleimide-sensitive factor, a key regulator of vesicle fusion. Here we address the question of how PTPMEG2 is targeted to this subcellular location. Using a series of deletion mutants, we pinpointed the N-terminal Sec14p homology (SEC14) domain of PTPMEG2, residues 1-261, as the region containing the secretory vesicle targeting signal. This domain, alone or appended to a heterologous protein, was localized to intracellular vesicle membranes. Yeast two-hybrid screening identified a number of secretory vesicle proteins that interacted directly with the SEC14 domain of PTPMEG2, providing a mechanism for PTPMEG2 targeting to secretory vesicles. Two such proteins, mannose 6-phosphate receptor-interacting protein TIP47 and Arfaptin2, were found to alter PTPMEG2 localization when overexpressed, and elimination of TIP47 resulted in loss of PTPMEG2 function. We conclude that the N terminus of PTPMEG2 is necessary for the targeting of this phosphatase to the secretory vesicle compartment by association with other proteins involved in intracellular transport.     

In vivo imaging of retrogradely transported synaptic vesicle proteins in Caenorhabditis elegans neurons

Axonal transport is an essential process that carries cargoes in the anterograde direction to the synapse and in the retrograde direction back to the cell body. We have developed a novel in vivo method to exclusively mark and dynamically track retrogradely moving compartments carrying specific endogenous synaptic vesicle proteins in the Caenorhabditis elegans model. Our method is based on the uptake of a fluorescently labeled anti-green fluorescent protein (GFP) antibody delivered in an animal expressing the synaptic vesicle protein synaptobrevin-1::GFP in neurons. We show that this method largely labels retrogradely moving compartments. Very little labeling is observed upon blocking vesicle exocytosis or if the synapse is physically separated from the cell body. The extent of labeling is also dependent on the dyenin-dynactin complex. These data support the interpretation that the labeling of synaptobrevin-1::GFP largely occurs after vesicle fusion and the major labeling likely takes place at the synapse. Further, we observe that the retrograde compartment carrying synaptobrevin contains synaptotagmin but lacks the endosomal marker RAB-5. This labeling method is very general and can be readily adapted to any transmembrane protein on synaptic vesicles with a GFP tag inside the vesicle and can also be extended to other model systems.     

Most Synaptic Vesicles Isolated from Rat Brain Carry Three Membrane Proteins, SV2, Synaptophysin, and p65

We have prepared highly purified synaptic vesicles from rat brain by subjecting vesicles purified by our previous method to a further fractionation step, i.e., equilibrium centrifugation on a Ficoll gradient. Monoclonal antibodies to three membrane proteins enriched in synaptic vesicles--SV2, synaptophysin, and p65--each were able to immunoprecipitate specifically approximately 90% of the total membrane protein from Ficoll-purified synaptic vesicle preparations. Anti-SV2 precipitated 96% of protein, anti-synaptophysin 92%, and anti-p65 83%. These results demonstrate two points: (1) Ficoll-purified synaptic vesicles appear to be greater than 90% pure, i.e., less than 10% of membranes in the preparation do not carry synaptic vesicle-associated proteins. These very pure synaptic vesicles may be useful for direct biochemical analyses of mammalian synaptic vesicle composition and function. (2) SV2, synaptophysin, and p65 coexist on most rat brain synaptic vesicles. This result suggests that the functions of these proteins are common to most brain synaptic vesicles. However, if SV2, synaptophysin, or p65 is involved in synaptic vesicle dynamics, e.g., in vesicle trafficking or exocytosis, separate cellular systems are very likely required to modulate the activity of such proteins in a temporally or spatially specific manner.     

Emerging Role of the Scaffolding Protein Dlg1 in Vesicle Trafficking

Discs large 1 (Dlg1) is a modular scaffolding protein implicated in the control of cell polarity through assembly of specific multiprotein complexes, including receptors, ion channels and signaling proteins, at specialized zones of the plasma membrane. Recent data have shown that in addition to these well‐known interaction partners, Dlg1 may also recruit components of the vesicle trafficking machinery either to the plasma membrane or to transport vesicles. Here, we discuss Dlg1 function in vesicle formation, targeting, tethering and fusion, in both the exocytotic and endocytotic pathways. These pathways contribute to cell functions as major and diverse as glutamatergic activity in the neurons, membrane homeostasis in Schwann cell myelination, insulin stimulation of glucose transport in adipocytes, or endothelial secretion of the hemostatic protein, von Willebrand factor (VWF).