An update on transport vesicle tethering

Abstract

Membrane trafficking involves the collection of cargo into nascent transport vesicles that bud off from a donor compartment, translocate along cytoskeletal tracks, and then dock and fuse with their target membranes. Docking and fusion involve initial interaction at a distance (tethering), followed by a closer interaction that leads to pairing of vesicle SNARE proteins (v-SNAREs) with target membrane SNAREs (t-SNAREs), thereby catalyzing vesicle fusion. When tethering cannot take place, transport vesicles accumulate in the cytoplasm. Tethering is generally carried out by two broad classes of molecules: extended, coiled-coil proteins such as the so-called Golgin proteins, or multi-subunit complexes such as the Exocyst, COG or Dsl complexes. This review will focus on the most recent advances in terms of our understanding of the mechanism by which tethers carry out their roles, and new structural insights into tethering complex transactions.     

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.     

Myelin proteolipid protein-induced aggregation of lipid vesicles: efficacy of the various molecular species

The different molecular species that form the myelin proteolipid protein family were isolated by size-exclusion and ion-exchange chromatography in organic solvents and their adhesive properties were tested using a vesicle aggregation assay. Addition of the major proteolipid (PLP) to phosphatidylcholine-cholesterol vesicles caused their clustering as determined by increase in O.D._{450 nm} and by transmission electron microscopy. A small fraction of the aggregated vesicles underwent fusion as determined by resonance energy transfer experiments. Vesicle aggregation by PLP, but not the dissociation of the aggregates, was influenced by pH suggesting that electrostatic interactions are important only during cluster formation. Cleavage of disulfide bonds and methylation of carboxyl groups in PLP greatly reduced the aggregating activity, indicating that the process is dependent on the protein's conformation. Unexpectedly, the proteolipid DM-20 was also effective at inducing the clustering of neutral lipid vesicles. In contrast, three protein fractions comprising the naturally-occurring PLP fragments 1-107/112, 113/125-276 and 129/131-276, bearing different net charges, displayed a much lower activity. In addition, trypsin digestion of PLP resulted in a progressive decrease in the protein's ability to induce vesicle aggregation which coincided with the disappearance of the full-length molecule. Together, these results suggest that even large PLP fragments cannot fulfill the adhesive function of the intact protein.     

Synaptic vesicle recycling: steps and principles

Synaptic vesicle recycling is one of the best‐studied cellular pathways. Many of the proteins involved are known, and their interactions are becoming increasingly clear. However, as for many other pathways, it is still difficult to understand synaptic vesicle recycling as a whole. While it is generally possible to point out how synaptic reactions take place, it is not always easy to understand what triggers or controls them. Also, it is often difficult to understand how the availability of the reaction partners is controlled: how the reaction partners manage to find each other in the right place, at the right time. I present here an overview of synaptic vesicle recycling, discussing the mechanisms that trigger different reactions, and those that ensure the availability of reaction partners. A central argument is that synaptic vesicles bind soluble cofactor proteins, with low affinity, and thus control their availability in the synapse, forming a buffer for cofactor proteins. The availability of cofactor proteins, in turn, regulates the different synaptic reactions. Similar mechanisms, in which one of the reaction partners buffers another, may apply to many other processes, from the biogenesis to the degradation of the synaptic vesicle.     

P2 Protein in Oligodendrocytes and Myelin of the Rabbit Central Nervous System

P2 protein, a myelin-specific protein, was detected immunocytochemically and biochemically in rabbit central nervous system (CNS) myelin. P2 protein was synthesized by rabbit oligodendrocytes and was present in varying amounts throughout the rabbit CNS. Comparison of P2 and myelin basic protein (MBP) stained sections revealed that P2 antiserum did not stain all myelin sheaths within the rabbit CNS. The proportion of myelin sheaths stained by P2 antiserum and the amount of P2 detected biochemically were greater in more caudal regions of the rabbit CNS. The highest concentration of P2 protein was found in rabbit spinal cord myelin, where P2 antiserum stained the majority of myelin sheaths. P2 protein was barely detectable biochemically in myelin isolated from frontal cortex, and in sections of frontal cortex only occasional myelin sheaths reacted with P2 antiserum. These results suggest the the regional variations in the amount of P2 protein are dut to regional differences in the number of myelin sheaths that contain P2 protein. P2 protein was detected immunocytochemically and biochemically in rabbit sciatic nerve myelin. Immunocytochemically, P2 antiserum only stained a portion of the myelin sheaths present. The myelin sheaths not reacting with P2 antiserum had small diameters and represented less than 10% of the total myelinated fibers.     

Axonal transport of synaptic vesicle proteins in the rat optic nerve

The optic nerve, as a part of the central nervous system (CNS), has been used to study axonal transport for decades. The present study has concentrated on the axonal transport of synaptic vesicle proteins in the optic nerve, using the “stop-flow/nerve crush” method. After blocking fast axonal transport, distinct accumulations of synaptic vesicle proteins developed during the first hour after crush-operation and marked increases were observed up to 8 h postoperative. Semiquantitative analysis, using cytofluorimetric scanning (CFS) of immunoincubated sections, revealed that the ratio between distal accumulations (organelles in retrograde transport) and proximal accumulations (organelles in anterograde transport) was much higher (up to 80–90%) for the transmembrane proteins than that for surface adsorbed proteins (only 10–20%). The pattern of axonal transport in the optic nerve was comparable to that in the sciatic nerve. However, clathrin and Rab3a immunoreactivities were accumulated in much lower amounts than that in the sciatic nerve. Most synaptic vesicle proteins were colocalized in the axons proximal to the crush. A differential distribution of synaptobrevin I and II, however, was observed in the optic nerve axons; synaptobrevin I was present in large-sized axons, while synaptobrevin II immunoreactivity was present in most axons, including the large ones. The two isoforms were, thus, partially colocalized. The results demonstrate that (1) cytofluorimetric scanning techniques could be successfully used to study axonal transport not only in peripheral nerves, but also in the CNS; (2) synaptic vesicles are transported with fast axonal transport in this nerve; and (3) some differences were noted compared with the sciatic nerve, especially for Rab3a and clathrin. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 237–250, 1997.     

SM蛋白在膜泡运输中的功能

大多数细胞内都包含靶向不同细胞器的各种运输囊泡,其运输机制在进化上是高度保守的。Sec1/Munc-18(SM)蛋白在膜泡运输中起着重要的调控作用,它能够与SNARE(Soluble N-ethylmaleimide-sensitive factorattachment protein receptor)蛋白结合,共同在细胞内各个膜融合发生部位发挥重要作用。SM蛋白和SNARE复合体中的Syntaxin蛋白结合,调节SNARE复合体的装配,并与SNARE协同作用促进整个膜融合过程。文章对SM蛋白在结构和功能分析方面的最新研究进展进行了概述。     

Membrane transport in the endocytic pathway: Animal versus plant cells

Summary The endocytic pathway is a well established process in animal cells, but it is not well understood in plant cells. At the morphological level, all the compartments involved in endocytosis in animal cells seem to have counterparts in plant cells, and the organization of the pathway appears to share some striking similarities. Several Rab homologues have been found in plant cells, including homologues of Rab5, Rab7, and Rab11, markers of endocytic compartments in animal cells. Coat proteins are also present in plant cells, including clathrin, adaptins, and ADP ribosylation factor proteins. However, endocytic compartments in plant cells also exhibit specific features both in organization and function. The molecular composition of these compartments remains to be established, and future work will be necessary to identify the key regulators of endocytic trafficking in plant cells.Abbreviations EE early endosome - LE late endosome - ECV-MVB endosomal carrier vesicle-multivesicular body - PCR partially coated reticulum - MPR mannose 6-phosphate receptor - TGN trans-Golgi network     

Myelin Basic Protein Is a Zinc-Binding Protein in Brain: Possible Role in Myelin Compaction

The zinc-binding proteins (ZnBPs) in porcine brain were characterized by the radioactive zinc-blot technique. Three ZnBPs of molecular weights about 53 kDa, 42 kDa, and 21 kDa were identified. The 53 kDa and 42 kDa ZnBPs were found in all subcellular fractions while the 21 kDa ZnBP was mainly associated with particulate fractions. This 21 kDa ZnBP was identified by internal protein sequence data as the myelin basic protein. Further characterization of its electrophoretic properties and cyanogen bromide cleavage pattern with the authentic protein confirmed its identity. The zinc binding properties of myelin basic protein are metal specific, concentration dependent and pH dependent. The zinc binding property is conferred by the histidine residues since modification of these residues by diethyl-pyrocarbonate would abolish this activity. Furthermore, zinc ion was found to potentiate myelin basic protein-induced phospholipid vesicle aggregation. It is likely that zinc plays an important role in myelin compaction by interacting with myelin basic protein.     

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.     

缺铁水稻根转录本组及与膜泡运输相关基因的分析

随着大规模芯片技术(即cDNA微点阵)的开展,这一技术已用于胁迫基因表达的转录本组分析。本论文重点阐述转录 本图谱分析的方法,以及缺铁胁迫下,水稻的基因组表达差异,其中重点是与膜泡运输相关的几个基因。     

Mechanisms of myelin basic protein and proteolipid protein targeting in oligodendrocytes (Review)

Summary

The segregation of proteins to specific cellular membranes is recognized as a common phenomenon. In oligodendrocytes of the central nervous system, localization of certain proteins to select regions of the plasma membrane gives rise to the myelin membrane. Whilst the fundamental structure and composition of myelin is well understood, less is known of the mechanisms by which the constituent proteins are specifically recruited to those regions of plasma membrane that are forming myelin. The two principal proteins of myelin, the myelin basic protein and proteolipid protein, differ greatly in character and sites of synthesis. The message for myelin basic protein is selectively translocated to the ends of the cell processes, where it is translated on free ribosomes and is incorporated directly into the membrane. Proteolipid protein synthesized at the rough endoplasmic reticulum, processed through the Golgi apparatus, and presumably transported via vesicles to the myelin membrane. This review examines the mechanisms by which these two proteins are targeted to the myelin membrane.     

SNARE membrane trafficking dynamics in vivo

The ER/Golgi soluble NSF attachment protein receptor (SNARE) membrin, rsec22b, and rbet1 are enriched in approximately 1-micrometer cytoplasmic structures that lie very close to the ER. These appear to be ER exit sites since secretory cargo concentrates in and exits from these structures. rsec22b and rbet1 fused to fluorescent proteins are enriched at approximately 1-micrometer ER exit sites that remained more or less stationary, but periodically emitted streaks of fluorescence that traveled generally in the direction of the Golgi complex. These exit sites were reused and subsequent tubules or streams of vesicles followed similar trajectories. Fluorescent membrin- enriched approximately 1-micrometer peripheral structures were more mobile and appeared to translocate through the cytoplasm back and forth, between the periphery and the Golgi area. These mobile structures could serve to collect secretory cargo by fusing with ER-derived vesicles and ferrying the cargo to the Golgi. The post-Golgi SNAREs, syntaxin 6 and syntaxin 13, when fused to fluorescent proteins each displayed characteristic patterns of movement. However, syntaxin 13 was the only SNARE whose life cycle appeared to involve interactions with the plasma membrane. These studies reveal the in vivo spatiotemporal dynamics of SNARE proteins and provide new insight into their roles in membrane trafficking.     

On the biogenesis of the myelin sheath: Cognate polarized trafficking pathways in oligodendrocytes

Oligodendrocytes, the myelinating cells of the central nervous system, are capable of transporting vast quantities of proteins and of lipids, in particular galactosphingolipids, to the myelin sheath. The sheath is continuous with the plasma membrane of the oligodendrocyte, but the composition of both membrane domains differs substantially. Given its high glycosphingolipid and cholesterol content the myelin sheath bears similarity to the lipid composition of the apical domain of a polarized cell. The question thus arises whether myelin components, like typical apical membrane proteins are transported by an apical-like trafficking mechanism to the sheath, involving a raft-mediated mechanism. Indeed, the evidence indicates the presence of cognate apical and basolateral pathways in oligodendrocytes. However, all major myelin proteins do not participate in this pathway, and remarkably apical-like trafficking seems to be restricted to the oligodendrocyte cell body. In this review, we summarize the evidence on the existence of different trafficking pathways in the oligodendrocyte, and discuss possible mechanisms separating the oligodendrocyte's membrane domains.     

植物囊泡运输与抗病性

细胞内的囊泡运输是生命活动中一个极其复杂的动态生物学过程,参与各种植物发育过程和对环境的响应,包括植物组织细胞特异性和防御响应。该文从蛋白质分选、分泌蛋白的合成和囊泡运输的特异性对植物囊泡运输与植物的先天性免疫的关系进行了详细阐述。     

Tau protein expression in adult bovine oligodendrocytes: functional and pathological significance

In tauopathies, overexpression of tau exon 10 is linked to degeneration and abnormal tau deposition in neurons and oligodendroglia (OLGs). To compare exon 10 expression in normal neurons and OLGs, adult bovine brain was examined for the expression of tau in gray matter and cultured OLGs isolated from white matter. Using exon-specific antibodies, we found that both types of tissues abundantly expressed exon 2 but isolated OLGs had a lower expression of exons 3 and 10 when compared to gray matter. Relative expression of exons 3 and 10 did not change significantly during the in vitro maturation of OLGs for 39 days. Using a panel of well-characterized antibodies against tau, we determined that isolated OLGs contained tau phosphorylated at the Tau-1, 12E8, and PHF-1 but not the AT8, AT100, AT180, and AT270 epitopes. Tau phosphorylation status diminished during in vitro maturation, suggesting that healthy OLG processes require regulated phosphorylation of tau at specific sites. We propose that the tau isoform profile and phosphorylation status contribute to the vulnerability of OLGs in degenerative diseases linked to overexpression of exon 10.     

Immunoisolation of two synaptic vesicle pools from synaptosomes: a proteomics analysis

The nerve terminal proteome governs neurotransmitter release as well as the structural and functional dynamics of the presynaptic compartment. In order to further define specific presynaptic subproteomes we used subcellular fractionation and a monoclonal antibody against the synaptic vesicle protein SV2 for immunoaffinity purification of two major synaptosome-derived synaptic vesicle-containing fractions: one sedimenting at lower and one sedimenting at higher sucrose density. The less dense fraction contains free synaptic vesicles, the denser fraction synaptic vesicles as well as components of the presynaptic membrane compartment. These immunoisolated fractions were analyzed using the cationic benzyldimethyl-n-hexadecylammonium chloride (BAC) polyacrylamide gel system in the first and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Protein spots were subjected to analysis by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS). We identified 72 proteins in the free vesicle fraction and 81 proteins in the plasma membrane-containing denser fraction. Synaptic vesicles contain a considerably larger number of protein constituents than previously anticipated. The plasma membrane-containing fraction contains synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery and numerous other proteins potentially involved in regulating the functional and structural dynamics of the nerve terminal.     

Protein Composition of PNS Myelin: Developmental Comparison of Control and Quaking Mice

Protein compositions were determined for sciatic nerve myelin isolated from young and adult control and quaking (Qk) mice. Age-related changes in the relative amounts of large (Pl) and small (Pr) basic proteins were found. In control animals, the ratio Pr/Pl increased with age, a change similar to that observed for the large (Bl) and small (Bs) CNS myelin basic proteins of adult mice. Pr/Pl also increased with age in the Qk mouse sciatic nerve, but only to the point that the value in the adult Qk mouse was similar to that observed for young control animals, a situation reminiscent of the effect of the Qk mutation on CNS basic proteins. Thus, our data suggest that the Qk mutation has a similar effect on peripheral nervous system (PNS) and CNS basic proteins. Our findings are consistent with recent electrophoretic and immunochemical data showing that PNS and CNS myelin basic proteins in rodents are analogous, and they suggest that the genetic program controlling basic protein expression is common to oligodendroglia and Schwann cells.