How peptide hormone vesicles are transported to the secretion site for exocytosis

Post-Golgi transport of peptide hormone-containing vesicles from the site of genesis at the trans-Golgi network to the release site at the plasma membrane is essential for activity-dependent hormone secretion to mediate various endocrinological functions. It is known that these vesicles are transported on microtubules to the proximity of the release site, and they are then loaded onto an actin/myosin system for distal transport through the actin cortex to just below the plasma membrane. The vesicles are then tethered to the plasma membrane, and a subpopulation of them are docked and primed to become the readily releasable pool. Cytoplasmic tails of vesicular transmembrane proteins, as well as many cytosolic proteins including adaptor proteins, motor proteins, and guanosine triphosphatases, are involved in vesicle budding, the anchoring of the vesicles, and the facilitation of movement along the transport systems. In addition, a set of cytosolic proteins is also necessary for tethering/docking of the vesicles to the plasma membrane. Many of these proteins have been identified from different types of (neuro)endocrine cells. Here, we summarize the proteins known to be involved in the mechanisms of sorting various cargo proteins into regulated secretory pathway hormone-containing vesicles, movement of these vesicles along microtubules and actin filaments, and their eventual tethering/docking to the plasma membrane for hormone secretion.     

Quantitative composition and characterization of the proteins in membrane vesicles released from erythrocytes by dimyristoylphosphatidylcholine. A membrane system without cytoskeleton

Membrane vesicles were prepared by incubation of human erythrocytes with dimyristoylphosphatidylcholine [3] and isolated by isopycnic centrifugation on Dextran density gradients. Protein analyses were carried out with crossed immunoelectrophoresis and dodecylsulfate polyacrylamide gel electrophoresis. The right-side-out-oriented membrane vesicles contained membrane and cytoplasmic proteins of the erythrocyte but lacked cytoskeletal components. Comparison of proteins in vesicles and erythrocyte membranes showed that acetylcholinesterase was enriched two to six times in the vesicles relative to both membrane-spanning proteins, band 3, and glycophorin. Two further, hitherto unidentified, sialic acid-containing membrane antigens were found in the vesicles. Both faced the outside of the membranes and were enriched two to seven times. Ankyrin was not present in the membrane vesicles and spectrin could not be detected by dodecylsulfate polyacrylamide gel electrophoresis. We suggest that the redistribution of proteins in the vesicles reflects differences in their interactions with other membrane components and their relative mobility within the erythrocyte membrane.     

Visualization of the Dynamics of Synaptic Vesicle and Plasma Membrane Proteins in Living Axons

Newly synthesized membrane proteins are transported by fast axonal flow to their targets such as the plasma membrane and synaptic vesicles. However, their transporting vesicles have not yet been identified. We have successfully visualized the transporting vesicles of plasma membrane proteins, synaptic vesicle proteins, and the trans-Golgi network residual proteins in living axons at high resolution using laser scan microscopy of green fluorescent protein-tagged proteins after photobleaching. We found that all of these proteins are transported by tubulovesicular organelles of various sizes and shapes that circulate within axons from branch to branch and switch the direction of movement. These organelles are distinct from the endosomal compartments and constitute a new entity of membrane organelles that mediate the transport of newly synthesized proteins from the trans-Golgi network to the plasma membrane.     

Reconstitution of membrane proteins. Spontaneous incorporation of integral membrane proteins into preformed bilayers of pure phospholipid

The spontaneous reconstitution of lipid-protein complexes was examined by mixing bacteriorhodopsin or UDP-glucuronosyltransferase with preformed, unilamellar bilayers of pure dimyristoylphosphatidylcholine. Spontaneous insertion of these proteins into vesicles of dimyristoylphosphatidylcholine was facilitated by resonicating the vesicles at 4 degrees C. The property of resonicated vesicles that led to spontaneous reconstitution could be annealed by melting the bilayers, which slowed down reconstitution. The overall process of reconstitution consisted, however, of two steps. There was an initial insertion of proteins into a small portion of vesicles followed by subsequent fusion between protein-free vesicles and vesicles containing lipid-protein complexes. The first step appeared to proceed rapidly in all vesicles in a gel phase, whether or not they were resonicated or whether or not resonicated vesicles were annealed. The rate of the second step was sensitive to these treatments. The membrane proteins also inserted into preformed vesicles in a liquid crystalline phase, but this step was slower than for vesicles in a gel phase. Fusion between protein-free and protein-containing vesicles in a liquid crystalline phase was extremely slow. The data show that the spontaneous insertion of pure membrane proteins into preformed vesicles can be a facile event and that the overall reconstitution of membrane proteins into preformed unilamellar vesicles may be simpler to achieve than has been appreciated.     

Low molecular weight GTP-binding proteins are associated with neuronal organelles involved in rapid axonal transport and exocytosis

Recent evidence suggests that low molecular weight GTP-binding proteins may play important roles in a variety of membrane transport processes. In order to address the question of whether these proteins are involved in transport processes in the nerve axon, we have assessed their presence in rapid transport membranes from rabbit optic nerve. We report the characterization of a group of low molecular weight GTP-binding proteins which are constituents of rapid transport vesicles. Although these proteins are components of rapid transport vesicles, they are apparently not major rapidly transported species. They are localized in cytosolic as well as in membrane fractions of axons, and the membrane-associated form behaves as an integral membrane protein(s). These proteins are also found in association with a variety of vesicular and organellar components of neurons including coated vesicles, synaptic vesicles, synaptic plasma membranes, and mitochondria. We discuss the possible roles of these proteins in rapid axonal transport and exocytosis.     

Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins

To identify the membrane regions through which yeast mitochondria import proteins from the cytoplasm, we have tagged these regions with two different partly translocated precursor proteins. One of these was bound to the mitochondrial surface of ATP-depleted mitochondria and could subsequently be chased into mitochondria upon addition of ATP. The other intermediate was irreversibly stuck across both mitochondrial membranes at protein import sites. Upon subfraction of the mitochondria, both intermediates cofractionated with membrane vesicles whose buoyant density was between that of inner and outer membranes. When these vesicles were prepared from mitochondria containing the chaseable intermediate, they internalized it upon addition of ATP. A non-hydrolyzable ATP analogue was inactive. This vesicle fraction contained closed, right-side-out inner membrane vesicles attached to leaky outer membrane vesicles. The vesicles contained the mitochondrial binding sites for cytoplasmic ribosomes and contained several mitochondrial proteins that were enriched relative to markers of inner or outer membranes. By immunoelectron microscopy, two of these proteins were concentrated at sites where mitochondrial inner and outer membranes are closely apposed. We conclude that these vesicles contain contact sites between the two mitochondrial membranes, that these sites are the entry point for proteins into mitochondria, and that the isolated vesicles are still translocation competent.     

Evidence for a primary endocytic vesicle involved in synaptic vesicle biogenesis

The regulated release of neurotransmitters at synapses is mediated by the fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane. Continuous synaptic activity relies on the constant recycling of synaptic vesicle proteins into newly formed synaptic vesicles. At least two different mechanisms are presumed to mediate synaptic vesicle biogenesis at the synapse as follows: direct retrieval of synaptic vesicle proteins and lipids from the plasma membrane, and indirect passage of synaptic vesicle proteins through an endosomal intermediate. We have identified a vesicle population with the characteristics of a primary endocytic vesicle responsible for the recycling of synaptic vesicle proteins through the indirect pathway. We find that synaptic vesicle proteins colocalize in this vesicle with a variety of proteins known to recycle from the plasma membrane through the endocytic pathway, including three different glucose transporters, GLUT1, GLUT3, and GLUT4, and the transferrin receptor. These vesicles differ from "classical" synaptic vesicles in their size and their generic protein content, indicating that they do not discriminate between synaptic vesicle-specific proteins and other recycling proteins. We propose that these vesicles deliver synaptic vesicle proteins that have escaped internalization by the direct pathway to endosomes, where they are sorted from other recycling proteins and packaged into synaptic vesicles.     

Proteomic study of the mucin granulae in an intestinal goblet cell model

Goblet cells specialize in producing and secreting mucus with its main component, mucins. An inducible goblet-like cell line was used for the purification of the mucus vesicles stored in these cells by density gradient ultracentrifugation, and their proteome was analyzed by nanoLC-MS and MS/MS. Although the density of these vesicles coincides with others, it was possible to reveal a number of proteins that after immunolocalization on colon tissue and functional analyses were likely to be linked to the MUC2 vesicles. Most of the proteins were associated with the vesicle membrane or their outer surface. The ATP6AP2, previously suggested to be associated with vesicular proton pumps, was colocalized with MUC2 without other V-ATPase proteins and, thus, probably has roles in mucin vesicle function yet to be discovered. FAM62B, known to be a calcium-sensitive protein involved in vesicle fusion, also colocalized with the MUC2 vesicles and is probably involved in unknown ways in the later events of the MUC2 vesicles and their secretion.     

Vectorial proteomics

Vectorial proteomics is a methodology for the differential identification and characterization of proteins and their domains exposed to the opposite sides of biological membranes. Proteomics of membrane vesicles from defined isolated membranes automatically determine cellular localization of the identified proteins and reduce complexity of protein characterizations. The enzymatic shaving of naturally-oriented, or specifically-inverted sealed membrane vesicles, release the surface-exposed peptides from membrane proteins. These soluble peptides are amenable to various chromatographic separations and to sequencing by mass spectrometry, which provides information on the topology of membrane proteins and on their posttranslational modifications. The membrane shaving techniques have made a breakthrough in the identification of in vivo protein phosphorylation sites in membrane proteins form plant photosynthetic and plasma membranes, and from caveolae membrane vesicles of human fat cells. This approach has also allowed investigation of dynamics for in vivo protein phosphorylation in membranes from cells exposed to different conditions. Vectorial proteomics of membrane vesicles with retained peripheral proteins identify extrinsic proteins associated with distinct membrane surfaces, as well as a variety of posttranslational modifications in these proteins. The rapid integration of versatile vectorial proteomics techniques in the functional characterization of biological membranes is anticipated to bring significant insights in cell biology.     

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。     

NgCAM and VAMP2 reveal that direct delivery and dendritic degradation maintain axonal polarity

Neurons are polarized cells of extreme scale and compartmentalization. To fulfill their role in electrochemical signaling, axons must maintain a specific complement of membrane proteins. Despite being the subject of considerable attention, the trafficking pathway of axonal membrane proteins is not well understood. Two pathways, direct delivery and transcytosis, have been proposed. Previous studies reached contradictory conclusions about which of these mediates delivery of axonal membrane proteins to their destination, in part because they evaluated long-term distribution changes and not vesicle transport. We developed a novel strategy to selectively label vesicles in different trafficking pathways and determined the trafficking of two canonical axonal membrane proteins, neuron–glia cell adhesion molecule and vesicle-associated membrane protein-2. Results from detailed quantitative analyses of transporting vesicles differed substantially from previous studies and found that axonal membrane proteins overwhelmingly undergo direct delivery. Transcytosis plays only a minor role in axonal delivery of these proteins. In addition, we identified a novel pathway by which wayward axonal proteins that reach the dendritic plasma membrane are targeted to lysosomes. These results redefine how axonal proteins achieve their polarized distribution, a crucial requirement for elucidating the underlying molecular mechanisms.     

Nuclear membrane vesicle targeting to chromatin in a Drosophila embryo cell-free system.

A Drosophila cell-free system was used to characterize proteins that are required for targeting vesicles to chromatin and for fusion of vesicles to form nuclear envelopes. Treatment of vesicles with 1 M NaCl abolished their ability to bind to chromatin. Binding of salt-treated vesicles to chromatin could be restored by adding the dialyzed salt extract. Lamin Dm is one of the peripheral proteins whose activity was required, since supplying interphase lamin isoforms Dm1, and Dm2 to the assembly extract restored binding. As opposed to the findings in Xenopus, okadaic acid had no effect on vesicle binding. Trypsin digestion of the salt-stripped vesicles eliminated their association with chromatin even in the presence of the dialyzed salt extract. One vesicles attached to chromatin surface, fusion events took place were found to be sensitive to guanosine 5'-[gamma-thio]triphosphate (GTP gamma S). These chromatin-attached vesicles contained lamin Dm and otefin but not gp210. Thus, these results show that in Drosophila there are two populations of nuclear vesicles. The population that interacts first with chromatin contains lamin and otefin and requires both peripheral and integral membrane proteins, whereas fusion of vesicles requires GTPase activity.     

Bakterielle Membranvesikel

Bacterial membrane vesicles Cells of all three domains of our life (eukaryotes, bacteria and archaea) produce and segregate membrane vesicles surrounded by a lipid double membrane. Most of them are spherical with a diameter of 20–500 nm and can contain in their interior, the lumen, different types of molecules called cargo. In most cases they contain different proteins, polysaccharides and metabolites and sometimes nucleic acids (DNA, RNA) as well as misfolded proteins. Membrane vesicles play an important role in the horizontal gene transfer and in pathogenesis. Furthermore, it has been shown quite recently that membrane vesicles can transfer phage receptors to phage resistant cells and even closely related species. Worldwide several companies investigate their application as vaccines. In addition, investigations are going on to find out whether membrane vesicles can be used in genomic engineering.     

The effect of the inhibitor diphenylene iodonium on the superoxide-generating system of neutrophils. Specific labelling of a component polypeptide of the oxidase.

Horse kidney brush border membrane proteins were incorporated into phosphatidylcholine vesicles. Structural analysis of proteoliposomes prepared with various lipid:protein ratios showed that: (a) only a few of the proteins present in the crude brush border extract are integrated, (b) all known membrane hydrolases are integrated, and (c) these proteoliposomes are homogeneous vesicles. Papain solubilization of brush border membrane hydrolases, i.e. aminopeptidase M, neutral alpha-glucosidase, gamma-glutamyltransferase and alkaline phosphatase, performed in parallel on native membrane vesicles and proteoliposomes, revealed similar kinetics. Analysis of membrane vesicles and proteoliposomes on sucrose density gradients either without any treatment, or after papain treatment showed that: (a) in proteoliposomes, neutral alpha-glucosidase is associated with radiolabelled phosphatidylcholine, and (b) papain-treated vesicles and proteoliposomes released enzyme activity in the same way. These results suggest that the integration mechanism of brush border membrane proteins may be similar in proteoliposomes and native membrane vesicles. Transport experiments under equilibrium exchange conditions showed that the uptake properties of proteoliposomes are similar to those of brush border membrane vesicles.     

Wolbachia bacteria reside in host Golgi-related vesicles whose position is regulated by polarity proteins

Wolbachia pipientis are intracellular symbiotic bacteria extremely common in various organisms including Drosophila melanogaster, and are known for their ability to induce changes in host reproduction. These bacteria are present in astral microtubule-associated vesicular structures in host cytoplasm, but little is known about the identity of these vesicles. We report here that Wolbachia are restricted only to a group of Golgi-related vesicles concentrated near the site of membrane biogenesis and minus-ends of microtubules. The Wolbachia vesicles were significantly mislocalized in mutant embryos defective in cell/planar polarity genes suggesting that cell/tissue polarity genes are required for apical localization of these Golgi-related vesicles. Furthermore, two of the polarity proteins, Van Gogh/Strabismus and Scribble, appeared to be present in these Golgi-related vesicles. Thus, establishment of polarity may be closely linked to the precise insertion of Golgi vesicles into the new membrane addition site.     

Isolation and characterization of large (0.5 - 1.0 micron) cytoskeleton-free vesicles from human and rabbit erythrocytes

Large (0.5 - 1.0 micron) cytoskeleton-free vesicles were obtained, by 'budding', from fresh human and rabbit erythrocytes incubated at 45 degrees C and titrated with EDTA and CaCl2. This process occurs without hemolysis. The isolated vesicles maintain their cytoplasmic integrity and normal membrane orientation, and are resistant to hemolysis over the pH range 5.0 - 11.0 and temperature range 4-50 degrees C. The only membrane proteins detected in vesicles from human erythrocytes were band 3 region polypeptides and bands PAS-1, PAS-2 and PAS-3. Vesicles obtained from rabbit erythrocytes were similarly simple. Because of their size and stability these vesicles are amenable to both kinetic and quantitative analysis using the same experimental techniques employed in studies of synthetic lipid membranes. The results obtained in this study indicate that these vesicles are essentially markedly simplified biological cells, and thus may be useful as a biologically relevant model membrane system for examining the molecular interactions which occur within, across and between cell membranes.     

Molecular assemblies and membrane domains in multivesicular endosome dynamics

Along the degradation pathway, endosomes exhibit a characteristic multivesicular organization, resulting from the budding of vesicles into the endosomal lumen. After endocytosis and transport to early endosomes, activated signaling receptors are incorporated into these intralumenal vesicles through the action of the ESCRT machinery, a process that contributes to terminate signaling. Then, the vesicles and their protein cargo are further transported towards lysosomes for degradation. Evidence also shows that intralumenal vesicles can undergo “back-fusion” with the late endosome limiting membrane, a route exploited by some pathogens and presumably followed by proteins and lipids that need to be recycled from within the endosomal lumen. This process depends on the late endosomal lipid lysobisphosphatidic acid and its putative effector Alix/AIP1, and is presumably coupled to the invagination of the endosomal limiting membrane at the molecular level via ESCRT proteins. In this review, we discuss the intra-endosomal transport routes in mammalian cells, and in particular the different mechanisms involved in membrane invagination, vesicle formation and fusion in a space inaccessible to proteins known to control intracellular membrane traffic.     

A budding and fusing journey through the secretory pathway. Genetic and In Vitro Analysis of Cell Compartmentalization: A UCLA symposium, Taos, NM, USA, February 3-9, 1990

Reconstitution of vesicular transport events and the molecular and genetic analysis of the secretory pathway have taken the field of membrane traffic into a new era. Already, proteins have been discovered that facilitate multiple transport steps, and studies of the identities and modes of action of additional transport components, such as those that specify the targets of transport vesicles, will soon follow. Even after we understand how transport vesicles form, find their targets, and then fuse, other fundamental questions will still remain. How are proteins sorted into distinct transport vesicles? How is the directionality of protein transport achieved? How do organelles maintain their identities in the face of large volumes of membrane traffic? Finally, how is membrane traffic regulated? Answers to each of these fundamental questions are likely to be available in the not-too-distant future.     

Signals involved in targeting membrane proteins to synaptic vesicles

1. Synaptic vesicles (SVs) mediate fast regulated secretion of classical neurotransmitters. In order to perform their task SVs rely on a restrict set of membrane proteins. The mechanisms responsible for targeting these proteins to the SV membrane are still poorly understood.2. Likewise, little is known about the intracellular routes taken by these proteins in their way to SV membrane. Recently, several domains and motifs necessary for correct localization of SV proteins have been identified.3. In this review we summarize the sequence motifs that have been identified in the cytoplasmic domains of SV proteins that are involved in endocytosis and targeting of SVs. We suggest that the vesicular acetylcholine transporter, a protein found predominantly in synaptic vesicles, is perhaps a model protein to understand the pathways and interactions that are used for synaptic vesicle targeting.     

Hen oviduct signal peptidase is an integral membrane protein

Membrane preparations from rough endoplasmic reticulum of hen oviduct resemble those of dog pancreas in their capacity to translocate nascent secretory proteins into membrane vesicles present during cell-free protein synthesis. As with the dog membranes, the precursor form of human placental lactogen is transported into the vesicles and processed to the native secretory form by an associated "signal peptidase." The oviduct microsomal membranes glycosylate nascent ovomucoid and ovalbumin in vitro. Attempts to extract the signal peptidase from these membrane vesicles revealed that it is one of the least easily solubilized proteins. A protocol for enrichment of signal peptidase was developed that took advantage of its tight association with these vesicles. These studies indicate that the enzyme has the characteristics of an integral membrane protein which remains active in membrane vesicles even after extraction with low concentrations of detergent that do not dissolve the lipid bilayer or after disruption of membrane vesicles in ice-cold 0.1 M Na2CO3, pH 11.5 (Fujiki, Y., Hubbard, A. L., Fowler, S., and Lazarow, P.B. (1982) J. Cell Biol. 93, 97-102), which releases the majority of membrane-associated proteins. Solubilization requires concentrations of nondenaturing detergents that totally dissolve the lipid bilayer. The detergent-solubilized enzyme retains the activity and the characteristic specificity of the membrane-bound form.