Thy-1 Is a Component Common to Multiple Populations of Synaptic Vesicles

Jeng, Cherng-Jye; McCarroll, Steven A.; Martin, Thomas F.J.; Floor, Erik; Adams, James K.; Krantz, David E.; Butz, Stefan; Edwards, Robert H.; Schweitzer, Erik S.

doi:10.1083/jcb.140.3.685

Cited by 50 publications

(46 citation statements)

References 90 publications

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“…However, Thy-1 does colocalize with the major peak of synaptophysin characteristic of synaptic vesicles, consistent with a previous report demonstrating that Thy-1 is present in synaptic vesicle preparations from rat brain and PC12 cells (30) (Fig. 9).…”

Section: Fig 5 Distributions Of Vesicle Populations Containing Endosupporting

confidence: 78%

“…Finally, Thy-1, an abundant cell-surface marker of neurons and PC12 cells (29,30), is not detectable in the GLUT1-containing fractions of our glycerol gradient. However, Thy-1 does colocalize with the major peak of synaptophysin characteristic of synaptic vesicles, consistent with a previous report demonstrating that Thy-1 is present in synaptic vesicle preparations from rat brain and PC12 cells (30) (Fig.…”

Section: Fig 5 Distributions Of Vesicle Populations Containing Endomentioning

confidence: 99%

“…Thy-1, a plasma membrane protein that is also targeted to synaptic vesicles, is not enriched in the GLUT1 vesicle population. A, Western blot illustrating the distribution of Thy-1, a glycophosphatidylinositol-linked protein that is found in both the plasma membrane and synaptic vesicles (30). PC12 cells that were transfected with GLUT1HA cDNA were homogenized and analyzed in glycerol velocity gradients for the relative distributions of GLUT1HA, Thy-1, and synaptophysin.…”

Section: Fig 5 Distributions Of Vesicle Populations Containing Endomentioning

confidence: 99%

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Evidence for a Primary Endocytic Vesicle Involved in Synaptic Vesicle Biogenesis

Provoda¹,

Waring

Buckley

2000

Journal of Biological Chemistry

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The regulated release of neurotransmitters at synapses is mediated by the fusion of neurotransmitterfilled 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.The biogenesis of synaptic vesicles is a complex orchestration of events culminating in a vesicle population responsible for the uptake, storage, and regulated secretion of neurotransmitters. Current models of synaptic vesicle biogenesis suggest that at least two pathways exist for the formation of new synaptic vesicles after exocytosis: directly from the plasma membrane and indirectly via an intermediate endosomal compartment (1, 2). In neurons, both a direct pathway, which is at the active zone, and a more distal indirect pathway have been demonstrated at synapses in the Drosophila shibire ts1 mutant (3). Studies of membrane recycling in hippocampal synapses using the fluorescent dye FM1-43 also suggest that synaptic vesicles formed by endocytosis can fuse with the plasma membrane directly, without passing through an endosomal compartment (4). In the PC12 neuroendocrine cell line, synaptic vesicles are also derived from two different pathways as follows: directly from the plasma membrane (5, 6) as well as from an endosomal intermediate (7)(8)(9)(10). Together these data form an emerging model of multiple pathways to recycle both synaptic vesicle proteins and synaptic vesicle membrane.Although presumably all synaptic vesicle proteins undergo endocytic trafficking, relatively little is known about the intermediate steps in the pathway to mature synaptic vesicles. In order to understand fully the mechanisms underlying synaptic vesicle biogenesis, it is critical that the synaptic vesicle protein trafficking pathways are fully elucidated. In neurons, synaptic vesicle proteins are presen...

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Section: Fig 5 Distributions Of Vesicle Populations Containing Endosupporting

confidence: 78%

Section: Fig 5 Distributions Of Vesicle Populations Containing Endomentioning

confidence: 99%

Section: Fig 5 Distributions Of Vesicle Populations Containing Endomentioning

confidence: 99%

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Evidence for a Primary Endocytic Vesicle Involved in Synaptic Vesicle Biogenesis

Provoda¹,

Waring

Buckley

2000

Journal of Biological Chemistry

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“…Among the polypeptides identified as DRM or DRM-associated proteins we found a variety of bona fide SV proteins including synaptobrevin 2, synaptotagmin 1, synaptophysin, synapsins, SV2a, Rab3A, and the V-ATPase, some of which were identified based on as many as 36 peptides in total (Table I, top). The glycosylphosphatidylinositol-linked "raft" marker protein Thy-1 identified here as a component of synaptic DRMs has also been demonstrated to be a common component of SVs (32). In addition, we found factors involved in exocytic membrane fusion such as SNAP-25a, Munc18-1, or NSF and in SV recycling including clathrin (both heavy and light chains), the ␣ and ␤2 subunits of AP-2, neuronal AP-3B (2, 3), uncoating ATPase (Hsc70) (33) ).…”

Section: Quantitative Proteomics Analysis Of Drms Isolated Frommentioning

confidence: 99%

Quantitative Proteomics Analysis of Detergent-resistant Membranes from Chemical Synapses

Jia

Lamer

Schümann

et al. 2006

Molecular & Cellular Proteomics

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Synaptic vesicles (SVs) in the central nervous system upon stimulation undergo rapid calcium-triggered exoendocytic cycling within the nerve terminal that at least in part depends on components of the clathrin-and dynamindependent endocytosis machinery. How exocytic SV fusion and endocytic retrieval are temporally and spatially coordinated is still an open question. One possibility is that specialized membrane microdomains characterized by their high content in membrane cholesterol may assist in the spatial coordination of synaptic membrane protein recycling. Quantitative proteomics analysis of detergentresistant membranes (DRMs) isolated from rat brain synapses or cholesterol-depleted control samples by liquid chromatography-tandem mass spectrometry identified a total of 159 proteins. Among these 122 proteins were classified as cholesterol-dependent DRM or DRM-associated proteins, many of which with proven or hypothesized functions in exoendocytic vesicle cycling including clathrin, the clathrin adaptor complex AP-2, and a variety of SV proteins. In agreement with this, SV membrane and endocytic proteins displayed a partial resistance to extraction with cold Triton X-100 in cultured rat hippocampal neurons where they co-localized with labeled cholera toxin B, a marker for cholesterol-enriched DRMs. Moreover SV proteins formed cholesterol-dependent complexes in CHAPS-extracted synaptic membrane lysates. Our combined data suggest that lipid microdomains may act as spatial coordinators for exoendocytic vesicle cycling at synapses.

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“…Proteins were included for analysis 1) if represented by at least two peptides with a MASCOT score Ͼ45 or one peptide with a score Ͼ70 (defined from the antigen Thy1, entry 33; Supplemental Table 1, already known to be present in PC12 microvesicles; Jeng et al, 1998), and/or 2) if represented in two of the three spectrometry analyses. Peptides identified in a single MS run also were included if the corresponding proteins were part of a complex in which other subunits were identified in a distinct MS run (vacATPase, AP-3, synaptic vesicle proteins, and BLOCI).…”

Section: Mass Spectrometrymentioning

confidence: 99%

Phosphatidylinositol-4-Kinase Type II α Is a Component of Adaptor Protein-3-derived Vesicles

Salazar

Craige

Wainer

et al. 2005

MBoC

100

136

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A membrane fraction enriched in vesicles containing the adaptor protein (AP) -3 cargo zinc transporter 3 was generated from PC12 cells and was used to identify new components of these organelles by mass spectrometry. Proteins prominently represented in the fraction included AP-3 subunits, synaptic vesicle proteins, and lysosomal proteins known to be sorted in an AP-3-dependent way or to interact genetically with AP-3. A protein enriched in this fraction was phosphatidylinositol-4-kinase type II␣ (PI4KII␣). Biochemical, pharmacological, and morphological analyses supported the presence of PI4KII␣ in AP-3-positive organelles. Furthermore, the subcellular localization of PI4KII␣ was altered in cells from AP-3-deficient mocha mutant mice. The PI4KII␣ normally present both in perinuclear and peripheral organelles was substantially decreased in the peripheral membranes of AP-3-deficient mocha fibroblasts. In addition, as is the case for other proteins sorted in an AP-3-dependent way, PI4KII␣ content was strongly reduced in nerve terminals of mocha hippocampal mossy fibers. The functional relationship between AP-3 and PI4KII␣ was further explored by PI4KII␣ knockdown experiments. Reduction of the cellular content of PI4KII␣ strongly decreased the punctate distribution of AP-3 observed in PC12 cells. These results indicate that PI4KII␣ is present on AP-3 organelles where it regulates AP-3 function. INTRODUCTIONMembrane-enclosed organelles possess distinctive protein compositions that are dynamically maintained by inbound and outbound vesicle carriers. These vesicles selectively concentrate appropriate membrane proteins while leaving behind resident proteins found in the donor organelle, a process called sorting (Bonifacino and Glick, 2004). Central to membrane protein sorting and vesiculation are a family of cytosolic coat complexes that mediate vesicle budding and function as cargo-specific adaptors. These include monomeric proteins and the heterotetrameric adaptor proteins (AP)-1, -2, -3, and -4 (Bonifacino and Glick, 2004;Robinson, 2004). These coats participate in the generation of vesicles that carry a unique array of membrane protein "cargoes." The characterization of these carriers has played a major role in the functional dissection of coat-dependent sorting and vesiculation mechanisms. For example, vesicles "in a basket" isolated from brain led to the biochemical identification of the first sorting machinery, clathrin and the AP-1 and AP-2 adaptors (Kanaseki and Kadota, 1969;Pearse, 1975;Pfeffer and Kelly, 1981;Pearse and Crowther, 1987). Subsequent studies of cargo molecules in brain clathrin-coated vesicles were crucial in revealing mechanisms of synaptic vesicle recycling (Pfeffer and Kelly, 1985;Maycox et al., 1992;Blondeau et al., 2004). The advent of complete genome sequencing and the concomitant development of informatics tools has led to the rapid identification of proteins by mass spectrometry (Taylor et al., 2003). Application of these methodologies to clathrin-coated vesicles has allowed the identifi...

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Thy-1 Is a Component Common to Multiple Populations of Synaptic Vesicles

Cited by 50 publications

References 90 publications

Evidence for a Primary Endocytic Vesicle Involved in Synaptic Vesicle Biogenesis

Evidence for a Primary Endocytic Vesicle Involved in Synaptic Vesicle Biogenesis

Quantitative Proteomics Analysis of Detergent-resistant Membranes from Chemical Synapses

Phosphatidylinositol-4-Kinase Type II α Is a Component of Adaptor Protein-3-derived Vesicles

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