Phosphorylation of the Saccharomyces cerevisiae La protein does not appear to be required for its functions in tRNA maturation and nascent RNA stabilization.

An abundant nuclear phosphoprotein, the La autoantigen, is the first protein to bind all newly synthesized RNA polymerase III transcripts. Binding by the La protein to the 3' ends of these RNAs stabilizes the nascent transcripts from exonucleolytic degradation. In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the La protein is required for the normal pathway of tRNA maturation. Experiments in which the human protein was expressed in S. pombe have suggested that phosphorylation of the La protein regulates tRNA maturation. To dissect the role of phosphorylation in La protein function, we used mass spectrometry to identify three sites of serine phosphorylation in the S. cerevisiae La protein Lhp1p. Mutant versions of Lhp1p, in which each of the serines was mutated to alanine, were expressed in yeast cells lacking Lhp1p. Using two-dimensional gel electrophoresis, we determined that we had identified and mutated all major sites of phosphorylation in Lhp1p. Lhp1p lacking all three phosphorylation sites was functional in several yeast strains that require Lhp1p for growth. Northern blotting revealed no effects of Lhp1p phosphorylation status on either pre-tRNA maturation or stabilization of nascent RNAs. Both wild-type and mutant Lhp1 proteins localized to both nucleoplasm and nucleoli, demonstrating that phosphorylation does not affect subcellular location. Thus, although La proteins from yeast to humans are phosphoproteins, phosphorylation does not appear to be required for any of the identified functions of the S. cerevisiae protein.

The role of the COOH terminus of Sec2p in the transport of post-Golgi vesicles.

Sec2p is required for the polarized transport of secretory vesicles in S. cerevisiae. The Sec2p NH(2) terminus encodes an exchange factor for the Rab protein Sec4p. Sec2p associates with vesicles and in Sec2p COOH-terminal mutants Sec4p and vesicles no longer accumulate at bud tips. Thus, the Sec2p COOH terminus functions in targeting vesicles, however, the mechanism of function is unknown. We found comparable exchange activity for truncated and full-length Sec2 proteins, implying that the COOH terminus does not alter the exchange rate. Full-length Sec2-GFP, similar to Sec4p, concentrates at bud tips. A COOH-terminal 58-amino acid domain is necessary but not sufficient for localization. Sec2p localization depends on actin, Myo2p and Sec1p, Sec6p, and Sec9p function. Full-length, but not COOH-terminally truncated Sec2 proteins are enriched on membranes. Membrane association of full-length Sec2p is reduced in sec6-4 and sec9-4 backgrounds at 37 degrees C but unaffected at 25 degrees C. Taken together, these data correlate loss of localization of Sec2 proteins with reduced membrane association. In addition, Sec2p membrane attachment is substantially Sec4p independent, supporting the notion that Sec2p interacts with membranes via an unidentified Sec2p receptor, which would increase the accessibility of Sec2p exchange activity for Sec4p.

The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis.

Polarized secretion requires proper targeting of secretory vesicles to specific sites on the plasma membrane. Here we report that the exocyst complex plays a key role in vesicle targeting. Sec15p, an exocyst component, can associate with secretory vesicles and interact specifically with the rab GTPase, Sec4p, in its GTP-bound form. A chain of protein-protein interactions leads from Sec4p and Sec15p on the vesicle, through various subunits of the exocyst, to Sec3p, which marks the sites of exocytosis on the plasma membrane. Sec4p may control the assembly of the exocyst. The exocyst may therefore function as a rab effector system for targeted secretion.

Sec2p mediates nucleotide exchange on Sec4p and is involved in polarized delivery of post-Golgi vesicles.

The small GTPase Sec4p is required for vesicular transport at the post-Golgi stage of yeast secretion. Here we present evidence that mutations in SEC2, itself an essential gene that acts at the same stage of the secretory pathway, cause Sec4p to mislocalize as a result of a random rather than a polarized accumulation of vesicles. Sec2p and Sec4p interact directly, with the nucleotide-free conformation of Sec4p being the preferred state for interaction with Sec2p. Sec2p functions as an exchange protein, catalyzing the dissociation of GDP from Sec4 and promoting the binding of GTP. We propose that Sec2p functions to couple the activation of Sec4p to the polarized delivery of vesicles to the site of exocytosis.

The t-SNAREs syntaxin 1 and SNAP-25 are present on organelles that participate in synaptic vesicle recycling.

Syntaxin 1 and synaptosome-associated protein of 25 kD (SNAP-25) are neuronal plasmalemma proteins that appear to be essential for exocytosis of synaptic vesicles (SVs). Both proteins form a complex with synaptobrevin, an intrinsic membrane protein of SVs. This binding is thought to be responsible for vesicle docking and apparently precedes membrane fusion. According to the current concept, syntaxin 1 and SNAP-25 are members of larger protein families, collectively designated as target-SNAP receptors (t-SNAREs), whose specific localization to subcellular membranes define where transport vesicles bind and fuse. Here we demonstrate that major pools of syntaxin 1 and SNAP-25 recycle with SVs. Both proteins cofractionate with SVs and clathrin-coated vesicles upon subcellular fractionation. Using recombinant proteins as standards for quantitation, we found that syntaxin 1 and SNAP-25 each comprise approximately 3% of the total protein in highly purified SVs. Thus, both proteins are significant components of SVs although less abundant than synaptobrevin (8.7% of the total protein). Immunoisolation of vesicles using synaptophysin and syntaxin specific antibodies revealed that most SVs contain syntaxin 1. The widespread distribution of both syntaxin 1 and SNAP-25 on SVs was further confirmed by immunogold electron microscopy. Botulinum neurotoxin C1, a toxin that blocks exocytosis by proteolyzing syntaxin 1, preferentially cleaves vesicular syntaxin 1. We conclude that t-SNAREs participate in SV recycling in what may be functionally distinct forms.

Localization of Rab5 to synaptic vesicles identifies endosomal intermediate in synaptic vesicle recycling pathway.

After exocytosis, synaptic vesicles rapidly endocytose and recycle but little is known about the molecular mechanisms involved. Rab5 is a ubiquitous low molecular weight GTP-binding protein required for endosomal fusion in fibroblasts. We have now raised polyclonal and monoclonal antibodies to rat Rab5 and show that in rat brain, Rab5 is a major synaptic vesicle protein. Immunoisolation of vesicular organelles from brain with antibodies to either Rab3A and Rab5 as small GTP-binding proteins or with synaptophysin as general synaptic vesicle marker demonstrates that there are overlapping populations of synaptic vesicles containing either Rab5 or Rab3A or both, suggesting a stage-specific association of these low-molecular weight GTP-binding proteins with synaptic vesicles. Our data provide the first biochemical evidence that synaptic vesicle recycling involves an endosomal intermediate similar to that of the receptor-mediated endocytosis pathway.

GTP cleavage by the small GTP-binding protein Rab3A is associated with exocytosis of synaptic vesicles induced by alpha-latrotoxin.

Neurotransmitter release from presynaptic nerve terminals is a highly regulated form of exocytosis. Small GTP-binding proteins of the Rab family have been proposed to act as central regulators in this process that cycle between a GTP- and GDP-bound form. Previous work has shown that the synaptic vesicle protein Rab3A undergoes a membrane association-dissociation cycle that is associated with neurotransmitter release. Using isolated nerve terminals as our model system, we have now analyzed the GDP/GTP status of Rab3A. Synaptic vesicle-bound Rab3A was almost exclusively in the GTP form whereas cytosolic Rab3A contained only GDP. Approximately equal amounts of GTP and GDP were found in the pool of Rab3A localized to a membrane fraction containing plasma membrane-synaptic vesicle complexes. In contrast to Rab3A, Rab5 (an endosomal G-protein) was predominantly GDP-bound in all analyzed compartments. To analyze whether Rab3A-bound GTP is cleaved during exocytosis, synaptosomes were stimulated with alpha-latrotoxin, the active component of black widow spider venom. This resulted in massive exocytosis. A significant increase of the GDP/GTP ratio of Rab3A was observed under these conditions that was not due to a nonspecific loss of high energy nucleotides. Our findings suggest that cleavage of Rab3A-bound GTP is a crucial step in regulated exocytosis of synaptic vesicles.

Relative properties and localizations of synaptic vesicle protein isoforms: the case of the synaptophysins.

Synaptophysins are abundant synaptic vesicle proteins present in two forms: synaptophysin, also referred to as synaptophysin I (abbreviated syp I), and synaptoporin, also referred to as synaptophysin II (abbreviated syp II). In the present study, the properties and localizations of syp I and syp II were investigated to shed light on their relative functions. Our results reveal that syp II, similar to syp I, is an abundant, N-glycosylated membrane protein that is part of a heteromultimeric complex in synaptic vesicle membranes. Cross-linking studies indicate that syp II is linked to a low-molecular-weight protein in this complex as has been observed before for syp I. Furthermore, after transfection into CHO cells, syp II, similar to syp I, is targeted to the receptor-mediated endocytosis pathway. Immunocytochemistry of rat brain sections reveals that syp II expression is highly heterogeneous, with high concentrations of syp II only in selected neuronal populations, whereas syp I is more homogeneously expressed in most nerve terminals. In general, nerve terminals expressing syp II also express syp I. In addition to high levels of syp II observed in selected neurons, a rostrocaudal gradient of syp II expression was observed in the cerebellar cortex. Immunoelectron microscopy confirmed that syp II is localized to synaptic vesicles. Immunoprecipitations of synaptic vesicles from rat brain with antibodies to syp I demonstrated that syp II is colocalized with syp I on the same vesicles. However, after detergent solubilization, no coimmunoprecipitations of the two proteins were observed, suggesting that they are not complexed with each other although they are on the same vesicles. Together our results demonstrate that syp I and syp II have similar properties and are present on the same synaptic vesicles but do not coassemble. The presence of the two proteins in the same nerve terminal suggests that they have similar but nonidentical functions and that the relative abundance of the two proteins may contribute to the functional heterogeneity of nerve terminals.

Synaptotagmin: a membrane constituent of neuropeptide-containing large dense-core vesicles.

Synaptotagmin is known to be a major membrane protein of synaptic vesicles (SVs) in neurons. We have now used an immunoisolation procedure to demonstrate that synaptotagmin is also present in the membranes of peptide containing large dense-core vesicles (LDCVs) of rat hypothalamus and bovine posterior pituitary. Synaptotagmin bead-immunoisolated organelles from these tissues primarily consisted of SVs but contained occasionally larger structures reminiscent of LDCVs that were absent from vesicle populations immunoisolated with a synaptophysin antibody. Furthermore, the vesicles immunoisolated with synaptotagmin beads contained significant amounts of neuropeptide Y (NPY). In contrast, vesicles immunoisolated with synaptophysin beads did not contain detectable levels of NPY. Sucrose density gradient fractionation of postnuclear supernatants obtained from the bovine posterior pituitary resulted in a bimodal distribution of synaptotagmin, corresponding to the positions of both SVs and neurosecretory granules. A similar distribution was found for cytochrome b561 and the 116 kDa subunit of the vacuolar proton pump. In contrast, the SV proteins synaptophysin, SV2, and p29 were restricted to the SV-containing fractions. Immunoisolation of small and large vesicles from the sucrose gradient confirmed the differential distribution of synaptotagmin and synaptophysin in the two types of secretory vesicles in nerve endings of the posterior pituitary. We conclude that synaptotagmin is a constituent of both SVs and peptide-containing secretory vesicles in the nervous system. Since both types of organelles undergo Ca(2+)-dependent exocytosis, these findings support a general role of synaptotagmin as an exocytotic Ca2+ receptor.

Synaptic vesicle proteins in exocytosis: what do we know?

Synaptic release of neurotransmitters is a fast process that is mediated by Ca(2+)-dependent exocytosis of synaptic vesicles. Several abundant membrane proteins of synaptic vesicles have been characterized at the molecular level but their function in synaptic vesicle traffic is poorly understood. Recent evidence suggests that some of these proteins are involved in exocytotic membrane fusion.

A gamma-aminobutyric acid transporter driven by a proton pump is present in synaptic-like microvesicles of pancreatic beta cells.

A variety of peptide-secreting endocrine cells contain a population of recycling microvesicles that share several major membrane polypeptides with neuronal synaptic vesicles (SVs). The function of these synaptic-like microvesicles (SLMVs) remains to be elucidated. It was previously suggested that SLMVs of pancreatic beta cells may store and secrete gamma-aminobutyric acid (GABA). GABA, the major nonpeptide inhibitory neurotransmitter of the central nervous system, is stored in and secreted from SVs. GABA uptake into SVs is mediated by a transporter that is driven by a vacuolar proton ATPase. GABA is also present at high concentration in the endocrine pancreas where it is selectively localized in insulin-secreting beta cells, the core cells of pancreatic islets. GABA is not present in peripheral islet cells (mantle cells), represented primarily by glucagon-secreting alpha cells. In this study, an immunoisolation procedure was used to purify SLMVs from cell lines derived from mouse beta cells and alpha cells. SLMVs obtained from the beta-cell line, but not those obtained from the alpha-cell line, displayed a GABA-transport activity dependent upon a proton electrochemical gradient generated by a vacuolar proton ATPase. These data support the hypotheses that (i) SLMVs have a secretory function similar to that of SVs and (ii) beta-cell SLMVs are involved in the secretion of GABA, which in turn may have a paracrine function on mantle cells of the islet.