The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast.
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Attachment of ubiquitin to cellular proteins frequently targets them to the 26S proteasome for degradation. In addition, ubiquitination of cell surface proteins stimulates their endocytosis and eventual degradation in the vacuole or lysosome. In the yeast Saccharomyces cerevisiae, ubiquitin is a long-lived protein, so it must be efficiently recycled from the proteolytic intermediates to which it becomes linked. We identified previously a yeast deubiquitinating enzyme, Doa4, that plays a central role in ubiquitin-dependent proteolysis by the proteasome. Biochemical and genetic data suggest that Doa4 action is closely linked to that of the proteasome. Here we provide evidence that Doa4 is required for recycling ubiquitin from ubiquitinated substrates targeted to the proteasome and, surprisingly, to the vacuole as well. In the doa4Delta mutant, ubiquitin is strongly depleted under certain conditions, most notably as cells approach stationary phase. Ubiquitin depletion precedes a striking loss of cell viability in stationary phase doa4Delta cells. This loss of viability and several other defects of doa4Delta cells are rescued by provision of additional ubiquitin. Ubiquitin becomes depleted in the mutant because it is degraded much more rapidly than in wild-type cells. Aberrant ubiquitin degradation can be partially suppressed by mutation of the proteasome or by inactivation of vacuolar proteolysis or endocytosis. We propose that Doa4 helps recycle ubiquitin from both proteasome-bound ubiquitinated intermediates and membrane proteins destined for destruction in the vacuole. (+info)
Tetanus toxin blocks the exocytosis of synaptic vesicles clustered at synapses but not of synaptic vesicles in isolated axons.
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Recycling synaptic vesicles are already present in isolated axons of developing neurons (Matteoli et al., Zakharenko et al., 1999). This vesicle recycling is distinct from the vesicular traffic implicated in axon outgrowth. Formation of synaptic contacts coincides with a clustering of synaptic vesicles at the contact site and with a downregulation of their basal rate of exo-endocytosis (Kraszewski et al, 1995; Coco et al., 1998) We report here that tetanus toxin-mediated cleavage of synaptobrevin/vesicle-associated membrane protein (VAMP2), previously shown not to affect axon outgrowth, also does not inhibit synaptic vesicle exocytosis in isolated axons, despite its potent blocking effect on their exocytosis at synapses. This differential effect of tetanus toxin could be seen even on different branches of a same neuron. In contrast, botulinum toxins A and E [which cleave synaptosome-associated protein of 25 kDa. (SNAP-25)] and F (which cleaves synaptobrevin/VAMP1 and 2) blocked synaptic vesicle exocytosis both in isolated axons and at synapses, strongly suggesting that this process is dependent on "classical" synaptic SNAP receptor (SNARE) complexes both before and after synaptogenesis. A tetanus toxin-resistant form of synaptic vesicle recycling, which proceeds in the absence of external stimuli and is sensitive to botulinum toxin F, E, and A, persists at mature synapses. These data suggest the involvement of a tetanus toxin-resistant, but botulinum F-sensitive, isoform of synaptobrevin/VAMP in synaptic vesicle exocytosis before synapse formation and the partial persistence of this form of exocytosis at mature synaptic contacts. (+info)
Presence of a complex containing vesicle-associated membrane protein 2 in rat parotid acinar cells and its disassembly upon activation of cAMP-dependent protein kinase.
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Amylase release from parotid acinar cells is mainly induced by the accumulation of intracellular cAMP, presumably through the phosphorylation of substrates by cAMP-dependent protein kinase (PKA). However, the molecular mechanisms of this process are not clear. In a previous study (Fujita-Yoshigaki, J., Dohke, Y., Hara-Yokoyama, M., Kamata, Y., Kozaki, S., Furuyama, S., and Sugiya, H. (1996) J. Biol. Chem. 271, 13130-13134), we reported that vesicle-associated membrane protein 2 (VAMP2) is localized at the secretory granule membrane and is involved in cAMP-induced amylase secretion. To study the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex containing VAMP2 in parotid acinar cells, we prepared rabbit polyclonal antibody against the peptide corresponding to Arg(47)-Asp(64) of VAMP2 (anti-SER4256). The recognition site of anti-SER4256 overlaps the domain involved in binding target membrane SNAREs (t-SNARES). Then we examined the condition of VAMP2 by immunoprecipitation with anti-SER4256. VAMP2 was not included in the immunoprecipitate from solubilized granule membrane fraction under the control conditions, but incubation with cytosolic fraction and cAMP caused immunoprecipitation of VAMP2. The effect of cytosolic fraction and cAMP was reduced by addition of PKA inhibitor H89. Addition of both the catalytic subunit of PKA and the cytosolic fraction allowed immunoprecipitation of VAMP2, whereas the PKA catalytic subunit alone did not. These results suggest that () the t-SNARE binding region of VAMP2 is masked by some protein X and activation of PKA caused the dissociation of X from VAMP2; and () the effect of PKA is not direct phosphorylation of X, but works through phosphorylation of some other cytosolic protein. (+info)
A critical role for N-ethylmaleimide-sensitive fusion protein (NSF) in platelet granule secretion.
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The molecular mechanisms that regulate membrane targeting/fusion during platelet granule secretion are not yet understood. N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAREs (SNAP receptors) are elements of a conserved molecular machinery for membrane targeting/fusion that have been detected in platelets. We examined whether NSF, an ATPase that has been shown to play a critical role in membrane targeting/fusion in many cell types, is necessary for platelet granule secretion. Peptides that mimic NSF sequence motifs inhibited both alpha-granule and dense-granule secretion in permeabilized human platelets. This inhibitory effect was sequence-specific, because neither proteinase K-digested peptides nor peptides containing similar amino acids in a scrambled sequence inhibited platelet secretion. The peptides that inhibited platelet granule secretion also inhibited the human recombinant alpha-SNAP-stimulated ATPase activity of recombinant NSF. It was also found that anti-NSF antibodies, which inhibited recombinant alpha-SNAP-stimulated ATPase activity of NSF, inhibited platelet granule secretion in permeabilized cells. The inhibition by anti-NSF antibodies was abolished by the addition of recombinant NSF. These data provide the first functional evidence that NSF plays an important role in platelet granule secretion. (+info)
Crystal structure of the vesicular transport protein Sec17: implications for SNAP function in SNARE complex disassembly.
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SNAP proteins play an essential role in membrane trafficking in eukaryotic cells. They activate and recycle SNARE proteins by serving as adaptors between SNAREs and the cytosolic chaperone NSF. We have determined the crystal structure of Sec17, the yeast homolog of alpha-SNAP, to 2.9 A resolution. Sec17 is composed of an N-terminal twisted sheet of alpha-helical hairpins and a C-terminal alpha-helical bundle. The N-terminal sheet has local similarity to the tetratricopeptide repeats from protein phosphatase 5 but has a different overall twist. Sec17 also shares structural features with HEAT and clathrin heavy chain repeats. Possible models of SNAP:SNARE binding suggest that SNAPs may function as lever arms, transmitting forces generated by conformational changes in NSF/Sec18 to drive disassembly of SNARE complexes. (+info)
NSF N-terminal domain crystal structure: models of NSF function.
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N-ethylmaleimide-sensitive factor (NSF) is a hexameric ATPase essential for eukaryotic vesicle fusion. Along with SNAP proteins, it disassembles cis-SNARE complexes upon ATP hydrolysis, preparing SNAREs for trans complex formation. We have determined the crystal structure of the N-terminal domain of NSF (N) to 1.9 A resolution. N contains two subdomains which form a groove that is a likely SNAP interaction site. Unexpectedly, both N subdomains are structurally similar to domains in EF-Tu. Based on this similarity, we propose a model for a large conformational change in NSF that drives SNARE complex disassembly. (+info)
A conformational switch in syntaxin during exocytosis: role of munc18.
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Syntaxin 1, an essential protein in synaptic membrane fusion, contains a helical autonomously folded N-terminal domain, a C-terminal SNARE motif and a transmembrane region. The SNARE motif binds to synaptobrevin and SNAP-25 to assemble the core complex, whereas almost the entire cytoplasmic sequence participates in a complex with munc18-1, a neuronal Sec1 homolog. We now demonstrate by NMR spectroscopy that, in isolation, syntaxin adopts a 'closed' conformation. This default conformation of syntaxin is incompatible with core complex assembly which requires an 'open' syntaxin conformation. Using site-directed mutagenesis, we find that disruption of the closed conformation abolishes the ability of syntaxin to bind to munc18-1 and to inhibit secretion in PC12 cells. These results indicate that syntaxin binds to munc18-1 in a closed conformation and suggest that this conformation represents an essential intermediate in exocytosis. Our data suggest a model whereby, during exocytosis, syntaxin undergoes a large conformational switch that mediates the transition between the syntaxin-munc18-1 complex and the core complex. (+info)
Yeast epsins contain an essential N-terminal ENTH domain, bind clathrin and are required for endocytosis.
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The mammalian protein epsin is required for endocytosis. In this study, we have characterized two homologous yeast proteins, Ent1p and Ent2p, which are similar to mammalian epsin. An essential function for the highly conserved N-terminal epsin N-terminal homology (ENTH) domain was revealed using deletions and randomly generated temperature-sensitive ent1 alleles. Changes in conserved ENTH domain residues in ent1(ts) cells revealed defects in endocytosis and actin cytoskeleton structure. The Ent1 protein was localized to peripheral and internal punctate structures, and biochemical fractionation studies found the protein associated with a large, Triton X-100-insoluble pellet. Finally, an Ent1p clathrin-binding domain was mapped to the final eight amino acids (RGYTLIDL*) in the Ent1 protein sequence. Based on these and other data, we propose that the yeast epsin-like proteins are essential components of an endocytic complex that may act at multiple stages in the endocytic pathway. (+info)