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1.
The Sec1/Munc18 protein, Vps33p, functions at the endosome and the vacuole of Saccharomyces cerevisiae
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The Sec1/Munc18 (SM) family of proteins is thought to impart compartmental specificity to vesicle fusion reactions. Here we report characterization of Vps33p, an SM family member previously thought to act exclusively at the vacuolar membrane with the vacuolar syntaxin Vam3p. Vacuolar morphology of vps33Delta cells resembles that of cells lacking both Vam3p and the endosomal syntaxin Pep12p, suggesting that Vps33p may function with these syntaxins at the vacuole and the endosome. Consistent with this, vps33 mutants secrete the Golgi precursor form of the vacuolar hydrolase CPY into the medium. We also demonstrate that Vps33p acts at other steps, for vps33 mutants show severe defects in endocytosis at the late endosome. At the endosome, Vps33p and other class C members exist as a complex with Vps8p, a protein previously known to act in transport between the late Golgi and the endosome. Vps33p also interacts with Pep12p, a known interactor of the SM protein Vps45p. High copy PEP7/VAC1 suppresses vacuolar morphology defects of vps33 mutants. These findings demonstrate that Vps33p functions at multiple trafficking steps and is not limited to action at the vacuolar membrane. This is the first report demonstrating the involvement of a single syntaxin with two SM proteins at the same organelle. 相似文献
2.
Cytoplasm to vacuole trafficking of aminopeptidase I requires a t-SNARE-Sec1p complex composed of Tlg2p and Vps45p.
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Aminopeptidase I (API) is imported into the yeast vacuole/lysosome by a constitutive non-classical vesicular transport mechanism, the cytoplasm to vacuole targeting (Cvt) pathway. Newly synthesized precursor API is sequestered in double-membrane cytoplasmic Cvt vesicles. The Cvt vesicles fuse with the vacuole, releasing single-membrane Cvt bodies containing proAPI into the vacuolar lumen, and maturation of API occurs when the Cvt body is degraded, releasing mature API. Under starvation conditions, API is transported to the vacuole by macroautophagy, an inducible, non-selective mechanism that shares many similarities with the Cvt pathway. Here we show that Tlg2p, a member of the syntaxin family of t-SNARE proteins, and Vps45p, a Sec1p homologue, are required in the constitutive Cvt pathway, but not in inducible macroautophagy. Fractionation and protease protection experiments indicate that Tlg2p is required prior to or at the step of API segregation into the Cvt vesicle. Thus, the early Vps45-Tlg2p-dependent step of the Cvt pathway appears to be mechanistically distinct from the comparable stage in macroautophagy. Vps45p associates with both the Tlg2p and Pep12p t-SNAREs, but API maturation is not blocked in a pep12(ts) mutant, indicating that Vps45p independently regulates the function of multiple t-SNARES at distinct trafficking steps. 相似文献
3.
Luv1p/Rki1p/Tcs3p/Vps54p, a yeast protein that localizes to the late Golgi and early endosome, is required for normal vacuolar morphology
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We have characterized LUV1/RKI1/TCS3/VPS54, a novel yeast gene required to maintain normal vacuolar morphology. The luv1 mutant was identified in a genetic screen for mutants requiring the phosphatase calcineurin for vegetative growth. luv1 mutants lack a morphologically intact vacuole and instead accumulate small vesicles that are acidified and contain the vacuolar proteins alkaline phosphatase and carboxypeptidase Y and the vacuolar membrane H(+)-ATPase. Endocytosis appears qualitatively normal in luv1 mutants, but some portion (28%) of carboxypeptidase Y is secreted. luv1 mutants are sensitive to several ions (Zn(2+), Mn(2+), and Cd(2+)) and to pH extremes. These mutants are also sensitive to hygromycin B, caffeine, and FK506, a specific inhibitor of calcineurin. Some vacuolar protein-sorting mutants display similar drug and ion sensitivities, including sensitivity to FK506. Luv1p sediments at 100,000 x g and can be solubilized by salt or carbonate, indicating that it is a peripheral membrane protein. A Green Fluorescent Protein-Luv1 fusion protein colocalizes with the dye FM 4-64 at the endosome, and hemagglutinin-tagged Luv1p colocalizes with the trans-Golgi network/endosomal protease Kex2p. Computer analysis predicts a short coiled-coil domain in Luv1p. We propose that this protein maintains traffic through or the integrity of the early endosome and that this function is required for proper vacuolar morphology. 相似文献
4.
Vps52p, Vps53p, and Vps54p form a novel multisubunit complex required for protein sorting at the yeast late Golgi
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The late Golgi of the yeast Saccharomyces cerevisiae receives membrane traffic from the secretory pathway as well as retrograde traffic from post-Golgi compartments, but the machinery that regulates these vesicle-docking and fusion events has not been characterized. We have identified three components of a novel protein complex that is required for protein sorting at the yeast late Golgi compartment. Mutation of VPS52, VPS53, or VPS54 results in the missorting of 70% of the vacuolar hydrolase carboxypeptidase Y as well as the mislocalization of late Golgi membrane proteins to the vacuole, whereas protein traffic through the early part of the Golgi complex is unaffected. A vps52/53/54 triple mutant strain is phenotypically indistinguishable from each of the single mutants, consistent with the model that all three are required for a common step in membrane transport. Native coimmunoprecipitation experiments indicate that Vps52p, Vps53p, and Vps54p are associated in a 1:1:1 complex that sediments as a single peak on sucrose velocity gradients. This complex, which exists both in a soluble pool and as a peripheral component of a membrane fraction, colocalizes with markers of the yeast late Golgi by immunofluorescence microscopy. Together, the phenotypic and biochemical data suggest that VPS52, VPS53, and VPS54 are required for the retrograde transport of Golgi membrane proteins from an endosomal/prevacuolar compartment. The Vps52/53/54 complex joins a growing list of distinct multisubunit complexes that regulate membrane-trafficking events. 相似文献
5.
Characterization of yeast Vps33p, a protein required for vacuolar protein sorting and vacuole biogenesis. 总被引:18,自引:2,他引:16
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vps33 mutants missort and secrete multiple vacuolar hydrolases and exhibit extreme defects in vacuolar morphology. Toward a molecular understanding of the role of the VPS33 gene in vacuole biogenesis, we have cloned this gene from a yeast genomic library by complementation of a temperature-sensitive vps33 mutation. Gene disruption demonstrated that VPS33 was not essential but was required for growth at high temperatures. At the permissive temperature, vps33 null mutants exhibited defects in vacuolar protein localization and vacuole morphology similar to those seen in most of the original mutant alleles. Sequence analysis revealed a putative open reading frame sufficient to encode a protein of 691 amino acids. Hydropathy analysis indicated that the deduced product of the VPS33 gene is generally hydrophilic, contains no obvious signal sequence or transmembrane domains, and is therefore unlikely to enter the secretory pathway. Polyclonal antisera raised against TrpE-Vps33 fusion proteins recognized a protein in yeast cells of the expected molecular weight, approximately 75,000. In cell fractionation studies, Vps33p behaved as a cytosolic protein. The predicted VPS33 gene product possessed sequence similarity with a number of ATPases and ATP-binding proteins specifically in their ATP-binding domains. One vps33 temperature-sensitive mutant contained a missense mutation near this region of sequence similarity; the mutation resulted in a Leu-646----Pro substitution in Vps33p. This temperature-sensitive mutant strain contained normal vacuoles at the permissive temperature but lacked vacuoles specifically in the bud at the nonpermissive temperature. Our data suggest that Vps33p acts in the cytoplasm to facilitate Golgi-to-vacuole protein delivery. We propose that as a consequence of the vps33 protein-sorting defects, abnormalities in vacuolar morphology and vacuole assembly result. 相似文献
6.
Vps4p is an AAA-type ATPase required for efficient transport of biosynthetic and endocytic cargo from an endosome to the lysosome-like vacuole of Saccharomyces cerevisiae. Vps4p mutants that do not bind ATP or are defective in ATP hydrolysis were characterized both in vivo and in vitro. The nucleotide-free or ADP-bound form of Vps4p existed as a dimer, whereas in the ATP-locked state, Vps4p dimers assembled into a decameric complex. This suggests that ATP hydrolysis drives a cycle of association and dissociation of Vps4p dimers/decamers. Nucleotide binding also regulated the association of Vps4p with an endosomal compartment in vivo. This membrane association required the N-terminal coiled-coil motif of Vps4p, but deletion of the coiled-coil domain did not affect ATPase activity or oligomeric assembly of the protein. Membrane association of two previously uncharacterized class E Vps proteins, Vps24p and Vps32p/Snf7p, was also affected by mutations in VPS4. Upon inactivation of a temperature-conditional vps4 mutant, Vps24p and Vps32p/Snf7p rapidly accumulated in a large membrane-bound complex. Immunofluorescence indicated that both proteins function with Vps4p at a common endosomal compartment. Together, the data suggest that the Vps4 ATPase catalyzes the release (uncoating) of an endosomal membrane-associated class E protein complex(es) required for normal morphology and sorting activity of the endosome. 相似文献
7.
AtRMR1 functions as a cargo receptor for protein trafficking to the protein storage vacuole
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Organellar proteins are sorted by cargo receptors on the way to their final destination. However, receptors for proteins that are destined for the protein storage vacuole (PSV) are largely unknown. In this study, we investigated the biological role that Arabidopsis thaliana receptor homology region transmembrane domain ring H2 motif protein (AtRMR) 1 plays in protein trafficking to the PSV. AtRMR1 mainly colocalized to the prevacuolar compartment of the PSV, but a minor portion also localized to the Golgi complex. The coexpression of AtRMR1 mutants that were localized to the Golgi complex strongly inhibited the trafficking of phaseolin to the PSV and caused accumulation of phaseolin in the Golgi complex or its secretion. Co-immunoprecipitation and in vitro binding assays revealed that the lumenal domain of AtRMR1 interacts with the COOH-terminal sorting signal of phaseolin at acidic pH. Furthermore, phaseolin colocalized with AtRMR1 on its way to the PSV. Based on these results, we propose that AtRMR1 functions as the sorting receptor of phaseolin for its trafficking to the PSV. 相似文献
8.
Vacuolar protein sorting (vps) mutants of Saccharomyces cerevisiae missort and secrete vacuolar hydrolases. The gene affected in one of these mutants, VPS21, encodes a member of the Sec4/Ypt/Rab family of small GTPases. Rab proteins play an essential role in vesicle-mediated protein transport. Using both yeast two-hybrid assays and chemical cross-linking, we have identified another VPS gene product, Vps9p, that preferentially interacts with a mutant form of Vps21p-S21N that binds GDP but not GTP. In vitro purified Vps9p was found to stimulate GDP release from Vps21p in a dose-dependent manner. Vps9p also stimulated GTP association as a result of facilitated GDP release. However, Vps9p did not stimulate guanine nucleotide exchange of GTP-bound Vps21p or GTP hydrolysis. We tested the ability of Vps9p to stimulate the intrinsic guanine nucleotide exchange activity of Rab5, which is a mammalian sequence homologue of Vps21p, and Ypt7p, which is another yeast Rab protein involved in vacuolar protein transport. Rab5, but not Ypt7p was responsive to Vps9p, which indicates that Vps9p recognizes sequence variation among Rab proteins. We conclude that Vps9p is a novel guanine nucleotide exchange factor that is specific for Vps21p/Rab5. Since there are no obvious Vps9p sequence homologues in yeast, Vps9p may also possess unique regulatory functions required for vacuolar protein transport. 相似文献
9.
Involvement of specific COPI subunits in protein sorting from the late endosome to the vacuole in yeast
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Although COPI function on the early secretory pathway in eukaryotes is well established, earlier studies also proposed a nonconventional role for this coat complex in endocytosis in mammalian cells. Here we present results that suggest an involvement for specific COPI subunits in the late steps of endosomal protein sorting in Saccharomyces cerevisiae. First, we found that carboxypeptidase Y (CPY) was partially missorted to the cell surface in certain mutants of the COPIB subcomplex (COPIb; Sec27, Sec28, and possibly Sec33), which indicates an impairment in endosomal transport. Second, integral membrane proteins destined for the vacuolar lumen (i.e., carboxypeptidase S [CPS1]; Fur4, Ste2, and Ste3) accumulated at an aberrant late endosomal compartment in these mutants. The observed phenotypes for COPIb mutants resemble those of class E vacuolar protein sorting (vps) mutants that are impaired in multivesicular body (MVB) protein sorting and biogenesis. Third, we observed physical interactions and colocalization between COPIb subunits and an MVB-associated protein, Vps27. Together, our findings suggest that certain COPI subunits could have a direct role in vacuolar protein sorting to the MVB compartment. 相似文献
10.
Trioulier Y Torch S Blot B Cristina N Chatellard-Causse C Verna JM Sadoul R 《The Journal of biological chemistry》2004,279(3):2046-2052
Alix/AIP1 is a cytoplasmic protein, which was first characterized as an interactor of ALG-2, a calcium-binding protein necessary for cell death. Alix has also recently been defined as a regulator of the endo-lysosomal system. Here we have used post-mitotic cerebellar neurons to test Alix function in caspase-dependent and -independent cell death. Indeed, these neurons survived when cultured in 25 mm potassium-containing medium but underwent apoptosis soon after the extracellular potassium was lowered to 5 mm. In agreement with other studies, we show that caspases are activated after K+ deprivation, but that inhibition of these proteases, using the pancaspase inhibitor boc-aspartyl(OMe)-fluoromethylketone, has no effect on cell survival. Transfection experiments demonstrated that Alix overexpression is sufficient to induce caspase activation, whereas overexpression of its C-terminal half, Alix-CT, blocks caspase activation and cell death after K+ deprivation. We also define a 12-amino acid PXY repeat of the C-terminal proline-rich domain necessary for binding ALG-2. Deletion of this domain in Alix or in Alix-CT abolished the effects of the overexpressed proteins on neuronal survival, demonstrating that the ALG-2-binding region is crucial for the death-modulating function of Alix. Overall, these findings define the Alix/ALG-2 complex as a regulator of cell death controlling both caspase-dependent and -independent pathways. They also suggest a molecular link between the endo-lysosomal system and the effectors of the cell death machinery. 相似文献
11.
Yeast functional analysis: identification of two essential genes involved in ER to Golgi trafficking 总被引:3,自引:0,他引:3
Belgareh-Touzé N Corral-Debrinski M Launhardt H Galan JM Munder T Le Panse S Haguenauer-Tsapis R 《Traffic (Copenhagen, Denmark)》2003,4(9):607-617
We screened for genes potentially involved in the secretory and vacuolar pathways a collection of 61 yeast strains, each bearing an essential orphan gene regulated by the tetO7 -CYC1 promoter that can be down-regulated by doxycycline. After down-regulating the expression of these genes, we performed systematic Western blot analysis for markers of the secretory and vacuolar pathways that undergo post-translational modifications in their intracellular trafficking. Accumulation of protein precursors, revealed by Western immunoblot analysis, indicates defects in the secretory pathway or in associated biochemical modifications. After screening the whole collection, we identified two genes involved in ER to Golgi trafficking: RER2 , a cis -prenyl transferase, and USE1 , the function of which was unknown . We demonstrated that repression of USE1 also leads to BiP secretion, and therefore likely affects retrograde, in addition to anterograde, ER to Golgi trafficking. The collection also includes two essential genes involved in intracellular trafficking that were conveniently repressed without resulting growth or trafficking defects. 相似文献
12.
Soi3p/Rav1p functions at the early endosome to regulate endocytic trafficking to the vacuole and localization of trans-Golgi network transmembrane proteins
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Sipos G Brickner JH Brace EJ Chen L Rambourg A Kepes F Fuller RS 《Molecular biology of the cell》2004,15(7):3196-3209
SOI3 was identified by a mutation, soi3-1, that suppressed a mutant trans-Golgi network (TGN) localization signal in the Kex2p cytosolic tail. SOI3, identical to RAV1, encodes a protein important for regulated assembly of vacuolar ATPase. Here, we show that Soi3/Rav1p is required for transport between the early endosome and the late endosome/prevacuolar compartment (PVC). By electron microscopy, soi3-1 mutants massively accumulated structures that resembled early endosomes. soi3Delta mutants exhibited a kinetic delay in transfer of the endocytic tracer dye FM4-64, from the 14 degrees C endocytic intermediate to the vacuole. The soi3Delta mutation delayed vacuolar degradation but not internalization of the a-factor receptor Ste3p. By density gradient fractionation, Soi3/Rav1p associated as a peripheral protein with membranes of a density characteristic of early endosomes. The soi3 null mutation markedly reduced the rate of Kex2p transport from the TGN to the PVC but had no effect on vacuolar protein sorting or cycling of Vps10p. These results suggest that assembly of vacuolar ATPase at the early endosome is required for transport of both Ste3p and Kex2p from the early endosome to the PVC and support a model in which cycling through the early endosome is part of the normal itinerary of Kex2p and other TGN-resident proteins. 相似文献
13.
Role for dynamin in late endosome dynamics and trafficking of the cation-independent mannose 6-phosphate receptor
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Nicoziani P Vilhardt F Llorente A Hilout L Courtoy PJ Sandvig K van Deurs B 《Molecular biology of the cell》2000,11(2):481-495
It is well established that dynamin is involved in clathrin-dependent endocytosis, but relatively little is known about possible intracellular functions of this GTPase. Using confocal imaging, we found that endogenous dynamin was associated with the plasma membrane, the trans-Golgi network, and a perinuclear cluster of cation-independent mannose 6-phosphate receptor (CI-MPR)-containing structures. By electron microscopy (EM), it was shown that these structures were late endosomes and that the endogenous dynamin was preferentially localized to tubulo-vesicular appendices on these late endosomes. Upon induction of the dominant-negative dynK44A mutant, confocal microscopy demonstrated a redistribution of the CI-MPR in mutant-expressing cells. Quantitative EM analysis of the ratio of CI-MPR to lysosome-associated membrane protein-1 in endosome profiles revealed a higher colocalization of the two markers in dynK44A-expressing cells than in control cells. Western blot analysis showed that dynK44A-expressing cells had an increased cellular procathepsin D content. Finally, EM revealed that in dynK44A-expressing cells, endosomal tubules containing CI-MPR were formed. These results are in contrast to recent reports that dynamin-2 is exclusively associated with endocytic structures at the plasma membrane. They suggest instead that endogenous dynamin also plays an important role in the molecular machinery behind the recycling of the CI-MPR from endosomes to the trans-Golgi network, and we propose that dynamin is required for the final scission of vesicles budding from endosome tubules. 相似文献
14.
Yeast Rrp14p is a nucleolar protein involved in both ribosome biogenesis and cell polarity
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We previously cloned RRP14/YKL082c, whose product exhibits two-hybrid interaction with Ebp2p, a regulatory factor of assembly of 60S ribosomal subunits. Depletion of Rrp14p results in shortage of 60S ribosomal subunits and retardation of processing from 27S pre-rRNA to 25S rRNA. Furthermore, 35S pre-rRNA synthesis appears to decline in Rrp14p-depleted cells. Rrp14p interacts with regulatory factors of 60S subunit assembly and also with Utp11p and Faf1p, which are regulatory factors required for assembly of 40S ribosomal subunits. We propose that Rrp14p is involved in ribosome synthesis from the beginning of 35S pre-rRNA synthesis to assembly of the 60S ribosomal subunit. Disruption of RRP14 causes an extremely slow growth rate of the cell, a severe defect in ribosome synthesis, and a depolarized localization of cortical actin patches throughout the cell cycle. These results suggest that Rrp14p has dual functions in ribosome synthesis and polarized cell growth. 相似文献
15.
Membrane-associated RING-CH (MARCH) is a recently identified member of the mammalian E3 ubiquitin ligase family, some members of which down-regulate the expression of immune recognition molecules. Here, we have identified MARCH-II, which is ubiquitously expressed and localized to endosomal vesicles and the plasma membrane. Immunoprecipitation and in vitro binding studies established that MARCH-II directly associates with syntaxin 6. Overexpression of MARCH-II resulted in redistribution of syntaxin 6 as well as some syntaxin-6-interacting soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) into the MARCH-II-positive vesicles. In addition, the retrograde transport of TGN38 and a chimeric version of furin to trans-Golgi network (TGN) was perturbed--without affecting the endocytic degradative and biosynthetic secretory pathways--similar to effects caused by a syntaxin 6 mutant lacking the transmembrane domain. MARCH-II overexpression markedly reduced the cell surface expression of transferrin (Tf) receptor and Tf uptake and interfered with delivery of internalized Tf to perinuclear recycling endosomes. Depletion of MARCH-II by small interfering RNA perturbed the TGN localization of syntaxin 6 and TGN38/46. MARCH-II is thus likely a regulator of trafficking between the TGN and endosomes, which is a novel function for the MARCH family. 相似文献
16.
Background
Mammalian receptors that couple to effectors via heterotrimeric G proteins (e.g., beta 2-adrenergic receptors) and receptors with intrinsic tyrosine kinase activity (e.g., insulin and IGF-I receptors) constitute the proximal points of two dominant cell signaling pathways. Receptors coupled to G proteins can be substrates for tyrosine kinases, integrating signals from both pathways. Yeast cells, in contrast, display G protein-coupled receptors (e.g., alpha-factor pheromone receptor Ste2) that have evolved in the absence of receptor tyrosine kinases, such as those found in higher organisms. We sought to understand the motifs in G protein-coupled receptors that act as substrates for receptor tyrosine kinases and the functional consequence of such phosphorylation on receptor biology. We expressed in human HEK 293 cells yeast wild-type Ste2 as well as a Ste2 chimera engineered with cytoplasmic domains of the beta2-adrenergic receptor and tested receptor sequestration in response to activation of the insulin receptor tyrosine kinase.Results
The yeast Ste2 was successfully expressed in HEK 293 cells. In response to alpha-factor, Ste2 signals to the mitogen-activated protein kinase pathway and internalizes. Wash out of agonist and addition of antagonist does not lead to Ste2 recycling to the cell membrane. Internalized Ste2 is not significantly degraded. Beta2-adrenergic receptors display internalization in response to agonist (isoproterenol), but rapidly recycle to the cell membrane following wash out of agonist and addition of antagonist. Beta2-adrenergic receptors display internalization in response to activation of insulin receptors (i.e., cross-regulation), whereas Ste2 does not. Substitution of the cytoplasmic domains of the β2-adrenergic receptor for those of Ste2 creates a Ste2/beta2-adrenergic receptor chimera displaying insulin-stimulated internalization.Conclusion
Chimera composed of yeast Ste2 into which domains of mammalian G protein-coupled receptors have been substituted, when expressed in animal cells, provide a unique tool for study of the regulation of G protein-coupled receptor trafficking by mammalian receptor tyrosine kinases and adaptor proteins. 相似文献17.
The yeast vacuolar enzyme aminopeptidase I (API) is synthesized in the cytoplasm as a precursor (pAPI). Upon its assembly into dodecamers, pAPI is wrapped by double-membrane saccular structures for its further transport within vesicles that fuse with the vacuolar membrane and release their content in the vacuolar lumen. Targeting of API to the vacuole occurs by two alternative transport routes, the cvt and the autophagy pathways, which although mechanistically similar specifically operate under vegetative growth or nitrogen starvation conditions, respectively. We have studied the role of Yol082p, a protein identified by its ability to interact with API, in the transport of its precursor to the vacuole. We show that Yol082p interacts with mature API, an interaction that is strengthened by the amino extension of the API protein. Yol082p is required for targeting of pAPI to the vacuole, both under growing and short term nitrogen starvation conditions. Absence of Yol082p does not impede the assembly of pAPI into dodecamers, but precludes the enclosure of pAPI within transport vesicles. Microscopy studies show that during vegetative growth Yol082p is distributed between a cytoplasmic pool and a variable number of 0.13--0.27-microm round, mobile structures, which are no longer observed under conditions of nitrogen starvation, and become larger in cells expressing the inactive Yol082 Delta C32p, or lacking Apg12p. In contrast to the autophagy mutants involved in API transport, a Delta yol082 strain does not lose viability under nitrogen starvation conditions, indicating normal function of the autophagy pathway. The data are consistent with a role of Yol082p in an early step of the API transport, after its assembly into dodecamers. Because Yol082p fulfills the functional requisites that define the CVT proteins, we propose to name it Cvt19. 相似文献
18.
Nbp2p is an Src homology 3 (SH3) domain-containing yeast protein that is involved in a variety of cellular processes. This small adaptor protein binds to a number of different proteins through its SH3 domain, and a region N-terminal to the SH3 domain binds to the protein phosphatase, Ptc1p. Despite its involvement in a large number of physical and genetic interactions, the only well characterized function of Nbp2p is to recruit Ptc1p to the high osmolarity glycerol pathway, which results in down-regulation of this pathway. In this study, we have discovered that Nbp2p orthologues exist in all Ascomycete and Basidiomycete fungal genomes and that all possess an SH3 domain and a conserved novel Ptc1p binding motif. The ubiquitous occurrence of these two features, which we have shown are both critical for Nbp2p function in Saccharomyces cerevisiae, implies that a conserved role of Nbp2p in all of these fungal species is the targeting of Ptc1p to proteins recognized by the SH3 domain. We also show that in a manner analogous to its role in the high osmolarity glycerol pathway, Nbp2p functions in the down-regulation of the cell wall integrity pathway through SH3 domain-mediated interaction with Bck1p, a component kinase of this pathway. Based on functional studies on the Schizosaccharomyces pombe and Neurospora crassa Nbp2p orthologues and the high conservation of the Nbp2p binding site in Bck1p orthologues, this function of Nbp2p appears to be conserved across Ascomycetes. Our results also clearly imply a function for the Nbp2p-Ptc1p complex other cellular processes. 相似文献
19.
The putative Arabidopsis homolog of yeast vps52p is required for pollen tube elongation, localizes to Golgi, and might be involved in vesicle trafficking 总被引:4,自引:0,他引:4
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The screening of the Versailles collection of Arabidopsis T-DNA transformants allowed us to identify several male gametophytic mutants, including poky pollen tube (pok). The pok mutant, which could only be isolated as a hemizygous line, exhibits very short pollen tubes, explaining the male-specific transmission defect observed in this line. We show that the POK gene is duplicated in the Arabidopsis genome and that the predicted POK protein sequence is highly conserved from lower to higher eukaryotes. The putative POK homolog in yeast (Saccharomyces cerevisiae), referred to as Vps52p/SAC2, has been shown to be located at the late Golgi and to function in a complex with other proteins, Vps53p, Vps54p, and Vps51p. This complex is involved in retrograde trafficking of vesicles between the early endosomal compartment and the trans-Golgi network. We present the expression patterns of the POK gene and its duplicate P2 in Arabidopsis, and of the putative Arabidopsis homologs of VPS53 and VPS54 of yeast. We show that a POK::GFP fusion protein localizes to Golgi in plant cells, supporting the possibility of a conserved function for Vps52p and POK proteins. These results, together with the expression pattern of the POK::GUS fusion and the lack of plants homozygous for the pok mutation, suggest a more general role for POK in polar growth beyond the pollen tube elongation process. 相似文献
20.
A yeast protein related to a mammalian Ras-binding protein, Vps9p, is required for localization of vacuolar proteins. 总被引:7,自引:0,他引:7
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In the yeast Saccharomyces cerevisiae, mutations in vacuolar protein sorting (VPS) genes result in secretion of proteins normally localized to the vacuole. Characterization of the VPS pathway has provided considerable insight into mechanisms of protein sorting and vesicle-mediated intracellular transport. We have cloned VPS9 by complementation of the vacuolar protein sorting defect of vps9 cells, characterized its gene product, and investigated its role in vacuolar protein sorting. Cells with a vps9 disruption exhibit severe vacuolar protein sorting defects and a temperature-sensitive growth defect at 38 degrees C. Electron microscopic examination of delta vps9 cells revealed the appearance of novel reticular membrane structures as well as an accumulation of 40- to 50-nm-diameter vesicles, suggesting that Vps9p may be required for the consumption of transport vesicles containing vacuolar protein precursors. A temperature-conditional allele of vps9 was constructed and used to investigate the function of Vps9p. Immediately upon shifting of temperature-conditional vps9 cells to the nonpermissive temperature, newly synthesized carboxypeptidase Y was secreted, indicating that Vps9p function is directly required in the VPS pathway. Antibodies raised against Vps9p immunoprecipitate a rare 52-kDa protein that fractionates with cytosolic proteins following cell lysis and centrifugation. Analysis of the VPS9 DNA sequence predicts that Vps9p is related to human proteins that bind Ras and negatively regulate Ras-mediated signaling. We term the related regions of Vps9p and these Ras-binding proteins a GTPase binding homology domain and suggest that it defines a family of proteins that bind monomeric GTPases. Vps9p may bind and serve as an effector of a rab GTPase, like Vps2lp, required for vacuolar protein sorting. 相似文献