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Scott D. Emr - One of the best experts on this subject based on the ideXlab platform.
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ligand recognition and domain structure of vps10p a Vacuolar Protein Sorting receptor in saccharomyces cerevisiae
FEBS Journal, 1999Co-Authors: Marianne U Jorgensen, Scott D. Emr, Jakob R WintherAbstract:Vps10p is a receptor that sorts several different Vacuolar Proteins by cycling between a late Golgi compartment and the endosome. The cytoplasmic tail of Vps10p is necessary for the recycling, whereas the lumenal domain is predicted to interact with the soluble ligands. We have studied ligand binding to Vps10p by introducing deletions in the lumenal region. This region contains two domains with homology to each other. Domain 2 binds carboxypeptidase Y (CPY), Proteinase A (PrA) and hybrids of these proteases with invertase. Moreover, we show that aminopeptidase Y (APY) is a ligand of Vps10p. The native proteases compete for binding to domain 2. Binding of CPY(156)-invertase or PrA(137)-invertase, on the other hand, do not interfere with binding of CPY to Vps10p. Furthermore, the Q24RPL27 sequence known to be important for Vacuolar Sorting of CPY, is of little importance in the Vps10p-dependent Sorting of CPY-invertase. Apparently, domain 2 contains two different binding sites; one for APY, CPY and PrA, and one for CPY-invertase and PrA-invertase. The latter interaction seems not to be sequence specific, and we suggest that an unfolded structure in these ligands is recognized by Vps10p.
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Protein traffic in the yeast endocytic and Vacuolar Protein Sorting pathways
Current Opinion in Cell Biology, 1998Co-Authors: Beverly Wendland, Scott D. Emr, Howard RiezmanAbstract:Endocytosis is a fundamental membrane trafficking event that occurs in all eukaryotes. The yeast Saccharomyces cerevisiae has been particularly useful in efforts to uncover novel Proteins that mediate endocytosis, and many of these factors share similarity with Proteins from higher eukaryotes. In the past two years, progress has centered on three major areas: modifications/signaling pathways that initiate or regulate internalization, Protein complexes that are implicated in the internalization process, and factors that are involved in regulation of traffic through late endosomal compartments. As the parallels between the mechanisms employed in yeast and higher eukaryotes are further explored, new insights into the complex process of endocytosis should emerge.
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a Sorting nexin 1 homologue vps5p forms a complex with vps17p and is required for recycling the Vacuolar Protein Sorting receptor
Molecular Biology of the Cell, 1997Co-Authors: Bruce F Horazdovsky, Brian A Davies, Matthew N J Seaman, Steven A Mclaughlin, Suk Hoon Yoon, Scott D. EmrAbstract:A number of the Saccharomyces cerevisiae Vacuolar Protein-Sorting (vps) mutants exhibit an altered Vacuolar morphology. Unlike wild-type cells that contain 1-3 large Vacuolar structures, the class B vps5 and vps17 mutant cells contain 10-20 smaller vacuole-like compartments. To explore the role of these VPS gene products in vacuole biogenesis, we cloned and sequenced VPS5 and characterized its Protein products. The VPS5 gene is predicted to encode a very hydrophilic Protein of 675 amino acids that shows significant sequence homology with mammalian Sorting nexin-1. Polyclonal antiserum directed against the VPS5 gene product detects a single, cytoplasmic Protein that is phosphorylated specifically on a serine residue(s). Subcellular fractionation studies indicate that Vps5p is associated peripherally with a dense membrane fraction distinct from Golgi, endosomal, and Vacuolar membranes. This association was found to be dependent on the presence of another class B VPS gene product, Vps17p. Biochemical cross-linking studies demonstrated that Vps5p and Vps17p physically interact. Gene disruption experiments show that the VPS5 genes product is not essential for cell viability; however, cells carrying the null allele contain fragmented vacuoles and exhibit defects in Vacuolar Protein-Sorting similar to vps17 null mutants. More than 95% of carboxypeptidase Y is secreted from these cells in its Golgi-modified p2 precursor form. Additionally, the Vps10p Vacuolar Protein-Sorting receptor is mislocalized to the vacuole in vps5 mutant cells. On the basis of these and other observations, we propose that the Vps17p Protein complex may participate in the intracellular trafficking of the Vps10p-Sorting receptor, as well as other later-Golgi Proteins.
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a novel sec18p nsf dependent complex required for golgi to endosome transport in yeast
Molecular Biology of the Cell, 1997Co-Authors: Christopher G Burd, Christopher R Cowles, Michael R Peterson, Scott D. EmrAbstract:The Vacuolar Protein-Sorting (VPS) pathway of Saccharomyces cerevisiae mediates localization of Proteins from the trans-Golgi to the vacuole via a preVacuolar endosome compartment. Mutations in class D Vacuolar Protein-Sorting (vps) genes affect vesicle-mediated Golgi-to-endosome transport and result in secretion of Vacuolar Proteins. Temperature-sensitive-for-function (tsf) and dominant negative mutations in PEP12, encoding a putative SNARE vesicle receptor on the endosome, and tsf mutations in VAC1, a gene implicated in vacuole inheritance and Vacuolar Protein Sorting, were constructed and used to demonstrate that Pep12p and Vac1p are components of the VPS pathway. The sequence of Vac1p contains two putative zinc-binding RING motifs, a zinc finger motif, and a coiled-coil motif. Site-directed mutations in the carboxyl-terminal RING motif strongly affected Vacuolar Protein Sorting. Vac1p was found to be tightly associated with membranes as a monomer and in a large SDS-resistant complex. By using Pep12p affinity chromatography, we found that Vac1p, Vps45p (SEC1 family member), and Sec18p (yeast N-ethyl maleimide-sensitive factor, NSF) bind Pep12p. Consistent with a functional role for this complex in Vacuolar Protein Sorting, double pep12tsfvac1tsf and pep12tsf vps45tsf mutants exhibited synthetic Vps- phenotypes, the tsf phenotype of the vac1tsf mutant was rescued by overexpression of VPS45 or PEP12, overexpression of a dominant pep12 allele in a sec18-1 strain resulted in a severe synthetic growth defect that was rescued by deletion of PEP12 or VAC1, and subcellular fractionation of vac1 delta cells revealed a striking change in the fractionation of Pep12p and Vps21p, a rab family GTPase required for Vacuolar Protein Sorting. The functions of Pep12p, Vps45p, and Vps21p indicate that key aspects of Golgi-to-endosome trafficking are similar to other vesicle-mediated transport steps, although the role of Vac1p suggests that there are also novel components of the VPS pathway.
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multilamellar endosome like compartment accumulates in the yeast vps28 Vacuolar Protein Sorting mutant
Molecular Biology of the Cell, 1996Co-Authors: Stephanie E Rieder, Lois M Banta, K Kohrer, J M Mccaffery, Scott D. EmrAbstract:In the yeast Saccharomyces cerevisiae, Vacuolar Proteins such as carboxypeptidase Y transit from the Golgi to the lysosome-like vacuole via an endosome-like intermediate compartment. The Vacuolar Protein Sorting (vps) mutant vps28, a member of the "class E" vps mutants, accumulates Vacuolar, endocytic, and late Golgi markers in an aberrant endosome-like class E compartment. Sequence analysis of VPS28 revealed an open reading frame predicted to encode a hydrophilic Protein of 242 amino acids. Consistent with this, polyclonal antiserum raised against Vps28p recognized a cytoplasmic Protein of 28 kDa. Disruption of VPS28 resulted in moderate defects in both biosynthetic traffic and endocytic traffic destined for the vacuole. The transport of soluble Vacuolar hydrolases to the vacuole was impaired in vps28 null mutant cells (approximately 40-50% carboxypeptidase Y missorted). Internalization of the endocytic marker FM 4-64, a vital lipophilic dye, resulted in intense staining of a small intracellular compartment adjacent to an enlarged vacuole in delta vps28 cells. Furthermore, the Vacuolar H+-ATPase accumulated in the periVacuolar class E compartment in delta vps28 cells, as did a-factor receptor Ste3p that was internalized from the plasma membrane. Electron microscopic analysis revealed the presence of a novel compartment consisting of stacks of curved membrane cisternae. Immunolocalization studies demonstrated that the Vacuolar H+-ATPase is associated with this cupped cisternal structure, indicating that it corresponds to the class E compartment observed by fluorescence microscopy. Our data indicate that kinetic defects in both anterograde and retrograde transport out of the preVacuolar compartment in vps28 mutants result in the accumulation of Protein and membrane in an exaggerated multilamellar endosomal compartment. We propose that Vps28p, as well as other class E Vps Proteins, may facilitate (possibly as coat Proteins) the formation of transport intermediates required for efficient transport out of the preVacuolar endosome.
Stephan Becker - One of the best experts on this subject based on the ideXlab platform.
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Vacuolar Protein Sorting pathway contributes to the release of marburg virus
Journal of Virology, 2009Co-Authors: Evamaria Mittler, Thomas Strecker, Larissa Kolesnikova, Eiji Morita, Florian Zielecki, Colin M. Crump, Stephan BeckerAbstract:VP40, the major matrix Protein of Marburg virus, is the main driving force for viral budding. Additionally, cellular factors are likely to play an important role in the release of progeny virus. In the present study, we characterized the influence of the Vacuolar Protein Sorting (VPS) pathway on the release of virus-like particles (VLPs), which are induced by Marburg virus VP40. In the supernatants of HEK 293 cells expressing VP40, different populations of VLPs with either a vesicular or a filamentous morphology were detected. While the filaments were almost completely composed of VP40, the vesicular particles additionally contained considerable amounts of cellular Proteins. In contrast to that in the vesicles, the VP40 in the filaments was regularly organized, probably inducing the elimination of cellular Proteins from the released VLPs. Vesicular particles were observed in the supernatants of cells even in the absence of VP40. Mutation of the late-domain motif in VP40 resulted in reduced release of filamentous particles, and likewise, inhibition of the VPS pathway by expression of a dominant-negative (DN) form of VPS4 inhibited the release of filamentous particles. In contrast, the release of vesicular particles did not respond significantly to the expression of DN VPS4. Like the budding of VLPs, the budding of Marburg virus particles was partially inhibited by the expression of DN VPS4. While the release of VLPs from VP40-expressing cells is a valuable tool with which to investigate the budding of Marburg virus particles, it is important to separate filamentous VLPs from vesicular particles, which contain many cellular Proteins and use a different budding mechanism.
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Vacuolar Protein Sorting pathway contributes to the release of marburg virus
Journal of Virology, 2009Co-Authors: Evamaria Mittler, Thomas Strecker, Larissa Kolesnikova, Eiji Morita, Florian Zielecki, Colin M. Crump, Stephan BeckerAbstract:VP40, the major matrix Protein of Marburg virus, is the main driving force for viral budding. Additionally, cellular factors are likely to play an important role in the release of progeny virus. In the present study, we characterized the influence of the Vacuolar Protein Sorting (VPS) pathway on the release of virus-like particles (VLPs), which are induced by Marburg virus VP40. In the supernatants of HEK 293 cells expressing VP40, different populations of VLPs with either a vesicular or a filamentous morphology were detected. While the filaments were almost completely composed of VP40, the vesicular particles additionally contained considerable amounts of cellular Proteins. In contrast to that in the vesicles, the VP40 in the filaments was regularly organized, probably inducing the elimination of cellular Proteins from the released VLPs. Vesicular particles were observed in the supernatants of cells even in the absence of VP40. Mutation of the late-domain motif in VP40 resulted in reduced release of filamentous particles, and likewise, inhibition of the VPS pathway by expression of a dominant-negative (DN) form of VPS4 inhibited the release of filamentous particles. In contrast, the release of vesicular particles did not respond significantly to the expression of DN VPS4. Like the budding of VLPs, the budding of Marburg virus particles was partially inhibited by the expression of DN VPS4. While the release of VLPs from VP40-expressing cells is a valuable tool with which to investigate the budding of Marburg virus particles, it is important to separate filamentous VLPs from vesicular particles, which contain many cellular Proteins and use a different budding mechanism.
Bruce F Horazdovsky - One of the best experts on this subject based on the ideXlab platform.
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the phosphatidylinositol 3 phosphate binding Protein vac1p interacts with a rab gtpase and a sec1p homologue to facilitate vesicle mediated Vacuolar Protein Sorting
Molecular Biology of the Cell, 1999Co-Authors: Gregory G Tall, Hiroko Hama, Daryll B Dewald, Bruce F HorazdovskyAbstract:Activated GTP-bound Rab Proteins are thought to interact with effectors to elicit vesicle targeting and fusion events. Vesicle-associated v-SNARE and target membrane t-SNARE Proteins are also involved in vesicular transport. Little is known about the functional relationship between Rabs and SNARE Protein complexes. We have constructed an activated allele of VPS21, a yeast Rab Protein involved in Vacuolar Protein Sorting, and demonstrated an allele-specific interaction between Vps21p and Vac1p. Vac1p was found to bind the Sec1p homologue Vps45p. Although no association between Vps21p and Vps45p was seen, a genetic interaction between VPS21 and VPS45 was observed. Vac1p contains a zinc-binding FYVE finger that may bind phosphatidylinositol 3-phosphate [PtdIns(3)P]. In other FYVE domain Proteins, this motif and PtdIns(3)P are necessary for membrane association. Vac1 Proteins with mutant FYVE fingers still associated with membranes but showed Vacuolar Protein Sorting defects and reduced interactions with Vps45p and activated Vps21p. Vac1p membrane association was not dependent on PtdIns(3)P, Pep12p, Vps21p, Vps45p, or the PtdIns 3-kinase, Vps34p. Vac1p FYVE finger mutant misSorting phenotypes were suppressed by a defective allele of VPS34. These data indicate that PtdIns(3)P may perform a regulatory role, possibly involved in mediating Vac1p Protein-Protein interactions. We propose that activated-Vps21p interacts with its effector, Vac1p, which interacts with Vps45p to regulate the Golgi to endosome SNARE complex.
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a Sorting nexin 1 homologue vps5p forms a complex with vps17p and is required for recycling the Vacuolar Protein Sorting receptor
Molecular Biology of the Cell, 1997Co-Authors: Bruce F Horazdovsky, Brian A Davies, Matthew N J Seaman, Steven A Mclaughlin, Suk Hoon Yoon, Scott D. EmrAbstract:A number of the Saccharomyces cerevisiae Vacuolar Protein-Sorting (vps) mutants exhibit an altered Vacuolar morphology. Unlike wild-type cells that contain 1-3 large Vacuolar structures, the class B vps5 and vps17 mutant cells contain 10-20 smaller vacuole-like compartments. To explore the role of these VPS gene products in vacuole biogenesis, we cloned and sequenced VPS5 and characterized its Protein products. The VPS5 gene is predicted to encode a very hydrophilic Protein of 675 amino acids that shows significant sequence homology with mammalian Sorting nexin-1. Polyclonal antiserum directed against the VPS5 gene product detects a single, cytoplasmic Protein that is phosphorylated specifically on a serine residue(s). Subcellular fractionation studies indicate that Vps5p is associated peripherally with a dense membrane fraction distinct from Golgi, endosomal, and Vacuolar membranes. This association was found to be dependent on the presence of another class B VPS gene product, Vps17p. Biochemical cross-linking studies demonstrated that Vps5p and Vps17p physically interact. Gene disruption experiments show that the VPS5 genes product is not essential for cell viability; however, cells carrying the null allele contain fragmented vacuoles and exhibit defects in Vacuolar Protein-Sorting similar to vps17 null mutants. More than 95% of carboxypeptidase Y is secreted from these cells in its Golgi-modified p2 precursor form. Additionally, the Vps10p Vacuolar Protein-Sorting receptor is mislocalized to the vacuole in vps5 mutant cells. On the basis of these and other observations, we propose that the Vps17p Protein complex may participate in the intracellular trafficking of the Vps10p-Sorting receptor, as well as other later-Golgi Proteins.
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mutations in the vps45 gene a sec1 homologue result in Vacuolar Protein Sorting defects and accumulation of membrane vesicles
Journal of Cell Science, 1994Co-Authors: Christopher R Cowles, Scott D. Emr, Bruce F HorazdovskyAbstract:Genetic analyses of Vacuolar Protein Sorting in Saccharomyces cerevisiae have uncovered a large number of mutants (vps) that missort and secrete Vacuolar hydrolases. A small subset of vps mutants exhibit a temperature-conditional growth phenotype and show a severe defect in the localization of soluble Vacuolar Proteins, yet maintain a near-normal vacuole structure. Here, we report on the cloning and characterization of the gene affected in one of these mutants, VPS45, which has been found to encode a member of a Protein family that includes the yeast Proteins Sec1p, Sly1p and Vps33p, as well as n-Sec1, UNC18 and Rop from other eukaryotic organisms. These Proteins are thought to participate in vesicle-mediated Protein transport events. Polyclonal antiserum raised against a TrpE-Vps45 fusion Protein specifically detects a stable 67 kDa Protein in labeled yeast cell extracts. Subcellular fractionation studies demonstrate that the majority of Vps45p is associated with a high-speed membrane pellet fraction that includes Golgi, transport vesicles and, potentially, endosomal membranes. Significantly, this fraction lacks ER, vacuole and plasma membranes. Overexpression of Vps45p saturates the sites with which Vps45p associates. A vps45 null mutant accumulates vesicles, many of which were found to be present in large clusters. This accumulation of potential transport vesicles indicates that Vps45p may facilitate the targeting and/or fusion of these vesicles in the Vacuolar Protein Sorting pathway.
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the vps16 gene product associates with a sedimentable Protein complex and is essential for Vacuolar Protein Sorting in yeast
Journal of Biological Chemistry, 1993Co-Authors: Bruce F Horazdovsky, Scott D. EmrAbstract:The Saccharomyces cerevisiae Vacuolar Protein Sorting mutant, vps16, exhibits pleiotropic defects in Vacuolar Protein targeting and vacuole morphology. To understand the role of the VPS16 gene in Vacuolar Protein Sorting and organelle assembly, a vps16 ts mutant was used to clone the wild-type gene. DNA sequence analysis identified a single open reading frame within a vps16 complementing DNA fragment, capable of encoding a Protein of 92,000 Da. Hydrophobicity analysis indicates that the Vps16 Protein (Vps16p) is hydrophilic and contains no obvious signal sequence or membrane spanning domains. Gene disruption experiments have shown that VPS16 is not essential. delta vps16 cells exhibit, 1) a severe defect in Vacuolar Protein Sorting; 2) a ts growth defect; 3) a grossly abnormal vacuole morphology, no normal vacuole compartment(s) is present; and 4) a defect in alpha-factor processing. A trpE-Vps16 fusion Protein has been used to generate polyclonal antiserum. This antiserum detects an unglycosylated Protein of 90,000 Da. Subcellular fractionation studies indicate that the vast majority of the VPS16 gene product is associated with a particulate cell fraction. This association is resistant to detergent and salt extractions, but Vps16p can be extracted with 6 M urea and alkali buffer. In addition, overexpression of Vps16p appears to saturate the association sites available in this sedimentable structure. These data indicate that Vps16p may be specifically associated with a large Protein complex, or with a limited number of sites on cytoskeletal elements of the cell.
Larissa Kolesnikova - One of the best experts on this subject based on the ideXlab platform.
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Vacuolar Protein Sorting pathway contributes to the release of marburg virus
Journal of Virology, 2009Co-Authors: Evamaria Mittler, Thomas Strecker, Larissa Kolesnikova, Eiji Morita, Florian Zielecki, Colin M. Crump, Stephan BeckerAbstract:VP40, the major matrix Protein of Marburg virus, is the main driving force for viral budding. Additionally, cellular factors are likely to play an important role in the release of progeny virus. In the present study, we characterized the influence of the Vacuolar Protein Sorting (VPS) pathway on the release of virus-like particles (VLPs), which are induced by Marburg virus VP40. In the supernatants of HEK 293 cells expressing VP40, different populations of VLPs with either a vesicular or a filamentous morphology were detected. While the filaments were almost completely composed of VP40, the vesicular particles additionally contained considerable amounts of cellular Proteins. In contrast to that in the vesicles, the VP40 in the filaments was regularly organized, probably inducing the elimination of cellular Proteins from the released VLPs. Vesicular particles were observed in the supernatants of cells even in the absence of VP40. Mutation of the late-domain motif in VP40 resulted in reduced release of filamentous particles, and likewise, inhibition of the VPS pathway by expression of a dominant-negative (DN) form of VPS4 inhibited the release of filamentous particles. In contrast, the release of vesicular particles did not respond significantly to the expression of DN VPS4. Like the budding of VLPs, the budding of Marburg virus particles was partially inhibited by the expression of DN VPS4. While the release of VLPs from VP40-expressing cells is a valuable tool with which to investigate the budding of Marburg virus particles, it is important to separate filamentous VLPs from vesicular particles, which contain many cellular Proteins and use a different budding mechanism.
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Vacuolar Protein Sorting pathway contributes to the release of marburg virus
Journal of Virology, 2009Co-Authors: Evamaria Mittler, Thomas Strecker, Larissa Kolesnikova, Eiji Morita, Florian Zielecki, Colin M. Crump, Stephan BeckerAbstract:VP40, the major matrix Protein of Marburg virus, is the main driving force for viral budding. Additionally, cellular factors are likely to play an important role in the release of progeny virus. In the present study, we characterized the influence of the Vacuolar Protein Sorting (VPS) pathway on the release of virus-like particles (VLPs), which are induced by Marburg virus VP40. In the supernatants of HEK 293 cells expressing VP40, different populations of VLPs with either a vesicular or a filamentous morphology were detected. While the filaments were almost completely composed of VP40, the vesicular particles additionally contained considerable amounts of cellular Proteins. In contrast to that in the vesicles, the VP40 in the filaments was regularly organized, probably inducing the elimination of cellular Proteins from the released VLPs. Vesicular particles were observed in the supernatants of cells even in the absence of VP40. Mutation of the late-domain motif in VP40 resulted in reduced release of filamentous particles, and likewise, inhibition of the VPS pathway by expression of a dominant-negative (DN) form of VPS4 inhibited the release of filamentous particles. In contrast, the release of vesicular particles did not respond significantly to the expression of DN VPS4. Like the budding of VLPs, the budding of Marburg virus particles was partially inhibited by the expression of DN VPS4. While the release of VLPs from VP40-expressing cells is a valuable tool with which to investigate the budding of Marburg virus particles, it is important to separate filamentous VLPs from vesicular particles, which contain many cellular Proteins and use a different budding mechanism.
Juan S. Bonifacino - One of the best experts on this subject based on the ideXlab platform.
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genomic screen for Vacuolar Protein Sorting genes in saccharomyces cerevisiae
Molecular Biology of the Cell, 2002Co-Authors: Cecilia J Bonangelino, Edna M Chavez, Juan S. BonifacinoAbstract:The biosynthetic Sorting of hydrolases to the yeast vacuole involves transport along two distinct routes referred to as the carboxypeptidase Y and alkaline phosphatase pathways. To identify genes involved in Sorting to the vacuole, we conducted a genome-wide screen of 4653 homozygous diploid gene deletion strains of Saccharomyces cerevisiae for misSorting of carboxypeptidase Y. We identified 146 mutant strains that secreted strong-to-moderate levels of carboxypeptidase Y. Of these, only 53 of the corresponding genes had been previously implicated in Vacuolar Protein Sorting, whereas the remaining 93 had either been identified in screens for other cellular processes or were only known as hypothetical open reading frames. Among these 93 were genes encoding: 1) the Ras-like GTP-binding Proteins Arl1p and Arl3p, 2) actin-related Proteins such as Arp5p and Arp6p, 3) the monensin and brefeldin A hypersensitivity Proteins Mon1p and Mon2p, and 4) 15 novel Proteins designated Vps61p-Vps75p. Most of the novel gene products were involved only in the carboxypeptidase Y pathway, whereas a few, including Mon1p, Mon2p, Vps61p, and Vps67p, appeared to be involved in both the carboxypeptidase Y and alkaline phosphatase pathways. Mutants lacking some of the novel gene products, including Arp5p, Arp6p, Vps64p, and Vps67p, were severely defective in secretion of mature alpha-factor. Others, such as Vps61p, Vps64p, and Vps67p, displayed defects in the actin cytoskeleton at 30 degrees C. The identification and phenotypic characterization of these novel mutants provide new insights into the mechanisms of Vacuolar Protein Sorting, most notably the probable involvement of the actin cytoskeleton in this process.
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Structural Requirements for Function of Yeast GGAs in Vacuolar Protein Sorting, α-Factor Maturation, and Interactions with Clathrin
Molecular and cellular biology, 2001Co-Authors: Chris Mullins, Juan S. BonifacinoAbstract:The GGAs (Golgi-localized, gamma-ear-containing, ARF-binding Proteins) are ubiquitous adaptor-like Proteins that associate with the cytoplasmic face of the trans-Golgi network (TGN) (4, 16, 22, 37, 47). Three GGAs exist in humans (GGA1, GGA2, and GGA3), one each exists in Drosophila melanogaster and Caenorhabditis elegans, and two exist in the budding yeast Saccharomyces cerevisiae (Gga1p and Gga2p). The GGAs are monomeric but have a multidomain structure consisting of VHS (Vps27, Hrs, and STAM), GAT (GGA and TOM), hinge, and GAE (gamma-adaptin ear) domains. Biochemical and immunocytochemical analyses have revealed that each of the four GGA domains serves a specific function. The VHS domain of the human GGAs functions as a recognition module for acidic cluster-dileucine Sorting signals contained within the cytosolic tails of sortilin (31) and the cation-independent (CI) and cation-dependent (CD) mannose 6-phosphate receptors (MPRs) that sort lysosomal hydrolases to lysosomes (38, 56). The GAT domain of human and yeast GGAs mediates interactions with the GTP-bound form of members of the ARF (ADP-ribosylation factor) family of Proteins (4, 16, 55). GAT-ARF interactions are responsible for the regulated recruitment of the GGAs from the cytosol to the Golgi complex (4, 16, 39). The hinge domain of all the GGAs contains putative clathrin-binding motifs composed of acidic and bulky hydrophobic amino acids (16, 39). For the human GGAs, these motifs have been shown to mediate interactions with clathrin in vitro and to promote recruitment of clathrin to the TGN in vivo (39). Finally, the GAE domain of the human GGAs binds Proteins such as γ-synergin and rabaptin-5, which may function to regulate assembly of GGA-containing coats or formation of coated vesicular carriers (22, 47). These properties of the human GGA domains indicate that they may mediate ARF-dependent recruitment of clathrin to the TGN in order to sort intracellular cargo receptors from the TGN to the endosomal system. Despite the detailed characterization of the properties of GGA domains, the significance of these properties for the function of the GGAs in vivo remains to be assessed. The existence of two GGAs in yeast provides an opportunity to perform analyses of the physiological roles of the GGAs in an organism easily amenable to genetic manipulation. In yeast, biosynthetic Protein Sorting from the Golgi complex to the vacuole, the equivalent of the mammalian lysosome, is mediated by two principal routes (for reviews, see references 7, 12, 26, and 29). The alkaline phosphatase (ALP) pathway sorts Vacuolar Proteins such as ALP and the t-SNARE Vam3p from the Golgi complex to the vacuole directly. Formation of Golgi complex-derived carrier vesicles and transport through this pathway require the adaptor Protein (AP) complex AP-3 (14, 46) and the VPS (Vacuolar Protein Sorting) gene products Vps41p/Vam2p, Vps39p/Vam6p (30), and Vps1p (32, 49), a member of the dynamin family of Proteins (49). In contrast, the carboxypeptidase Y (CPY) pathway sorts Vacuolar Proteins including CPY and Proteinase A (PrA) and subunits of the Vacuolar ATPase from the Golgi complex to the vacuole via a preVacuolar endosomal compartment (PVC). Sorting of both CPY and PrA through this pathway is mediated by interactions with the transmembrane receptor Vps10p, which delivers these Proteins to the PVC prior to recycling back to the Golgi complex (13, 27). Vesicle formation and transport in the CPY pathway involve the coat-scaffolding Protein clathrin (10, 45), the putative aminophospholipid translocase Drs2p (11), and the synaptojanin-like Proteins Inp52p and Inp53p (1, 33), as well as numerous VPS gene products, including Vps41p/Vam2p, Vps39p/Vam6p (30), and Vps1p (1, 49). Mutations in factors operating in these respective pathways result, to differing degrees, in impaired Vacuolar Sorting and defects in vacuole biogenesis. The yeast GGAs appear to play important, redundant roles in biosynthetic Sorting to the vacuole based on studies of a mutant gga1Δ gga2Δ strain containing disruptions of both yeast GGA genes. This mutant is defective for transport of pro-CPY to the vacuole and missorts pro-CPY to the periplasmic space (16, 22, 55). The gga1Δ gga2Δ mutant was also found to be defective in Sorting of the syntaxin Pep12p from the Golgi complex to late endosomes (2). The presence of a Vacuolar morphology defect in gga1Δ gga2Δ cells has, however, been debated, with one study reporting abnormal morphology (22) and another showing no morphological differences between wild-type and gga1Δ gga2Δ strains (55). As is evident from the well-defined phenotypes arising from mutations in the yeast GGA genes, these Proteins appear to play an important role in biosynthetic Protein Sorting. To expand our understanding of the GGAs' function and to assess the relative importance of the different GGA domains in vivo, we have performed a structure-function analysis of yeast Gga1p and Gga2p. First, we elaborate on the gga1Δ gga2Δ mutant phenotype by reporting defects in Sorting of additional Vacuolar Proteins and abnormal Vacuolar morphology and a strong defect in maturation of the mating pheromone α-factor. We then analyze the functional requirement of individual GGA domains and find that the VHS, GAT, and hinge domains are important for GGA-mediated pro-CPY Sorting and pro-α-factor processing, while the GAE domain appears less important. In addition, we show that, like their human counterparts, the yeast GGAs are capable of binding clathrin via acidic-bulky-hydrophobic motifs in their hinge domains. We also present evidence that these clathrin-binding motifs contribute to GGA-mediated Sorting in vivo. Finally, mutational analysis of the Gga2p VHS domain identifies a highly conserved sequence important for this Protein's function.
