Regulation of Fab1 Phosphatidylinositol 3-Phosphate 5-Kinase Pathway by Vac7 Protein and Fig4, a Polyphosphoinositide Phosphatase Family Member

The Saccharomyces cerevisiae FAB1 gene encodes the sole phosphatidylinositol 3-phosphate [PtdIns(3)P] 5-kinase responsible for synthesis of the polyphosphoinositide PtdIns(3,5)P(2). VAC7 encodes a 128-kDa transmembrane protein that localizes to vacuolar membranes. Both vac7 and fab1 null mutants have dramatically enlarged vacuoles and cannot grow at elevated temperatures. Additionally, vac7Δ mutants have nearly undetectable levels of PtdIns(3,5)P(2), suggesting that Vac7 functions to regulate Fab1 kinase activity. To test this hypothesis, we isolated a fab1 mutant allele that bypasses the requirement for Vac7 in PtdIns(3,5)P(2) production. Expression of this fab1 allele in vac7Δ mutant cells suppresses the temperature sensitivity, vacuolar morphology, and PtdIns(3,5)P(2) defects normally exhibited by vac7Δ mutants. We also identified a mutant allele of FIG4, whose gene product contains a Sac1 polyphosphoinositide phosphatase domain, which suppresses vac7Δ mutant phenotypes. Deletion of FIG4 in vac7Δ mutant cells suppresses the temperature sensitivity and vacuolar morphology defects, and dramatically restores PtdIns(3,5)P(2) levels. These results suggest that generation of PtdIns(3,5)P(2) by the Fab1 lipid kinase is regulated by Vac7, whereas turnover of PtdIns(3,5)P(2) is mediated in part by the Sac1 polyphosphoinositide phosphatase family member Fig4

Identification of the Functional Domains of Yeast Sorting Nexins Vps5p and Vps17p

Sorting nexins (Snxs) are a recently discovered family of conserved hydrophilic cytoplasmic proteins that have been found associated with membranes of the endocytic system and that are implicated in the trafficking of many endosomal membrane proteins, including the epidermal growth factor receptor and transferrin receptor. Snx proteins are partly defined by the presence of a p40 phox homology domain that has recently been shown to bind phosphatidylinositol 3-phosphate. Most Snx proteins also contain a predicted coiled-coils domain in the carboxyl-terminal half of the protein and have been shown to form dimers with other members of the Snx family. The yeast sorting nexins Vps5p and Vps17p form a dimer and are also components of the retromer complex that mediates endosome-to-Golgi transport of the carboxypeptidase Y receptor Vps10p. To functionally define the different domains of the yeast sorting nexins Vps5p and Vps17p, we have generated various truncations to examine the role that the different domains of Vps5p/Vps17p play in their respective functions. Herein, we show that the C-terminal halves of Vps5p and Vps17p, which contain the coiled-coils domains, are necessary and sufficient for their interaction. We have also mapped the retromer assembly domain to the N-terminal half of Vps5p and found that binding of Vps5p by Vps17p synergizes the interaction between Vps5p and other retromer components. Additionally, we have examined which domain(s) of Vps5p is necessary for membrane association

Genomic Analysis of Homotypic Vacuole Fusion

Yeast vacuoles undergo fission and homotypic fusion, yielding one to three vacuoles per cell at steady state. Defects in vacuole fusion result in vacuole fragmentation. We have screened 4828 yeast strains, each with a deletion of a nonessential gene, for vacuole morphology defects. Fragmented vacuoles were found in strains deleted for genes encoding known fusion catalysts as well as 19 enzymes of lipid metabolism, 4 SNAREs, 12 GTPases and GTPase effectors, 9 additional known vacuole protein-sorting genes, 16 protein kinases, 2 phosphatases, 11 cytoskeletal proteins, and 28 genes of unknown function. Vacuole fusion and vacuole protein sorting are catalyzed by distinct, but overlapping, sets of proteins. Novel pathways of vacuole priming and docking emerged from this deletion screen. These include ergosterol biosynthesis, phosphatidylinositol (4,5)-bisphosphate turnover, and signaling from Rho GTPases to actin remodeling. These pathways are supported by the sensitivity of the late stages of vacuole fusion to inhibitors of phospholipase C, calcium channels, and actin remodeling. Using databases of yeast protein interactions, we found that many nonessential genes identified in our deletion screen interact with essential genes that are directly involved in vacuole fusion. Our screen reveals regulatory pathways of vacuole docking and provides a genomic basis for studies of this reaction

Genomic Screen for Vacuolar Protein Sorting Genes in Saccharomyces cerevisiae

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 α-factor. Others, such as Vps61p, Vps64p, and Vps67p, displayed defects in the actin cytoskeleton at 30°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