Rho1p and Cdc42p act after Ypt7p to regulate vacuole docking.

Rho GTPases, which control polarized cell growth through cytoskeletal reorganization, have recently been implicated in the control of endo- and exocytosis. We now report that both Rho1p and Cdc42p have a direct role in mediating the docking stage of homotypic vacuole fusion. Vacuoles prepared from strains with temperature-sensitive alleles of either Rho1p or Cdc42p are thermolabile for fusion. RhoGDI (Rdi1p), which extracts Rho1p and Cdc42p from the vacuole membrane, blocks vacuole fusion. The Rho GTPases can not fulfill their function as long as priming and Ypt7p-dependent tethering are inhibited. However, reactions that are reversibly blocked after docking by the calcium chelator BAPTA have passed the point of sensitivity to Rdi1p. Extraction and removal of Ypt7p, Rho1p and Cdc42p from docked vacuoles (by Gdi1p, Gyp7p and Rdi1p) does not impede subsequent membrane fusion, which is still sensitive to GTPgammaS. Thus, multiple GTPases act in a defined sequence to regulate the docking steps of vacuole fusion.

Sequential action of two GTPases to promote vacuole docking and fusion.

Homotypic vacuole fusion occurs by sequential priming, docking and fusion reactions. Priming frees the HOPS complex (Vps 11, 16, 18, 33, 39 and 41) to activate Ypt7p for docking. Here we explore the roles of the GDP and GTP states of Ypt7p using Gdi1p (which extracts Ypt7:GDP), Gyp7p (a GTPase-activating protein for Ypt7p:GTP), GTPgammaS or GppNHp (non-hydrolyzable nucleotides), and mutant forms of Ypt7p that favor either GTP or GDP states. GDP-bound Ypt7p on isolated vacuoles can be extracted by Gdi1p, although only the GTP-bound state allows docking. Ypt7p is converted to the GTP-bound state after priming and stably associates with HOPS. Gyp7p can cause Ypt7p to hydrolyze bound GTP to GDP, driving HOPS release and accelerating Gdi1p-mediated release of Ypt7p. Ypt7p extraction does not inhibit the Ca(2+)-triggered cascade that leads to fusion. However, in the absence of Ypt7p, fusion is still sensitive to GTPgammaS and GppNHp, indicating that there is a second specific GTPase that regulates the calcium flux and hence fusion. Thus, two GTPases sequentially govern vacuole docking and fusion.

A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion.

Yeast vacuoles undergo priming, docking, and homotypic fusion, although little has been known of the connections between these reactions. Vacuole-associated Vam2p and Vam6p (Vam2/6p) are components of a 65S complex containing SNARE proteins. Upon priming by Sec18p/NSF and ATP, Vam2/6p is released as a 38S subcomplex that binds Ypt7p to initiate docking. We now report that the 38S complex consists of both Vam2/6p and the class C Vps proteins [Reider, S. E. and Emr, S. D. (1997) Mol. Biol. Cell 8, 2307-2327]. This complex includes Vps33p, a member of the Sec1 family of proteins that bind t-SNAREs. We term this 38S complex HOPS, for homotypic fusion and vacuole protein sorting. This unexpected finding explains how Vam2/6p associates with SNAREs and provides a mechanistic link of the class C Vps proteins to Ypt/Rab action. HOPS initially associates with vacuole SNAREs in "cis" and, after release by priming, hops to Ypt7p, activating this Ypt/Rab switch to initiate docking.

Regulation of peroxisome size and number by fatty acid beta -oxidation in the yeast yarrowia lipolytica.

The Yarrowia lipolytica MFE2 gene encodes peroxisomal beta-oxidation multifunctional enzyme type 2 (MFE2). MFE2 is peroxisomal in a wild-type strain but is cytosolic in a strain lacking the peroxisomal targeting signal-1 (PTS1) receptor. MFE2 has a PTS1, Ala-Lys-Leu, that is essential for targeting to peroxisomes. MFE2 lacking a PTS1 can apparently oligomerize with full-length MFE2 to enable targetting to peroxisomes. Peroxisomes of an oleic acid-induced MFE2 deletion strain, mfe2-KO, are larger and more abundant than those of the wild-type strain. Under growth conditions not requiring peroxisomes, peroxisomes of mfe2-KO are larger but less abundant than those of the wild-type strain, suggesting a role for MFE2 in the regulation of peroxisome size and number. A nonfunctional version of MFE2 did not restore normal peroxisome morphology to mfe2-KO cells, indicating that their phenotype is not due to the absence of MFE2. mfe2-KO cells contain higher amounts of beta-oxidation enzymes than do wild-type cells. We also show that increasing the level of the beta-oxidation enzyme thiolase results in enlarged peroxisomes. Our results implicate peroxisomal beta-oxidation in the control of peroxisome size and number in yeast.

Enlarged peroxisomes are present in oleic acid-grown Yarrowia lipolytica overexpressing the PEX16 gene encoding an intraperoxisomal peripheral membrane peroxin.

Pex mutants of the yeast Yarrowia lipolytica are defective in peroxisome assembly. The mutant strain pex16-1 lacks morphologically recognizable peroxisomes. Most peroxisomal proteins are mislocalized to a subcellular fraction enriched for cytosol in pex16 strains, but a subset of peroxisomal proteins is localized at, or near, wild-type levels to a fraction typically enriched for peroxisomes. The PEX16 gene was isolated by functional complementation of the pex16-1 strain and encodes a protein, Pex16p, of 391 amino acids (44,479 D). Pex16p has no known homologues. Pex16p is a peripheral protein located at the matrix face of the peroxisomal membrane. Substitution of the carboxylterminal tripeptide Ser-Thr-Leu, which is similar to the consensus sequence of peroxisomal targeting signal 1, does not affect targeting of Pex16p to peroxisomes. Pex16p is synthesized in wild-type cells grown in glucose-containing media, and its levels are modestly increased by growth of cells in oleic acid-containing medium. Overexpression of the PEX16 gene in oleic acid- grown Y. lipolytica leads to the appearance of a small number of enlarged peroxisomes, which contain the normal complement of peroxisomal proteins at levels approaching those of wild-type peroxisomes.

The Yarrowia lipolytica gene PAY5 encodes a peroxisomal integral membrane protein homologous to the mammalian peroxisome assembly factor PAF-1.

Pay mutants of the yeast Yarrowia lipolytica fail to assemble functional peroxisomes. One mutant strain, pay5-1, lacks normal peroxisomes and instead contains irregular vesicular structures surrounded by multiple unit membranes. The pay5-1 mutant is not totally deficient in peroxisomal matrix protein targeting, as a subset of matrix proteins continues to localize to a subcellular fraction enriched for peroxisomes. The functionally complementing gene PAY5 encodes a protein, Pay5p, of 380 amino acids (41,720 Da). Pay5p is a peroxisomal integral membrane protein homologous to mammalian PAF-1 proteins, which are essential for peroxisome assembly and whose mutation in humans results in Zellweger syndrome. Pay5p is targeted to mammalian peroxisomes, demonstrating the evolutionary conservation of the targeting mechanism for peroxisomal membrane proteins. Our results suggest that in pay5 mutants, normal peroxisome assembly is blocked, which leads to the accumulation of the membranous vesicular structures observed.

Mutations in the PAY5 gene of the yeast Yarrowia lipolytica cause the accumulation of multiple subpopulations of peroxisomes.

We previously reported the cloning of the PAY5 gene of the yeast Yarrowia lipolytica by complementation of the peroxisome assembly mutant pay5-1 (Eitzen, G. A., Titorenko, V. I., Smith, J. J., Veenhuis, M., Szilard, R. K., and Rachubinski, R. A. (1996) J. Biol. Chem. 271, 20300-20306). The peroxisomal integral membrane protein Pay5p is a homologue of mammalian PAF-1 proteins, which are essential for peroxisome assembly and whose mutation in humans results in peroxisome biogenesis disorders. Mutations in the PAY5 gene result in the accumulation of three distinct peroxisomal subpopulations. These subpopulations are characterized by differences in 1) buoyant density, 2) the relative distribution of peroxisomal matrix and membrane proteins, 3) the efficiency of import of several peroxisomal matrix proteins, and 4) the phospholipid levels of peroxisomal membranes. These data, together with the analysis of temporal changes in the relative abundance of individual peroxisomal subpopulations in pay5 mutants, suggest that these subpopulations represent intermediates in a multistep peroxisome assembly pathway normally operating in yeast cells.

The Yarrowia lipolytica gene PAY2 encodes a 42-kDa peroxisomal integral membrane protein essential for matrix protein import and peroxisome enlargement but not for peroxisome membrane proliferation.

PAY genes are required for peroxisome assembly in the yeast Yarrowia lipolytica. Here we show that a mutant strain, pay2, is disrupted for the import of proteins targeted by either peroxisomal targeting signal-1 or -2. Electron microscopy of pay2 cells revealed the presence of small peroxisomal "ghosts," similar to the vesicular structures found in fibroblasts of patients with the human peroxisome assembly disorder, Zellweger syndrome. Functional complementation of pay2 with a plasmid library of Y. lipolytica genomic DNA identified a gene, PAY2, that restores growth of pay2 on oleic acid, import of catalase and multifunctional enzyme into peroxisomes, and formation of wild type peroxisomes. The PAY2 gene encodes Pay2p, a hydrophobic polypeptide of 404 amino acids. An antibody raised against Pay2p recognizes a polypeptide of approximately 42-kDa whose synthesis is induced by growth of Y. lipolytica on oleic acid. Pay2p is a peroxisomal integral membrane protein, as it localizes to carbonate-stripped peroxisomal membranes. Pay2p shows no identity to any known protein. Our results suggest that Pay2p is essential for the activity of the peroxisomal import machinery but does not affect the initial steps of peroxisomal membrane proliferation.

PAY4, a gene required for peroxisome assembly in the yeast Yarrowia lipolytica, encodes a novel member of a family of putative ATPases.

PAY genes are required for peroxisome assembly in the yeast Yarrowia lipolytica. Here we characterize one mutant, pay4, and describe the cloning and sequencing of the PAY4 gene. The pay4 mutant shows no identifiable peroxisomes by biochemical and morphological criteria. The complementing PAY4 gene encodes a polypeptide, Pay4p, 1025 amino acids in length and having a predicted molecular mass of 112,258 Da. The predicted Pay4p sequence contains two putative ATP-binding domains and shows structural relationships to other potential ATP-binding proteins involved in biological processes as diverse as peroxisome biogenesis, vesicle-mediated protein transport, cell cycle control, and transcriptional regulation. These proteins all share a highly conserved stretch of approximately 175 amino acids that contains a consensus sequence for ATP binding. Pay4p shows sequence conservation with Pas1p and Pas5p, putative ATPases required for peroxisomal assembly in the yeasts Saccharomyces cerevisiae and Pichia pastoris, respectively. Pay4p, Pas1p, and Pas5p are presumably related members of a family of putative ATPases involved in peroxisome biogenesis. Pay4p is synthesized in low amounts in Y. lipolytica cells grown in glucose, and there is a rapid and pronounced increase in the levels of Pay4p upon transfer of the cells to a medium containing oleic acid as the sole carbon source.