Role of PUG1 in inducible porphyrin and heme transport in Saccharomyces cerevisiae

Unlike pathogenic fungi, the budding yeast Saccharomyces cerevisiae is not efficient at using heme as a nutritional source of iron. Here we report that for this yeast, heme uptake is induced under conditions of heme starvation. Heme synthesis requires oxygen, and yeast grown anaerobically exhibited an increased uptake of hemin. Similarly, a strain lacking aminolevulinate synthase exhibited a sixfold increase in hemin uptake when grown without 2-aminolevulinic acid. We used microarray analysis of cells grown under reduced oxygen tension or reduced intracellular heme conditions to identify candidate genes involved in heme uptake. Surprisingly, overexpression of PUG1 (protoporphyrin uptake gene 1) resulted in reduced utilization of exogenous heme by a heme-deficient strain and, conversely, increased the utilization of protoporphyrin IX. Pug1p was localized to the plasma membrane by indirect immunofluorescence and subcellular fractionation. Strains overexpressing PUG1 exhibited decreased accumulation of [(55)Fe]hemin but increased accumulation of protoporphyrin IX compared to the wild-type strain. To measure the effect of PUG1 overexpression on intracellular heme pools, we used a CYC1-lacZ reporter, which is activated in the presence of heme, and we monitored the activity of a heme-containing metalloreductase, Fre1p, expressed from a constitutive promoter. The data from these experiments were consistent with a role for Pug1p in inducible protoporphyrin IX influx and heme efflux.     

Two homologous genes,DCW1 (YKL046c) and DFG5, are essential for cell growth and encode glycosylphosphatidylinositol (GPI)-anchored membrane proteins required for cell wall biogenesis in Saccharomyces cerevisiae

The cell wall of Saccharomyces cerevisiae consists of glucan, chitin and various kinds of mannoproteins. Major parts of mannoproteins are synthesized as glycosylphosphatidylinositol (GPI)-anchored proteins and are then transferred to cell wall beta-1,6-glucan. A glycosyltransferase has been hypothesized to catalyse this transfer reaction. A database search revealed that the products of YKL046c and DFG5 are homologous to bacterial mannosidase. These genes are homologous to each other and have primary structures characteristic of GPI-anchored proteins. Although single disruptants of ykl046c and dfg5 were viable, ykl046cDelta was hypersensitive to a cell wall-digesting enzyme (zymolyase), suggesting that this gene is involved in cell wall biosynthesis. We therefore designated this gene as DCW1 (defective cell wall). A double disruptant of dcw1 and dfg5 was synthetically lethal, indicating that the functions of these gene products are redundant, and at least one of them is required for cell growth. Cells deficient in both Dcw1p and Dfg5p were round and large, had cell walls that contained an increased amount of chitin and secreted a major cell wall protein, Cwp1p, into the medium. Biochemical analyses showed that epitope-tagged Dcw1p is an N-glycosylated, GPI-anchored membrane protein and is localized in the membrane fraction including the cell surface. These results suggest that both Dcw1p and Dfg5p are GPI-anchored membrane proteins and are required for normal biosynthesis of the cell wall.     

The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import

Oxysterol binding proteins (OSBPs) comprise a large conserved family of proteins in eukaryotes. Their ubiquity notwithstanding, the functional activities of these proteins remain unknown. Kes1p, one of seven members of the yeast OSBP family, negatively regulates Golgi complex secretory functions that are dependent on the action of the major yeast phosphatidylinositol/phosphatidylcholine Sec14p. We now demonstrate that Kes1p is a peripheral membrane protein of the yeast Golgi complex, that localization to the Golgi complex is required for Kes1p function in vivo, and that targeting of Kes1p to the Golgi complex requires binding to a phosphoinositide pool generated via the action of the Pik1p, but not the Stt4p, PtdIns 4-kinase. Localization of Kes1p to yeast Golgi region also requires function of a conserved motif found in all members of the OSBP family. Finally, we present evidence to suggest that Kes1p may regulate adenosine diphosphate-ribosylation factor (ARF) function in yeast, and that it may be through altered regulation of ARF that Kes1p interfaces with Sec14p in controlling Golgi region secretory function.     

Nonvesicular sterol movement from plasma membrane to ER requires oxysterol-binding protein-related proteins and phosphoinositides

Sterols are moved between cellular membranes by nonvesicular pathways whose functions are poorly understood. In yeast, one such pathway transfers sterols from the plasma membrane (PM) to the endoplasmic reticulum (ER). We show that this transport requires oxysterol-binding protein (OSBP)-related proteins (ORPs), which are a large family of conserved lipid-binding proteins. We demonstrate that a representative member of this family, Osh4p/Kes1p, specifically facilitates the nonvesicular transfer of cholesterol and ergosterol between membranes in vitro. In addition, Osh4p transfers sterols more rapidly between membranes containing phosphoinositides (PIPs), suggesting that PIPs regulate sterol transport by ORPs. We confirmed this by showing that PM to ER sterol transport slows dramatically in mutants with conditional defects in PIP biosynthesis. Our findings argue that ORPs move sterols among cellular compartments and that sterol transport and intracellular distribution are regulated by PIPs.     

Disruption of YHC8, a member of the TSR1 gene family, reveals its direct involvement in yeast protein translocation

Genetic studies of Saccharomyces cerevisiae have identified many components acting to deliver specific proteins to their cellular locations. Genome analysis, however, has indicated that additional genes may also participate in such protein trafficking. The product of the yeast Yarrowia lipolytica TSR1 gene promotes the signal recognition particle-dependent translocation of secretory proteins through the endoplasmic reticulum. Here we describe the identification of a new gene family of proteins that is well conserved among different yeast species. The TSR1 genes encode polypeptides that share the same protein domain distribution and, like Tsr1p, may play an important role in the early steps of the signal recognition particle-dependent translocation pathway. We have identified five homologues of the TSR1 gene, four of them from the yeast Saccharomyces cerevisiae and the other from Hansenula polymorpha. We generated a null mutation in the S. cerevisiae YHC8 gene, the closest homologue to Y. lipolytica TSR1, and used different soluble (carboxypeptidase Y, alpha-factor, invertase) and membrane (dipeptidyl-aminopeptidase) secretory proteins to study its phenotype. A large accumulation of soluble protein precursors was detected in the mutant strain. Immunofluorescence experiments show that Yhc8p is localized in the endoplasmic reticulum. We propose that the YHC8 gene is a new and important component of the S. cerevisiae endoplasmic reticulum membrane and that it functions in protein translocation/insertion of secretory proteins through or into this compartment.     

Novel Predicted Peptidases with a Potential Role in the Ubiquitin Signaling Pathway

A multi-pronged strategy including extensive sequence searches, structuralmodeling, and analysis of contextual information extracted from domainarchitectures, genetic screens, and large-scale protein-protein interaction analyseswas employed to predict previously undetected components of the eukaryoticubiquitin signaling system. Two novel groups of proteins that are likely to function asde-ubiquitinating and de-SUMOylating peptidases (DUBs) were identified. The firstgroup of putative DUBs, designated PPPDE superfamily (after Permuted Papain foldPeptidases of DsRNA viruses and Eukaryotes), consists of predicted thiol peptidaseswith a circularly permuted papain-like fold. The inference of the likely DUB functionof the PPPDE superfamily proteins is based on the fusions of the catalytic domain toUb-binding PUG (PUB)/UBA domains and a novel alpha-helical Ub-associated domain(the PLAP, Ufd3p and Lub1p or PUL domain) amongst different members of thePPPDE supefamily. The presence of the PPPDE superfamily proteins in mosteukaryotic lineages, including basal ones, such as Giardia, suggest a role indeubiquitination of highly conserved proteins involved in key cellular functions, suchas cell cycle control. In addition to eukaryotic proteins, the PPPDE superfamilyincludes predicted proteases from several groups of double-stranded RNA virusesand one single-stranded DNA virus. The apparent recruitment of DUBs for viralpolyprotein processing seems to represent a common theme in evolution of viruses.The second group of putative DUBs identified in this study is the WLM (Wss1p-likemetalloproteases) family of Zincin-like superfamily of Zn-dependent peptidases,which are linked to the Ub -system by virtue of fusions with the UB-binding PUG(PUB), ubiquitin-like and Little Finger domains. More specifically on the basis ofgenetic evidence the WLM family is implicated in de-SUMOylation. If validatedexperimentally, the WLM family proteins will represent the first case of a Zincin-likemetalloprotease involvement in Ub-signaling.     

Identification and characterization of a Saccharomyces cerevisiae gene,RSB1, involved in sphingoid long-chain base release

Sphingoid long-chain bases (LCBs) and long-chain base phosphates (LCBPs) act as signaling molecules in eukaryotic cells. Accumulation of LCBPs results in cell growth inhibition in yeast, although the mechanism is unknown. Here, we identified a novel yeast gene, RSB1 (resistance to sphingoid long-chain base), by screening a multicopy suppressor of the LCB-sensitive phenotype of the LCBP lyase mutant. RSB1 encodes a polypeptide of 354 amino acids with a molecular mass of 40.4 kDa. Rsb1p is predicted to be an integral membrane protein with seven transmembrane-spanning domains. We demonstrated that cells overproducing Rsb1p showed a decrease in accumulation of exogenously added sphingosine and dihydrosphingosine because of their increased release. This release was ATP-dependent, and a mutant of the predicted ATP binding motif had no activity. Substrate specificity analysis of Rsb1p demonstrated that it is active on LCBs but not on LCBPs or other hydrophobic compounds. These results suggest that Rsb1p is a transporter or flippase that translocates LCBs from the cytoplasmic side toward the extracytoplasmic side of the membrane.     

Differential Regulation of Mitochondrial Pyruvate Carrier Genes Modulates Respiratory Capacity and Stress Tolerance in Yeast

Mpc proteins are highly conserved from yeast to humans and are necessary for the uptake of pyruvate at the inner mitochondrial membrane, which is used for leucine and valine biosynthesis and as a fuel for respiration. Our analysis of the yeast MPC gene family suggests that amino acid biosynthesis, respiration rate and oxidative stress tolerance are regulated by changes in the Mpc protein composition of the mitochondria. Mpc2 and Mpc3 are highly similar but functionally different: Mpc2 is most abundant under fermentative non stress conditions and important for amino acid biosynthesis, while Mpc3 is the most abundant family member upon salt stress or when high respiration rates are required. Accordingly, expression of the MPC3 gene is highly activated upon NaCl stress or during the transition from fermentation to respiration, both types of regulation depend on the Hog1 MAP kinase. Overexpression experiments show that gain of Mpc2 function leads to a severe respiration defect and ROS accumulation, while Mpc3 stimulates respiration and enhances tolerance to oxidative stress. Our results identify the regulated mitochondrial pyruvate uptake as an important determinant of respiration rate and stress resistance.