Utp9p Facilitates Msn5p-mediated Nuclear Reexport of Retrograded tRNAs in Saccharomyces cerevisiae

Utp9p is a nucleolar protein that is part of a subcomplex containing several U3 snoRNA-associated proteins including Utp8p, which is a protein that shuttles aminoacyl-tRNAs from the nucleolus to the nuclear tRNA export receptors Los1p and Msn5p in Saccharomyces cerevisiae. Here we show that Utp9p is also an intranuclear component of the Msn5p-mediated nuclear tRNA export pathway. Depletion of Utp9p caused nuclear accumulation of mature tRNAs derived from intron-containing precursors, but not tRNAs made from intronless pre-tRNAs. Utp9p binds tRNA directly and saturably, and copurifies with Utp8p, Gsp1p, and Msn5p, but not with Los1p or aminoacyl-tRNA synthetases. Utp9p interacts directly with Utp8p, Gsp1p, and Msn5p in vitro. Furthermore, Gsp1p forms a complex with Msn5p and Utp9p in a tRNA-dependent manner. However, Utp9p does not shuttle between the nucleus and the cytoplasm. Because tRNA splicing occurs in the cytoplasm and the spliced tRNAs are retrograded back to the nucleus, we propose that Utp9p facilitates nuclear reexport of retrograded tRNAs. Moreover, the data suggest that Utp9p together with Utp8p translocate aminoacyl-tRNAs from the nucleolus to Msn5p and assist with formation of the Msn5p-tRNA-Gsp1p-GTP export complex.     

Rsp5, a ubiquitin-protein ligase, is involved in degradation of the single-stranded-DNA binding protein rfa1 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, RAD1 and RAD52 are required for alternate pathways of mitotic recombination. Double-mutant strains exhibit a synergistic interaction that decreases direct repeat recombination rates dramatically. A mutation in RFA1, the largest subunit of a single-stranded DNA-binding protein complex (RP-A), suppresses the recombination deficiency of rad1 rad52 strains (J. Smith and R. Rothstein, Mol. Cell. Biol. 15:1632-1641, 1995). Previously, we hypothesized that this mutation, rfa1-D228Y, causes an increase in recombinogenic lesions as well as the activation of a RAD52-independent recombination pathway. To identify gene(s) acting in this pathway, temperature-sensitive (ts) mutations were screened for those that decrease recombination levels in a rad1 rad52 rfa1-D228Y strain. Three mutants were isolated. Each segregates as a single recessive gene. Two are allelic to RSP5, which encodes an essential ubiquitin-protein ligase. One allele, rsp5-25, contains two mutations within its open reading frame. The first mutation does not alter the amino acid sequence of Rsp5, but it decreases the amount of full-length protein in vivo. The second mutation results in the substitution of a tryptophan with a leucine residue in the ubiquitination domain. In rsp5-25 mutants, the UV sensitivity of rfa1-D228Y is suppressed to the same level as in strains overexpressing Rfa1-D228Y. Measurement of the relative rate of protein turnover demonstrated that the half-life of Rfa1-D228Y in rsp5-25 mutants was extended to 65 min compared to a 35-min half-life in wild-type strains. We propose that Rsp5 is involved in the degradation of Rfa1 linking ubiquitination with the replication-recombination machinery.     

Pex10p functions as an E3 ligase for the Ubc4p-dependent ubiquitination of Pex5p

The Saccharomyces cerevisiae (Sc) PTS1 import receptor Pex5p is modified by ubiquitin, both in an Ubc4p-dependent and a Pex4p (Ubc10p)-dependent manner. Both of these modifications require the RING domain-containing protein Pex10p in vivo, but the actual role this protein plays in the ubiquitination of Pex5p has so far, remained enigmatic. Here, we report that the RING domain of Pex10p exhibits E₃ ligase activity in vitro, in combination with the human E₂ enzyme UbcH5a, a homologue of ScUbc4p, but not when ScPex4p was used as an E₂ enzyme in the reaction. We have further characterised Pex10p’s E₃ ligase activity using mutants designed to disturb this activity and show that Pex10p acts as the E₃ ligase for Ubc4p-dependent ubiquitination of Pex5p but not Pex4p-dependent ubiquitination in vivo. These data imply that the two distinct Pex5p modifications require different E₃ ligases, as well as different E₂ enzymes.     

Enhancement of stress tolerance in Saccharomyces cerevisiae by overexpression of ubiquitin ligase Rsp5 and ubiquitin-conjugating enzymes

Yeast Saccharomyces cerevisiae cells overexpressing essential ubiquitin ligase Rsp5 or ubiquitin-conjugating enzymes (Ubc1-Ubc13) showed tolerance to various stresses. Co-overexpression of Rsp5 and Ubc1, Ubc2, Ubc3, Ubc5, Ubc6, Ubc9, Ubc10, Ubc11, Ubc12, or Ubc13 further enhanced stress tolerance. These results suggest that overexpression of ubiquitin-related enzymes might be a useful method for breeding novel stress-resistant strains.     

Modulation of Congo-red-induced aberrations in the yeast Saccharomyces cerevisiae by the general stress response protein Hsp12p

Previous studies have shown that in Saccharomyces cerevisiae HSP12, which codes for the small cell wall heat shock protein Hsp12p, was induced upon exposure to cell-wall-damaging agents such as Congo red. Here, we demonstrate that Hsp12p decreases the interaction between Congo red and chitin. A Deltahsp12 mutant strain displayed decreased viability, increased aggregation and sedimentation, as well as an altered morphology when grown in the presence of Congo red dye. The presence of Hsp12p was also necessary for the Congo-red-mediated invasion of agar plates.     

Localization of the Rsp5p ubiquitin-protein ligase at multiple sites within the endocytic pathway

The Saccharomyces cerevisiae RSP5 gene encodes an essential HECT E3 ubiquitin-protein ligase. Rsp5p contains an N-terminal C2 domain, three WW domains in the central portion of the molecule, and a C-terminal catalytic HECT domain. A diverse group of substrates of Rsp5p and vertebrate C2 WW-domain-containing HECT E3s have been identified, including both nuclear and membrane-associated proteins. We determined the intracellular localization of Rsp5p and the determinants necessary for localization, in order to better understand how Rsp5p activities are coordinated. Using both green fluorescent protein fusions to Rsp5p and immunogold electron microscopy, we found that Rsp5p was distributed in a punctate pattern at the plasma membrane, corresponding to membrane invaginations that are likely sites of endosome formation, as well as at perivacuolar sites. The latter appeared to correspond to endocytic intermediates, as these structures were not seen in a sla2/end4-1 mutant, and double-immunogold labeling demonstrated colocalization of Rsp5p with the endosomal markers Pep12p and Vps32p. The C2 domain was an important determinant of localization; however, mutations that disrupted HECT domain function also caused mislocalization of Rsp5p, indicating that enzymatic activity is linked to localization. Deletion of the C2 domain partially stabilized Fur4p, a protein previously shown to undergo Rsp5p- and ubiquitin-mediated endocytosis; however, Fur4p was still ubiquitinated at the plasma membrane when the C2 domain was deleted from the protein. Together, these results indicate that Rsp5p is located at multiple sites within the endocytic pathway and suggest that Rsp5p may function at multiple steps in the ubiquitin-mediated endocytosis pathway.     

Rsp5 ubiquitin ligase modulates translation accuracy in yeast Saccharomyces cerevisiae

Rsp5p is an essential yeast ubiquitin protein ligase that ubiquitinates multiple proteins involved in various processes. Recent studies indicate that ubiquitination also affects translation. Here, we show that the strain with the rsp5-13 mutation exhibits altered sensitivity to antibiotics and a slower rate of translation. Using a sensitive dual-gene reporter system, we demonstrate that stop codon readthrough efficiency is decreased in the rsp5-13 mutant, while both +1 and -1 frameshifting were unaffected. The effect of the rsp5-13 mutation on readthrough could be reversed by increased expression of ubiquitin and partially suppressed by overproduction of the elongation factor eEF1A. As assessed by fluorescence in situ hybridization, the rsp5-13 mutant cells accumulate tRNA nuclear pools, perhaps depleting tRNA from the cytoplasm. Nuclear accumulation of tRNA is observed only when rsp5-13 cells are grown in media with high amino acid content. This defect, also reversed by overproduction of the elongation factor eEF1A, may be the primary reason for altered translational decoding accuracy.     

Saccharomyces cerevisiae Ub-conjugating enzyme Ubc4 binds the proteasome in the presence of translationally damaged proteins

Surveillance mechanisms that monitor protein synthesis can promote rapid elimination of misfolded nascent proteins. We showed that the translation elongation factor eEF1A and the proteasome subunit Rpt1 play a central role in the translocation of nascent-damaged proteins to the proteasome. We show here that multiubiquitinated proteins, and the ubiquitin-conjugating (E2) enzyme Ubc4, are rapidly detected in the proteasome following translational damage. However, Ubc4 levels in the proteasome were reduced significantly in a strain that expressed a mutant Rpt1 subunit. Ubc4 and Ubc5 are functionally redundant E2 enzymes that represent ideal candidates for ubiquitinating damaged nascent proteins because they lack significant substrate specificity, are required for the degradation of bulk, damaged proteins, and contribute to cellular stress-tolerance mechanisms. In agreement with this hypothesis, we determined that ubc4Delta ubc5Delta is exceedingly sensitive to protein translation inhibitors. Collectively, these studies suggest a specific role for Ubc4 and Ubc5 in the degradation of cotranslationally damaged proteins that are targeted to the proteasome.     

The Saccharomyces cerevisiae 60 S ribosome biogenesis factor Tif6p is regulated by Hrr25p-mediated phosphorylation

The biosynthesis of 60 S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eIF6. This protein is necessary for the formation of 60 S ribosomal subunits because it is essential for the processing of 35 S pre-rRNA to the mature 25 S and 5.8 S rRNAs. In the present work, using molecular genetic and biochemical analyses, we show that Hrr25p, an isoform of yeast casein kinase I, phosphorylates Tif6p both in vitro and in vivo. Tryptic phosphopeptide mapping of in vitro phosphorylated Tif6p by Hrr25p and (32)P-labeled Tif6p isolated from yeast cells followed by mass spectrometric analysis revealed that phosphorylation occurred on a single tryptic peptide at Ser-174. Sucrose gradient fractionation and coimmunoprecipitation experiments demonstrate that a small but significant fraction of Hrr25p is bound to 66 S preribosomal particles that also contain bound Tif6p. Depletion of Hrr25p from a conditional yeast mutant that fails to phosphorylate Tif6p was unable to process pre-rRNAs efficiently, resulting in significant reduction in the formation of 25 S rRNA. These results along with our previous observations that phosphorylatable Ser-174 is required for yeast cell growth and viability, suggest that Hrr25p-mediated phosphorylation of Tif6p plays a critical role in the biogenesis of 60 S ribosomal subunits in yeast cells.     

The HECT ubiquitin ligase Rsp5p is required for proper nuclear export of mRNA in Saccharomyces cerevisiae

The nuclear transport of both proteins and RNAs has attracted considerable interest in recent years. However, regulation pathways of the nuclear transport machineries are still not well characterized. Previous studies indicated that ubiquitination is involved in poly(A)⁺ RNA nuclear export. For this reason, we systematically investigated ubiquitin-protein ligasess from the homologous to E6-AP carboxy terminus (HECT) family for potential individual roles in nuclear transport in Saccharomyces cerevisiae . Here we report that Rsp5, an essential yeast ubiquitin ligase involved in many cellular functions, when deleted or mutated in ligase activity, blocks the nuclear export of mRNAs. Affected messenger RNAs include both total poly(A)⁺ mRNA and heat-shock mRNAs. Mutation of Rsp5 does not affect nuclear protein import or export. Deletion of RSP5 blocks mRNA export, even under conditions where its essential role in unsaturated fatty acids biosynthesis is bypassed. Using domain mapping, we find that the ligase activity is required for proper mRNA export, indicating that ubiquitination by Rsp5 acts directly or indirectly to affect RNA export. The finding that Rsp5p ligase mutations cause a more pronounced defect at high temperatures suggests that ubiquitination of transport factors by Rsp5p may also be essential during stress conditions.     

Prokaryotic diversity of the Saccharomyces cerevisiae Atx1p-mediated copper pathway

MOTIVATION: Several genes involved in the cellular import of copper and its subsequent incorporation into the high-affinity iron transport complex in Saccharomyces cerevisiae are known to be conserved between eukaryotes and prokaryotes. However, the degree to which these genes share their functional context as members of the same pathway in the prokaryotic domain is less clear. RESULTS: The co-occurrence of gene families involved in Atx1p-mediated copper transport in the genomes and operon structures of 80 non-redundant prokaryotes was investigated. For this purpose, we developed a Web tool (SHOPS) to display the operon context for a given set of proteins. In total, a set of 43 putative operons was identified. These were found to be involved in a variety of pathways and indicate a large diversity in the functional context of the individual gene family members. AVAILABILITY: The SHOPS tool can be found at http://www.bioinformatics.med.uu.nl/shops supplemental data are available at http://humgen.med.uu.nl/publications/vanbakel/pathway/     

Degradation of the hexose transporter Hxt5p in Saccharomyces cerevisiae

BACKGROUND INFORMATION: Hxt5p is a member of a multigene family of hexose transporter proteins which translocate glucose across the plasma membrane of the yeast Saccharomyces cerevisiae. In contrast with other major hexose transporters of this family, Hxt5p expression is regulated by the growth rate of the cells and not by the external glucose concentration. Furthermore, Hxt5p is the only glucose transporter expressed during stationary phase. These observations suggest a different role for Hxt5p in S. cerevisiae. Therefore we studied the metabolism and localization of Hxt5p in more detail. RESULTS AND CONCLUSIONS: Inhibition of HXT5 expression in stationary-phase cells by the addition of glucose, which increases the growth rate, led to a decrease in the amount of Hxt5 protein within a few hours. Addition of glucose to stationary-phase cells resulted in a transient phosphorylation of Hxt5p on serine residues, but no ubiquitination was detected. The decrease in Hxt5p levels is caused by internalization of the protein, as observed by immunofluorescence microscopy. In stationary-phase cells, Hxt5p was localized predominantly at the cell periphery and upon addition of glucose to the cells the protein translocated to the cell interior. Electron microscopy demonstrated that the internalized Hxt5p-HA (haemagglutinin) protein was localized to small vesicles, multivesicular bodies and the vacuole. These results suggest that internalization and degradation of Hxt5p in the vacuole occur in an ubiquitination-independent manner via the endocytic pathway.     

tRNA thiolation links translation to stress responses in Saccharomyces cerevisiae

Although tRNA modifications have been well catalogued, the precise functions of many modifications and their roles in mediating gene expression are still being elucidated. Whereas tRNA modifications were long assumed to be constitutive, it is now apparent that the modification status of tRNAs changes in response to different environmental conditions. The URM1 pathway is required for thiolation of the cytoplasmic tRNAs tGlu^UUC, tGln^UUG, and tLys^UUU in Saccharomyces cerevisiae. We demonstrate that URM1 pathway mutants have impaired translation, which results in increased basal activation of the Hsf1-mediated heat shock response; we also find that tRNA thiolation levels in wild-type cells decrease when cells are grown at elevated temperature. We show that defects in tRNA thiolation can be conditionally advantageous, conferring resistance to endoplasmic reticulum stress. URM1 pathway proteins are unstable and hence are more sensitive to changes in the translational capacity of cells, which is decreased in cells experiencing stresses. We propose a model in which a stress-induced decrease in translation results in decreased levels of URM1 pathway components, which results in decreased tRNA thiolation levels, which further serves to decrease translation. This mechanism ensures that tRNA thiolation and translation are tightly coupled and coregulated according to need.     

Transcription regulation of the Saccharomyces cerevisiae PHO5 gene by the Ino2p and Ino4p basic helix-loop-helix proteins

The Saccharomyces cerevisiae PHO5 gene product accounts for a majority of the acid phosphatase activity. Its expression is induced by the basic helix-loop-helix (bHLH) protein, Pho4p, in response to phosphate depletion. Pho4p binds predominantly to two UAS elements (UASp1 at -356 and UASp2 at -247) in the PHO5 promoter. Previous studies from our lab have shown cross-regulation of different biological processes by bHLH proteins. This study tested the ability of all yeast bHLH proteins to regulate PHO5 expression and identified inositol-mediated regulation via the Ino2p/Ino4p bHLH proteins. Ino2p/Ino4p are known regulators of phospholipid biosynthetic genes. Genetic epistasis experiments showed that regulation by inositol required a third UAS site (UASp3 at -194). ChIP assays showed that Ino2p:Ino4p bind the PHO5 promoter and that this binding is dependent on Pho4p binding. These results demonstrate that phospholipid biosynthesis is co-ordinated with phosphate utilization via the bHLH proteins.