Eap1p, a novel eukaryotic translation initiation factor 4E-associated protein in Saccharomyces cerevisiae

Ribosome binding to eukaryotic mRNA is a multistep process which is mediated by the cap structure [m(7)G(5')ppp(5')N, where N is any nucleotide] present at the 5' termini of all cellular (with the exception of organellar) mRNAs. The heterotrimeric complex, eukaryotic initiation factor 4F (eIF4F), interacts directly with the cap structure via the eIF4E subunit and functions to assemble a ribosomal initiation complex on the mRNA. In mammalian cells, eIF4E activity is regulated in part by three related translational repressors (4E-BPs), which bind to eIF4E directly and preclude the assembly of eIF4F. No structural counterpart to 4E-BPs exists in the budding yeast, Saccharomyces cerevisiae. However, a functional homolog (named p20) has been described which blocks cap-dependent translation by a mechanism analogous to that of 4E-BPs. We report here on the characterization of a novel yeast eIF4E-associated protein (Eap1p) which can also regulate translation through binding to eIF4E. Eap1p shares limited homology to p20 in a region which contains the canonical eIF4E-binding motif. Deletion of this domain or point mutation abolishes the interaction of Eap1p with eIF4E. Eap1p competes with eIF4G (the large subunit of the cap-binding complex, eIF4F) and p20 for binding to eIF4E in vivo and inhibits cap-dependent translation in vitro. Targeted disruption of the EAP1 gene results in a temperature-sensitive phenotype and also confers partial resistance to growth inhibition by rapamycin. These data indicate that Eap1p plays a role in cell growth and implicates this protein in the TOR signaling cascade of S. cerevisiae.     

Modulation of efficiency of translation termination in Saccharomyces cerevisiae

Nonsense suppression is a readthrough of premature termination codons. It typically occurs either due to the recognition of stop codons by tRNAs with mutant anticodons, or due to a decrease in the fidelity of translation termination. In the latter case, suppressors usually promote the readthrough of different types of nonsense codons and are thus called omnipotent nonsense suppressors. Omnipotent nonsense suppressors were identified in yeast Saccharomyces cerevisiae in 1960s, and most of subsequent studies were performed in this model organism. Initially, omnipotent suppressors were localized by genetic analysis to different protein- and RNA-encoding genes, mostly the components of translational machinery. Later, nonsense suppression was found to be caused not only by genomic mutations, but also by epigenetic elements, prions. Prions are self-perpetuating protein conformations usually manifested by infectious protein aggregates. Modulation of translational accuracy by prions reflects changes in the activity of their structural proteins involved in different aspects of protein synthesis. Overall, nonsense suppression can be seen as a “phenotypic mirror” of events affecting the accuracy of the translational machine. However, the range of proteins participating in the modulation of translation termination fidelity is not fully elucidated. Recently, the list has been expanded significantly by findings that revealed a number of weak genetic and epigenetic nonsense suppressors, the effect of which can be detected only in specific genetic backgrounds. This review summarizes the data on the nonsense suppressors decreasing the fidelity of translation termination in S. cerevisiae, and discusses the functional significance of the modulation of translational accuracy.     

The identification and characterization of a novel splicing protein, Isy1p, of Saccharomyces cerevisiae

We have identified a novel splicing factor, Isy1p, through two-hybrid screens for interacting proteins involved in nuclear pre-mRNA splicing. Isy1p was tagged and demonstrated to be part of the splicing machinery, associated with spliceosomes throughout the splicing reactions. At least a portion of the Isy1 protein population is associated with snRNAs; low levels of U5 and U6 snRNAs are coimmunoprecipitated specifically with Isy1p. When the ISY1 gene was knocked out, no defect in vegetative growth was observed. Using a sensitive in vivo splicing assay, however, we observed lower splicing efficiency in the isy1 null mutant compared to wild-type, indicating that Isy1 p is important in the optimization of splicing.     

Ded1p, a DEAD-box protein required for translation initiation in Saccharomyces cerevisiae, is an RNA helicase.

The Ded1 protein (Ded1p), a member of the DEAD-box family, has recently been shown to be essential for translation initiation in Saccharomyces cerevisiae. Here, we show that Ded1p purified from Escherichia coli has an ATPase activity, which is stimulated by various RNA substrates. Using an RNA strand-displacement assay, we show that Ded1p has also an ATP-dependent RNA unwinding activity. Hydrolysis of ATP is required for this activity: the replacement of ATP by a nonhydrolyzable analog or a mutation in the DEAD motif abolishing ATPase activity results in loss of RNA unwinding. We find that cells harboring a Ded1 protein with this mutated DEAD motif are nonviable, suggesting that the ATPase and RNA helicase activities of this protein are essential to the cell. Finally, RNA binding measurements indicate that the presence of ATP, but not ADP, increases the affinity of Ded1p for duplex versus single-stranded RNA; we discuss how this differential effect might drive the unwinding reaction.     

Pag1p, a novel protein associated with protein kinase Cbk1p, is required for cell morphogenesis and proliferation in Saccharomyces cerevisiae

Protein kinases in the Cot-1/Orb6/Ndr/Warts family are important regulators of cell morphogenesis and proliferation. Cbk1p, a member of this family in Saccharomyces cerevisiae, has previously been shown to be required for normal morphogenesis in vegetatively growing cells and in haploid cells responding to mating pheromone. A mutant of PAG1, a novel gene in S. cerevisiae, displayed defects similar to those of cbk1 mutants. pag1 and cbk1 mutants share a common set of suppressors, including the disruption of SSD1, a gene encoding an RNA binding protein, and the overexpression of Sim1p, an extracellular protein. These genetic results suggest that PAG1 and CBK1 act in the same pathway. Furthermore, we found that Pag1p and Cbk1p localize to the same polarized peripheral sites and that they coimmunoprecipitate with each other. Pag1p is a conserved protein. The homologs of Pag1p in other organisms are likely to form complexes with the Cbk1p-related kinases and function with those kinases in the same biological processes.     

The C-terminus of eRF1 defines a functionally important domain for translation termination in Saccharomyces cerevisiae

Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3, which interact to form a heterodimer that mediates termination at all three stop codons. By C-terminal deletion analysis of eRF1 from the yeast Saccharomyces cerevisiae, we show that the extreme C-terminus of this 437-amino-acid protein defines a functionally important domain for translation termination. A strain encoding eRF1 lacking the C-terminal 32 amino acids is not viable, whereas deletion of the C-terminal 19 amino acids is viable but shows a termination defect in vivo causing an enhancement of nonsense suppression. Using a combination of two-hybrid analysis and in vitro binding studies, we demonstrate that deletions encompassing the C-terminus of eRF1 cause a significant reduction in eRF3 binding to eRF1. All of the C-terminally truncated eRF1 still bind the ribosome, suggesting that the C-terminus does not constitute a ribosome-binding domain and eRF1 does not need to form a stable complex with eRF3 in order to bind the ribosome. These data, together with previously published data, suggest that the region between amino acids 411 and 418 of yeast eRF1 defines an essential functional domain that is part of the major site of interaction with eRF3. However, a stable eRF1:eRF3 complex does not have to be formed to maintain viability or efficient translation termination. Alignment of the seven known eukaryotic eRF1 sequences indicates that a highly conserved motif, GFGGIGG/A is present within the region of the C-terminus, although our deletion studies suggest that it is sequences C-terminal to this region that are functionally important.     

Tpa1p is part of an mRNP complex that influences translation termination, mRNA deadenylation, and mRNA turnover in Saccharomyces cerevisiae

In this report, we show that the Saccharomyces cerevisiae protein Tpa1p (for termination and polyadenylation) influences translation termination efficiency, mRNA poly(A) tail length, and mRNA stability. Tpa1p is encoded by the previously uncharacterized open reading frame YER049W. Yeast strains carrying a deletion of the TPA1 gene (tpa1Delta) exhibited increased readthrough of stop codons, and coimmunoprecipitation assays revealed that Tpa1p interacts with the translation termination factors eRF1 and eRF3. In addition, the tpa1Delta mutation led to a 1.5- to 2-fold increase in the half-lives of mRNAs degraded by the general 5'-->3' pathway or the 3'-->5' nonstop decay pathway. In contrast, this mutation did not have any affect on the nonsense-mediated mRNA decay pathway. Examination of mRNA poly(A) tail length revealed that poly(A) tails are longer than normal in a tpa1Delta strain. Consistent with a potential role in regulating poly(A) tail length, Tpa1p was also found to coimmunoprecipitate with the yeast poly(A) binding protein Pab1p. These results suggest that Tpa1p is a component of a messenger ribonucleoprotein complex bound to the 3' untranslated region of mRNAs that affects translation termination, deadenylation, and mRNA decay.     

Characterization of Vta1p, a class E Vps protein in Saccharomyces cerevisiae

We identified VTA1 in a screen for mutations that result in altered vacuole morphology. Deletion of VTA1 resulted in delayed trafficking of the lipophilic dye FM4-64 to the vacuole and altered vacuolar morphology when cells were exposed to the dye 5-(and 6)-carboxy-2',7'-dichlorofluorescein diacetate (CDCFDA). Deletion of class E vacuolar protein sorting (VPS) genes, which encode proteins that affect multivesicular body formation, also showed altered vacuolar morphology upon exposure to high concentrations of CDCFDA. These results suggest a VPS defect for Deltavta1 cells. Deletion of VTA1 did not affect growth on raffinose and only mildly affected carboxypeptidase S sorting. Turnover of the surface protein Ste3p, the a-factor receptor, was affected in Deltavta1 cells with the protein accumulating on the vacuolar membrane. Likewise the alpha-factor receptor Ste2p accumulated on the vacuolar membrane in Deltavta1 cells. We demonstrated that many class E VPS deletion strains are hyper-resistant to the cell wall disruption agent calcofluor white. Deletion of VTA1 or VPS60, another putative class E gene, resulted in calcofluor white hypersensitivity. A Vta1p-green fluorescent protein fusion protein transiently associated with a Pep12p-positive compartment. This localization was altered by deletion of many of the class E VPS genes, indicating that Vta1p binds to endosomes in a manner dependent on the assembly of the endosomal sorting complexes required for transport. Membrane-associated Vta1p co-purified with Vps60p, suggesting that Vta1p is a class E Vps protein that interacts with Vps60p on a prevacuolar compartment.     

Crp1p,a new cruciform DNA-binding protein in the yeast Saccharomyces cerevisiae

A synthetic cruciform DNA (X-DNA) was used for screening cellular extracts of Saccharomyces cerevisiae for X-DNA-binding activity. Three X-DNA-binding proteins with apparent molecular mass of 28kDa, 26kDa and 24kDa, estimated by SDS-PAGE, were partially purified. They were identified as N-terminal fragments originating from the same putative protein, encoded by the open reading frame YHR146W, which we named CRP1 (cruciform DNA-recognising protein 1). Expression of CRP1 in Escherichia coli showed that Crp1p is subject to efficient proteolysis at one specific site. Cleavage leads to an N-terminal subpeptide of approximately 160 amino acid residues that is capable of binding specifically X-DNA with an estimated dissociation constant (K(d)) of 800nM, and a C-terminal subpeptide of approximately 305 residues without intrinsic X-DNA-binding activity. The N-terminal subpeptide is of a size similarly to that of the fragments identified in yeast, suggesting that the same cleavage process occurs in the yeast and the E.coli background. This makes the action of a site-specific protease unlikely and favours the possibility of an autoproteolytic activity of Crp1p. The DNA-binding domain of Crp1p was mapped to positions 120-141. This domain can act autonomously as an X-DNA-binding peptide and provides a new, lysine-rich DNA-binding domain different from those of known cruciform DNA-binding proteins (CBPs). As reported earlier for several other CBPs, Crp1p exerts an enhancing effect on the cleavage of X-DNA by endonuclease VII from bacteriophage T4.     

Association between defects of karyogamy and translation termination in yeast Saccharomyces cerevisiae

Mutants capable of a high frequency of cytoduction (Hfc+) were obtained in a haploid strain of Saccharomyces cerevisiae, suggesting impaired cytogamy. Nine of the 68 Hfc+ mutants showed the antisuppressor effect with respect to mutations of the SUP35 and SUP45 genes, which code for translation termination factors, or to the [PSI+] factor, which is the prion form of Sup35. Cosegregation of the characters "higher frequency of cytoduction" and "antisuppression" was demonstrated for three Hfc+ mutants. One (HFC12-2) of the Hfc+ mutations exerted a dominant antisuppressor effect with respect to [PSI+] and had no effect on [PSI+] maintenance. On the strength of the results, an interaction was assumed for translation termination components and cytoskeleton proteins, which play a role in karyogamy in yeasts.     

Saccharomyces cerevisiae Dap1p,a novel DNA damage response protein related to the mammalian membrane-associated progesterone receptor

The response to damage is crucial for cellular survival, and eukaryotic cells require a broad array of proteins for an intact damage response. We have found that the YPL170W (DAP1 [for damage response protein related to membrane-associated progesterone receptors]) gene is required for growth in the presence of the methylating agent methyl methanesulfonate (MMS). The DAP1 open reading frame shares homology with a broadly conserved family of membrane-associated progesterone receptors (MAPRs). Deletion of DAP1 leads to sensitivity to MMS, elongated telomeres, loss of mitochondrial function, and partial arrest in sterol synthesis. Sensitivity of dap1 strains to MMS is not due to loss of damage checkpoints. Instead, dap1 cells are arrested as unbudded cells after MMS treatment, suggesting that Dap1p is required for cell cycle progression following damage. Dap1p also directs resistance to itraconazole and fluconazole, inhibitors of sterol synthesis. We have found that dap1 cells have slightly decreased levels of ergosterol but increased levels of the ergosterol intermediates squalene and lanosterol, indicating that dap1 cells have a partial defect in sterol synthesis. This is the first evidence linking a MAPR family member to sterol regulation or the response to damage, and these functions are probably conserved in a variety of eukaryotes.     

Saccharomyces cerevisiae GTPase complex: Gtr1p-Gtr2p regulates cell-proliferation through Saccharomyces cerevisiae Ran-binding protein, Yrb2p

A Gtr1p GTPase, the GDP mutant of which suppresses both temperature-sensitive mutants of Saccharomyces cerevisiae RanGEF/Prp20p and RanGAP/Rna1p, was presently found to interact with Yrb2p, the S. cerevisiae homologue of mammalian Ran-binding protein 3. Gtr1p bound the Ran-binding domain of Yrb2p. In contrast, Gtr2p, a partner of Gtr1p, did not bind Yrb2p, although it bound Gtr1p. A triple mutant: yrb2delta gtr1delta gtr2delta was lethal, while a double mutant: gtr1delta gtr2delta survived well, indicating that Yrb2p protected cells from the killing effect of gtr1delta gtr2delta. Recombinant Gtr1p and Gtr2p were purified as a complex from Escherichia coli. The resulting Gtr1p-Gtr2p complex was comprised of an equal amount of Gtr1p and Gtr2p, which inhibited the Rna1p/Yrb2 dependent RanGAP activity. Thus, the Gtr1p-Gtr2p cycle was suggested to regulate the Ran cycle through Yrb2p.     

Yct1p, a novel, high-affinity, cysteine-specific transporter from the yeast Saccharomyces cerevisiae

Cysteine transport in the yeast Saccharomyces cerevisiae is mediated by at least eight different permeases, none of which are specific for cysteine. We describe a novel, high-affinity, (K(m) = 55 microM), cysteine-specific transporter encoded by the ORF YLL055w that was initially identified by a combined strategy of data mining, bioinformatics, and genetic analysis. Null mutants of YLL055w, but not of the other genes encoding for transporters that mediate cysteine uptake such as GAP1, GNP1, MUP1, or AGP1 in a met15Delta background, resulted in a growth defect when cysteine, at low concentrations, was provided as the sole sulfur source. Transport experiments further revealed that Yll055wp was the major contributor to cysteine transport under these conditions. The contributions of the other transporters became relevant only at higher concentrations of cysteine or when YLL055w was either deleted or repressed. YLL055w expression was repressed by organic sulfur sources and was mediated by the Met4p-dependent sulfur regulatory network. The results reveal that YLL055w encodes the principal cysteine transporter in S. cerevisiae, which we have named YCT1 (yeast cysteine transporter). Interestingly, Yct1p belongs to the Dal5p family of transporters rather than the amino acid permease family to which all the known amino acid transporters belong.     

Functional and physical interactions of Faf1p, a Saccharomyces cerevisiae nucleolar protein

We report the discovery and characterisation of a novel nucleolar protein of Saccharomyces cerevisiae. We identified this protein encoded by ORF YIL019w, designated in SGD base as Faf1p, in a two hybrid interaction screen using the known nucleolar protein Krr1 as bait. The presented data indicate that depletion of the Faf1 protein has an impact on the 40S ribosomal subunit biogenesis resulting from a decrease in the production of 18S rRNA. The primary defect is apparently due to inefficient processing of 35S rRNA at the A(0), A(1), and A(2) cleavage sites.     

Molecular characterization of Ypi1, a novel Saccharomyces cerevisiae type 1 protein phosphatase inhibitor

The Saccharomyces cerevisiae open reading frame YFR003c encodes a small (155-amino acid) hydrophilic protein that we identified as a novel, heat-stable inhibitor of type 1 protein phosphatase (Ypi1). Ypi1 interacts physically in vitro with both Glc7 and Ppz1 phosphatase catalytic subunits, as shown by pull-down assays. Ypi1 inhibits Glc7 but appears to be less effective toward Ppz1 phosphatase activity under the conditions tested. Ypi1 contains a 48RHNVRW53 sequence, which resembles the characteristic consensus PP1 phosphatase binding motif. A W53A mutation within this motif abolishes both binding to and inhibition of Glc7 and Ppz1 phosphatases. Deletion of YPI1 is lethal, suggesting a relevant role of the inhibitor in yeast physiology. Cells overexpressing Ypi1 display a number of phenotypes consistent with an inhibitory role of this protein on Glc7, such as decreased glycogen content and an increased growth defect in a slt2/mpk1 mitogen-activated protein kinase-deficient background. Taking together, these results define Ypi1 as the first inhibitory subunit of Glc7 identified in budding yeast.     

Identification of the tRNA-binding protein Arc1p as a novel target of in vivo biotinylation in Saccharomyces cerevisiae

Biotin is an essential cofactor of cell metabolism serving as a protein-bound coenzyme in ATP-dependent carboxylation, in transcarboxylation, and certain decarboxylation reactions. The involvement of biotinylated proteins in other cellular functions has been suggested occasionally, but available data on this are limited. In the present study, a Saccharomyces cerevisiae protein was identified that reacts with streptavidin on Western blots and is not identical to one of the known biotinylated yeast proteins. After affinity purification on monomeric avidin, the biotinylated protein was identified as Arc1p. Using 14C-labeled biotin, the cofactor was shown to be incorporated into Arc1p by covalent and alkali-stable linkage. Similar to the known carboxylases, Arc1p biotinylation is mediated by the yeast biotin:protein ligase, Bpl1p. Mutational studies revealed that biotinylation occurs at lysine 86 within the N-terminal domain of Arc1p. In contrast to the known carboxylases, however, in vitro biotinylation of Arc1p is incomplete and increases with BPL1 overexpression. In accordance to this fact, Arc1p lacks the canonical consensus sequence of known biotin binding domains, and the bacterial biotin:protein ligase, BirA, is unable to use Arc1p as a substrate. Arc1p was shown previously to organize the association of MetRS and GluRS tRNA synthetases with their cognate tRNAs thereby increasing the substrate affinity and catalytic efficiency of these enzymes. Remarkably, not only biotinylated but also the biotin-free Arc1p obtained by replacement of lysine 86 with arginine were capable of restoring Arc1p function in both arc1Delta and arc1Deltalos1Delta mutants, indicating that biotinylation of Arc1p is not essential for activity.