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.     

The ubiquitin ligase Rsp5 is required for ribosome stability in Saccharomyces cerevisiae

Rsp5p is a conserved HECT-domain ubiquitin ligase with diverse roles in cellular physiology. Here we report a previously unknown role of Rsp5p in facilitating the stability of the cytoplasmic ribosome pool in budding yeast. Yeast strains carrying temperature-sensitive mutations in RSP5 showed a progressive decline in levels of 18S and 25S rRNAs and accumulation of rRNA decay fragments when cells grown in rich medium were shifted to restrictive temperature. This was accompanied by a decreased number of translating ribosomes and the appearance of ribosomal subunits with an abnormally low sedimentation rate in polysome analysis. Abrogating Rsp5p function affected stability of other tested noncoding RNA species (tRNA and snoRNA), but to a lower extent than that of rRNA, and also inhibited processing of rRNA and tRNA precursors, in agreement with previous studies. The breakdown of cellular ribosomes was not affected by deletion of key genes involved in autophagy, previously implicated in ribosome turnover upon starvation. Our results suggest that functional Rsp5p is required to maintain the integrity of cytoplasmic ribosomes under rich nutrient conditions.     

Inhibition of receptor-mediated but not fluid-phase endocytosis in polymorphonuclear leukocytes

We have found that hypertonic medium inhibited the receptor-mediated uptake of the chemotactic peptide N-formylnorleucylleucylphenylalanine without affecting fluid-phase endocytosis by polymorphonuclear leukocytes (PMNs). Morphological and biochemical evidence demonstrated that cells in hypertonic medium did not accumulate peptide in a receptor-mediated manner. However, the cells continued to form endosomes containing fluid-phase markers. Furthermore, the content of these endosomes was processed normally, i.e., both digested and intact material were released into the medium. The inhibition of receptor- mediated uptake was a function of the tonicity. Partial inhibition occurred in 0.45 and 0.6 osmolar medium and maximal inhibition occurred in 0.75 osmolar medium. The inhibition was independent of the solute used to increase the tonicity: sodium chloride, sucrose, and lactose all inhibited uptake to similar extents. Hypertonic medium had little effect on saturable peptide binding. However, it did prevent the clustering of surface molecules as indicated by the inhibition of capping of fluorescent concanavalin A. In addition, hypertonic medium prevented the peptide-stimulated increase in cytosolic calcium levels as measured by quin 2 fluorescence. The tonicity dependence of the inhibition of quin 2 fluorescence paralleled the inhibition of receptor- mediated uptake.     

Rsp5 ubiquitin ligase is required for protein trafficking in Saccharomyces cerevisiae COPI mutants

Retrograde trafficking from the Golgi to the endoplasmic reticulum (ER) depends on the formation of vesicles coated with the multiprotein complex COPI. In Saccharomyces cerevisiae ubiquitinated derivatives of several COPI subunits have been identified. The importance of this modification of COPI proteins is unknown. With the exception of the Sec27 protein (β'COP) neither the ubiquitin ligase responsible for ubiquitination of COPI subunits nor the importance of this modification are known. Here we find that the ubiquitin ligase mutation, rsp5-1, has a negative effect that is additive with ret1-1 and sec28Δ mutations, in genes encoding α- and ε-COP, respectively. The double ret1-1 rsp5-1 mutant is also more severely defective in the Golgi-to-ER trafficking compared to the single ret1-1, secreting more of the ER chaperone Kar2p, localizing Rer1p mostly to the vacuole, and increasing sensitivity to neomycin. Overexpression of ubiquitin in ret1-1 rsp5-1 mutant suppresses vacuolar accumulation of Rer1p. We found that the effect of rsp5 mutation on the Golgi-to-ER trafficking is similar to that of sla1Δ mutation in a gene encoding actin cytoskeleton proteins, an Rsp5p substrate. Additionally, Rsp5 and Sla1 proteins were found by co-immunoprecipitation in a complex containing COPI subunits. Together, our results show that Rsp5 ligase plays a role in regulating retrograde Golgi-to-ER trafficking.     

Functional Domains of the Rsp5 Ubiquitin-Protein Ligase

RSP5, an essential gene of Saccharomyces cerevisiae, encodes a hect domain E3 ubiquitin-protein ligase. Hect E3 proteins have been proposed to consist of two broad functional domains: a conserved catalytic carboxyl-terminal domain of approximately 350 amino acids (the hect domain) and a large, nonconserved amino-terminal domain containing determinants of substrate specificity. We report here the mapping of the minimal region of Rsp5 necessary for its essential in vivo function, the minimal region necessary to stably interact with a substrate of Rsp5 (Rpb1, the large subunit of RNA polymerase II), and the finding that the hect domain, by itself, is sufficient for formation of the ubiquitin-thioester intermediate. Mutations within the hect domain that affect either the ability to form a ubiquitin-thioester or to catalyze substrate ubiquitination abrogate in vivo function, strongly suggesting that the ubiquitin-protein ligase activity of Rsp5 is intrinsically linked to its essential function. The amino-terminal region of Rsp5 contains three WW domains and a C2 calcium-binding domain. Two of the three WW domains are required for the essential in vivo function, while the C2 domain is not, and requirements for Rpb1 binding and ubiquitination lie within the region required for in vivo function. Together, these results support the two-domain model for hect E3 function and indicate that the WW domains play a role in the recognition of at least some of the substrates of Rsp5, including those related to its essential function. In addition, we show that haploid yeast strains bearing complete disruptions of either of two other hect E3 genes of yeast, designated HUL4 (YJR036C) and HUL5 (YGL141W), are viable.     

Multivesicular body sorting: ubiquitin ligase Rsp5 is required for the modification and sorting of carboxypeptidase S

The multivesicular body (MVB) sorting pathway provides a mechanism for delivering transmembrane proteins into the lumen of the lysosome/vacuole. Recent studies demonstrated that ubiquitin modification acts in cis as a signal for the sorting of cargoes into this pathway. Here, we present results from a genetic selection designed to identify mutants that missort MVB cargoes. This selection identified a point mutation in ubiquitin ligase Rsp5 (Rsp5-326). At the permissive temperature, this mutant is specifically defective for ubiquitination and sorting of the ubiquitin-dependent MVB cargo precursor carboxypeptidase S (pCPS), but not ligand-induced ubiquitination of Ste2. A previous study implicated Tul1 as the ubiquitin ligase responsible for MVB sorting of pCPS. However, we detected no defect in either the sorting or ubiquitination of pCPS in tul1 mutants. We had previously shown that Fab1 phosphatidylinositol 3-phosphate 5-kinase is also required for MVB sorting of pCPS, but not Ste2. However, our analyses reveal that fab1 mutants do not exhibit a defect in ubiquitination of pCPS. Thus, both Rsp5 and Fab1 play distinct and essential roles in the targeting of biosynthetic MVB cargoes. However, whereas Rsp5 seems to be responsible for cargo ubiquitination, the precise role for Fab1 remains to be elucidated.     

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.     

The ubiquitin ligase Rsp5p is required for modification and sorting of membrane proteins into multivesicular bodies

Precursor forms of vacuolar proteins with transmembrane domains, such as the carboxypeptidase S Cps1p and the polyphosphatase Phm5p, are selectively sorted in endosomal compartments to vesicles that invaginate, budding into the lumen of the late endosomes, resulting in the formation of multivesicular bodies (MVBs). These proteins are then delivered to the vacuolar lumen following fusion of the MVBs with the vacuole. The sorting of Cps1p and Phm5p to these structures is mediated by ubiquitylation, and in doa4 mutant cells, which have reduced level of free ubiquitin, these proteins are missorted to the vacuolar membrane. A RING-finger ubiquitin ligase Tul1p has been shown to participate in the ubiquitylation of Cps1p and Phm5p. We show here that the HECT-ubiquitin ligase Rsp5p is also required for the ubiquitylation of these proteins, and therefore for their sorting to MVBs. Rsp5p is an essential ubiquitin ligase containing an N-terminal C2 domain followed by three WW domains, and a C-terminal catalytic HECT domain. In cells with low levels of Rsp5p (npi1 mutant cells), vacuolar hydrolases do not reach the vacuolar lumen and are instead missorted to the vacuolar membrane. The C2 domain and both the second and third WW domains of Rsp5p are important determinants for sorting to MVBs. Ubiquitylation of Cps1p was strongly reduced in the npi1 mutant strain and ubiquitylation was completely abolished in the npi1 tul1Delta double mutant. These data demonstrate that Rsp5p plays a novel and key role in intracellular trafficking, and extend the currently very short list of substrates ubiquitylated in vivo by several different ubiquitin ligases acting cooperatively.     

ATP is required for receptor-mediated endocytosis in intact cells

We have demonstrated a requirement for cellular ATP in the receptor- mediated endocytosis of transferrin. This has been accomplished using a novel assay for endocytosis based on acquisition of resistance to the membrane impermeable reducing agent, glutathione (GSH). Diferric- transferrin was conjugated to biotin via a cleavable disulfide bond and iodinated. Internalization of 125I-biotin-S-S-transferrin (125I-BSST) was quantitated by adsorption to avidin-Sepharose after treatment of cells with GSH. Receptor-mediated endocytosis of 125I-BSST was severely inhibited in ATP-depleted cells. Similar results were obtained when ATP was depleted by incubation of cells either under a N2-atmosphere or in the presence of NaN3 and NaF. The latter treatment, alone, also resulted in a loss of surface transferrin receptors which could not be correlated to reductions in cellular ATP. In contrast to the acquisition of GSH resistance, the apparent internalization of 125I- BSST as assessed by inaccessibility to antitransferrin antibodies reached control levels in ATP-depleted cells. Our biochemical and morphological data suggested that, although ATP is required for receptor-mediated endocytosis, in ATP-depleted cells ligands can become efficiently sequestered into deeply invaginated pits that are inaccessible to large probes such as antibodies, but remain accessible to small molecules such as GSH.     

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.     

Stable variant of LM fibroblast defective in fluid-phase but competent in receptor-mediated endocytosis

The F-40 cell line, a stable variant of LM fibroblasts selected for its resistance to polyethylene glycol (PEG)-induced fusion (Roos and Davidson: Somatic Cell and Molecular Genetics 6:381-391, 1980), has a decreased capacity to internalize fluid-phase markers and nonspecifically surface-bound macromolecules. It is not defective in exocytosis since, after a short sucrose pulse, it releases the same fraction of ingested sucrose into the medium as does the parental line. F40 cells have a normal capacity to carry out receptor-mediated endocytosis, as tested with 125I-alpha-2 macroglobulin (alpha-2 MG) and 125I-transferrin (Tf), and to recycle Tf receptor to the cell surface. These data demonstrate that receptor-mediated and non-receptor mediated endocytosis are distinct processes that can be altered independently. Of the many membrane fusions occurring in the course of endocytosis, the only one that appears associated with the defect in cell fusion characteristic of F40 cells is the formation of primary endocytotic vesicles engaged in non-receptor-mediated internalizations.     

Multiple roles for Rsp5p-dependent ubiquitination at the internalization step of endocytosis

Ubiquitination of integral plasma membrane proteins triggers their rapid internalization into the endocytic pathway. The yeast ubiquitin ligase Rsp5p, a homologue of mammalian Nedd4 and Itch, is required for the ubiquitination and subsequent internalization of multiple plasma membrane proteins, including the alpha-factor receptor (Ste2p). Here we demonstrate that Rsp5p plays multiple roles at the internalization step of endocytosis. Temperature-sensitive rsp5 mutant cells were defective in the internalization of alpha-factor by a Ste2p-ubiquitin chimera, a receptor that does not require post-translational ubiquitination. Similarly, a modified version of Ste2p bearing a NPFXD linear peptide sequence as its only internalization signal was not internalized in rsp5 cells. Internalization of these variant receptors was dependent on the catalytic cysteine residue of Rsp5p and on ubiquitin-conjugating enzymes that bind Rsp5p. Thus, a Rsp5p-dependent ubiquitination event is required for internalization mediated by ubiquitin-dependent and -independent endocytosis signals. Constitutive Ste2p-ubiquitin internalization and fluid-phase endocytosis also required active ubiquitination machinery, including Rsp5p. These observations indicate that Rsp5p-dependent ubiquitination of a trans-acting protein component of the endocytosis machinery is required for the internalization step of endocytosis.     

Regulation of copper-dependent endocytosis and vacuolar degradation of the yeast copper transporter, Ctr1p, by the Rsp5 ubiquitin ligase

The Saccharomyces cerevisiae high-affinity copper transporter, Ctr1p, mediates cellular uptake of Cu(I). We report that when copper (50 microm CuSO(4)) is added to the growth medium of copper-starved cells, Ctr1p is rapidly internalized by endocytosis, delivered to the lumen of the lysosome-like vacuole and slowly degraded by vacuolar proteases. Through analysis of the trafficking and degradation of Ctr1p mutants, two lysine residues in the C-terminal cytoplasmic tail of Ctr1p, Lys340 and Lys345, were found to be critical for copper-dependent endocytosis and degradation. In response to copper addition, Ctr1p was found to be ubiquitylated and a mutation in the Rsp5 ubiquitin ligase largely abolished ubiquitylation, endocytosis and degradation. In a strain lacking the Rsp5p accessory factors Bul1p and Bul2p, endocytosis and degradation of Ctr1p-green fluorescent protein were substantially diminished. Surprisingly, a Ctr1p mutant that lacks Lys340 and Lys345 was still ubiquitylated in a copper-dependent manner, indicating that ubiquitylation of Ctr1p on other sites is insufficient to drive copper-dependent endocytosis and degradation. This study demonstrates that copper regulates turnover of Ctr1p by stimulating Rsp5p-dependent endocytosis and degradation of Ctr1p in the vacuole.     

A high throughput screen to identify substrates for the ubiquitin ligase Rsp5

Ubiquitin-protein ligases (E3s) are implicated in various human disorders and are attractive targets for therapeutic intervention. Although most cellular proteins are ubiquitinated, ubiquitination cannot be linked directly to a specific E3 for a large fraction of these proteins, and the substrates of most E3 enzymes are unknown. We have developed a luminescent assay to detect ubiquitination in vitro, which is more quantitative, effective, and sensitive than conventional ubiquitination assays. By taking advantage of the abundance of purified proteins made available by genomic efforts, we screened hundreds of purified yeast proteins for ubiquitination, and we identified previously reported and novel substrates of the yeast E3 ligase Rsp5. The relevance of these substrates was confirmed in vivo by showing that a number of them interact genetically with Rsp5, and some were ubiquitinated by Rsp5 in vivo. The combination of this sensitive assay and the availability of purified substrates will enable the identification of substrates for any purified E3 enzyme.     

Yeast ubiquitin ligase Rsp5 contains nuclear localization and export signals

The Rsp5 ubiquitin ligase regulates numerous cellular processes. Rsp5 is mainly localized to the cytoplasm but nuclear localization was also reported. A potential nuclear export signal was tested for activity by using a GFP(2) reporter. The 687-LIGGIAEIDI-696 sequence located in the Hect domain was identified as a nuclear export signal active in a Crm1-dependent manner, and its importance for the localization of Rsp5 was documented by using fluorescence microscopy and a lacZ-based reporter system. Analysis of the cellular location of other Rsp5 fragments fused with GFP(2) indicated two independent potential nuclear localization signals, both located in the Hect domain. We also uncovered Rsp5 fragments that are important to targeting/tethering Rsp5 to various regions in the cytoplasm. The presented data indicate that Rsp5 ligase is a shuttling protein whose distribution within the cytoplasm and partitioning between cytoplasmic and nuclear locations is determined by a balance between the actions of several targeting sequences and domains.     

Pharmacological attenuation of Paramecium fluid-phase endocytosis

Spectrophotometric quantification of fluid phase endocytosis in the presence of different pharmacological compounds was performed in the model unicellular eukaryote Paramecium. The kinetics of Lucifer Yellow Carbohydrazide (LY) uptake in cells exposed to forskolin and isoproterenol--known to stimulate phagocytosis in this cell--was analyzed. Reduction in both the rate of endocytosis and total accumulation of fluid phase marker was observed following the treatment. Forskolin diminished total LY accumulation by 11% and 21% after 5 min and 25 min of incubation, respectively, whereas the rate of uptake was lowered by 21% in comparison to control cells. The inhibitory effect ofisoproterenol was less pronounced than that of forskolin. The total accumulation of LY was decreased by 11% in 5 min as compared to the untreated cells and this effect was persistent upon further exposition to this reagent up to 25 min. To better understand these observations, the effect of inhibitors of PKA and cAMP phosphodiesterase on fluid phase uptake was tested. 3-isobutyl-1-methyl xanthine (IBMX) caused 12% decrease in LY accumulation after 5 min of incubation. In combination with isoproterenol or forskolin, IBMX enhanced their inhibitory effect on fluid endocytosis, which was lowered by 25% and 29%, respectively. The strongest inhibitory effect on fluid endocytosis was exerted by the 10 microM PKA inhibitor, which diminished endocytosis by 35% in 5 min. These results suggest that Paramecium fluid phase uptake may be regulated through activation of PKA, although the precise mechanism of this process has not yet been elucidated.     

Identification of determinants in E2 ubiquitin-conjugating enzymes required for hect E3 ubiquitin-protein ligase interaction

Members of the hect domain protein family are characterized by sequence similarity of their C-terminal regions to the C terminus of E6-AP, an E3 ubiquitin-protein ligase. An essential intermediate step in E6-AP-dependent ubiquitination is the formation of a thioester complex between E6-AP and ubiquitin in the presence of distinct E2 ubiquitin-conjugating enzymes including human UbcH5, a member of the UBC4/UBC5 subfamily of E2s. Similarly, several hect domain proteins, including Saccharomyces cerevisiae RSP5, form ubiquitin thioester complexes, indicating that hect domain proteins in general have E3 activity. We show here, by the use of chimeric E2s generated between UbcH5 and other E2s, that a region of UbcH5 encompassing the catalytic site cysteine residue is critical for its ability to interact with E6-AP and RSP5. Of particular importance is a phenylalanine residue at position 62 of UbcH5 that is conserved among the members of the UBC4/UBC5 subfamily but is not present in any of the other known E2s, whereas the N-terminal 60 amino acids do not contribute significantly to the specificity of these interactions. The conservation of this phenylalanine residue throughout evolution underlines the importance of the ability to interact with hect domain proteins for the cellular function of UBC4/UBC5 subfamily members.     

Yeast Rsp5 ubiquitin ligase affects the actin cytoskeleton in vivo and in vitro

Yeast Rsp5 ubiquitin ligase is involved in several cellular processes, including endocytosis. Actin patches are sites of endocytosis, a process involving actin assembly and disassembly. Here we show Rsp5 localization in cortical patches and demonstrate its involvement in actin cytoskeleton organization and dynamics. We found that the Rsp5-F1-GFP2 N-terminal fragment and full length GFP-Rsp5 were recruited to peripheral patches that temporarily co-localized with Abp1-mCherry, a marker of actin patches. Actin cytoskeleton organization was defective in a strain lacking RSP5 or overexpressing RSP5, and this phenotype was accompanied by morphological abnormalities. Overexpression of RSP5 caused hypersensitivity of cells to Latrunculin A, an actin-depolymerizing drug and was toxic to cells lacking Las17, an activator of actin nucleation. Moreover, Rsp5 was required for efficient actin polymerization in a whole cell extract based in vitro system. Rsp5 interacted with Las17 and Las17-binding proteins, Lsb1 and Lsb2, in a GST-Rsp5-WW2/3 pull down assay. Rsp5 ubiquitinated Lsb1-HA and Lsb2-HA without directing them for degradation. Overexpression of RSP5 increased the cellular level of HA-Las17 in wild type and in lsb1Δ lsb2Δ strains in which the basal level of Las17 was already elevated. This increase was prevented in a strain devoid of Las17-binding protein Sla1 which is also a target of Rsp5 ubiquitination. Thus, Rsp5 together with Lsb1, Lsb2 and Sla1 regulate the level of Las17, an important activator of actin polymerization.     

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.