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Genes Encoding Ribosomal Proteins Rps0a/B of Saccharomyces Cerevisiae Interact with TOM1 Mutants Defective in Ribosome Synthesis

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Genes Encoding Ribosomal Proteins Rps0a/B of Saccharomyces Cerevisiae Interact with TOM1 Mutants Defective in Ribosome Synthesis Copyright 2001 by the Genetics Society of America Genes Encoding Ribosomal Proteins Rps0A/B of Saccharomyces cerevisiae Interact With TOM1 Mutants Defective in Ribosome Synthesis Amy L. Tabb,* Takahiko Utsugi,² Clavia R. Wooten-Kee,* Takeshi Sasaki,² Steven A. Edling,* William Gump,* Yoshiko Kikuchi² and Steven R. Ellis* *Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, Kentucky 40292 and ²Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan Manuscript received September 15, 2000 Accepted for publication November 22, 2000 ABSTRACT The Saccharomyces cerevisiae RPS0A/B genes encode proteins of the 40S ribosomal subunit that are required for the maturation of 18S rRNA. We show here that the RPS0 genes interact genetically with TOM1. TOM1 encodes a member of the hect-domain-containing E3 ubiquitin-protein ligase family that is required for growth at elevated temperatures. Mutant alleles of the RPS0 and TOM1 genes have synergistic effects on cell growth at temperatures permissive for TOM1 mutants. Moreover, the growth arrest of TOM1 mutants at elevated temperatures is partially suppressed by overexpression of RPS0A/B. Strains with mutant alleles of TOM1 are defective in multiple steps in rRNA processing, and interactions between RPS0A/B and TOM1 stem, in part, from their roles in the maturation of ribosomal subunits. Ribosome synthesis is therefore included among the cellular processes governed by members of the hect-domain-containing E3 ubiquitin-protein ligase family. PS0A and RPS0B are duplicated genes of Saccharo- ence yeast cell growth, we undertook a synthetic lethal R myces cerevisiae that encode protein components of screen to identify genes that are critically sensitive to the 40S ribosomal subunit (Demianova et al. 1996). the level of Rps0 protein. A gene identi®ed in this screen Deletion of either RPS0 gene reduces growth rate, and was TOM1, which encodes a 3268-amino-acid protein deletion of both genes is lethal. Recent studies have containing a hect (homologous to E6-AP Cterminus) shown that the Rps0 proteins are required for pro- domain. Hect domains are characteristic of a family of cessing the 20S rRNA precursor to mature 18S rRNA, E3 ubiquitin-protein ligases that include the human E6- a late event in the maturation of 40S subunits (Ford et AP protein and the yeast Rsp5 (Huibregtse et al. 1995). al. 1999). Some of these proteins, including Tom1, interact with The Rps0 proteins have Ͼ60% sequence identity with transcription coactivator complexes (Saleh et al. 1998). the human p40/37-kD laminin-binding protein (37-LBP). Tom1 is required for full induction of the general stress The p40/37-LBP gene is overexpressed in a wide range and heat shock responses and is also required for ef®- of human tumors (Mafune et al. 1990; D'Errico et al. cient mRNA export (Utsugi et al. 1999; Duncan et al. 1991; Castronovo 1993; Vacca et al. 1993; Pei et al. 2000; Sasaki et al. 2000). Cells that have the hect domain 1996; Halatsch et al. 1997; Daheron et al. 1998). The of TOM1 deleted display temperature sensitivity and link between the overexpression of p40/37-LBP and arrest growth at the G2/M transition of the cell cycle tumorigenesis is frequently interpreted in terms of the (Utsugi et al. 1999). In addition to the synthetic lethal putative role for p40/37-LBP as a precursor for the 67- interaction between RPS0A/B and TOM1, growth arrest kD high af®nity laminin receptor (Liotta 1986; Cas- in TOM1 mutants can be partially suppressed by overex- tronovo 1993). This relationship is controversial, how- pression of RPS0 genes. This observation is particularly ever, and it is now known that, like the Rps0 proteins, intriguing in light of the link between overexpression p40/37-LBP is a component of the 40S ribosomal sub- of p40/37-LBP proteins and cancer. unit (Ardini et al. 1998). It is therefore important to Here, we show that mutant alleles of TOM1 affect determine if changes in expression of members of this the steady-state level of both large and small ribosomal family can in¯uence cell growth and proliferation in their subunits. Analysis of rRNA processing in TOM1 mutants roles as components of the translational machinery. revealed that the Tom1 protein in¯uences the matura- In an effort to understand how the level of expression tion of 18S rRNA by affecting several early steps in the of the RPS0 genes could impact other genes and in¯u- rRNA processing pathway. Therefore, the synthetic in- teraction between TOM1 and RPS0 mutants resides, in part, on a combined effect on the level of 40S subunits. Corresponding author: Steven R. Ellis, Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, KY 40292. TOM1 mutants also affect the level of 60S subunits, E-mail: [email protected] indicating that the Tom1 protein has one or more addi- Genetics 157: 1107±1116 (March 2001) 1108 A. L. Tabb et al. TABLE 1 Yeast strains used in this study Strain Genotype W3031B MAT␣␳ϩ ade2-1 can1-100 his3-11,15 ura3-1 leu2-3 trp1-1 W8-3dx MAT␣␳ϩ ade2-1 can1-100 his3-11,15 ura3-1 leu2-3 trp1-1 rps0A::URA3 RPS0B W8-3dy MATa ␳ϩ ade2-1 can1-100 his3-11,15 ura3-1 leu2-3 trp1-1 RPS0A rps0B::HIS3 Y505D MAT␣␳ϩ ade2 ade3 ura3 leu2 lys2 WYY5-2 MATa ␳ϩ ade2 ade3 ura3-1 lys2 RPS0A rps0B::HIS3 DOR1-111 MATa ␳ϩ ade2-1 ade3 ura3-1 lys2 RPS0A rps0B::HIS3 tom1-111 DOR1-111C MAT␣␳ϩ ade2-1 ade3 ura3-1 RPS0A rps0B::HIS3 tom1-111 DOR1-113 MATa ␳ϩ ade2-1 ade3 ura3-1 lys2 RPS0A rps0B::HIS3 tom1-113 1W29 MAT␣␳ϩ ade2-1 ura3-1 RPS0A RPS0B tom1-111 RAY3 MATa leu2 ura3 trp1 his3 tom1-2::LEU2 RAY5 MAT␣ leu2 ura3 trp1 his3 tom1-2::HIS3 tional roles in maturation/stability of ribosomal sub- sectors. Cells that did not sector and remained red could have units. These data add ribosome synthesis to the growing acquired a mutation that made the plasmid-borne RPS0A gene essential. Other mechanisms could also give a nonsectoring list of processes governed by members of the hect- phenotype such as integration of the plasmid into the chromo- domain-containing E3 ubiquitin ligase family. More- some or gene conversion between the wild-type ADE3 on the over, these data show that overexpression of a member plasmid with the mutant ade3 locus (Bender and Pringle of the p40/37-LBP family of proteins can in¯uence cell 1991). However, these latter mechanisms would not be ex- cycle progression in certain genetic backgrounds. pected to give a sectoring phenotype that was temperature sensitive. Therefore, only cells that did not sector at 37Њ but did sector at 30Њ were studied further. Two independent strains were characterized that exhibited a temperature-sensitive sec- MATERIALS AND METHODS toring phenotype. Each of these strains exhibited tempera- Yeast and bacterial strains: The yeast strains used in this ture-sensitive growth in the absence of the pTSV30A(RPS0A) work are listed in Table 1. Media used in cultivating yeast were plasmid, which could be complemented by RPS0A or RPS0B YPD (1% w/v yeast extract, 2% w/v peptone, and 2% w/v carried on plasmids other than pTSV30A. These strains were glucose) and synthetic (0.67% w/v yeast nitrogen base without labeled DOR for dependent on RPS0. amino acids and 2% w/v glucose). Where appropriate, nutri- Genetic procedures: DOR1-111 and DOR1-113 were crossed ents were added to synthetic media in amounts speci®ed by to the 8-3dy strain, which has a disrupted allele of RPS0B. The Sherman (1991). Diploids were sporulated on solid sporula- resulting diploids were tested for temperature-sensitive growth tion media (1% w/v potassium acetate, 0.1% w/v yeast extract, to determine if the loci responsible for the synthetic interac- 0.05% w/v glucose, and 2% w/v agar). Where appropriate, tions with the RPS0B deletion were recessive. Both loci were nutrients were added to sporulation media in 25% of the shown to be recessive (data not shown). Crosses were also amounts used in synthetic media. The Escherichia coli strain made to determine if the loci involved in the synthetic interac- used in this study was XL1-Blue (Stratagene, La Jolla, CA). tions with the RPS0B deletion formed a single complementa- DNA construction and sectoring assay: The RPS0A gene was tion group. Since DOR1-111 and DOR1-113 are the same inserted into the plasmid pTSV30A for use in a synthetic lethal mating type, DOR1-111 was outcrossed with 8-3dy to obtain screen for genes critically sensitive to RPS0 gene dosage. The a strain of opposite mating type that exhibited the synthetic plasmid pTSV30A contains the selectable marker LEU2 and a interaction with disrupted RPS0B. A haploid strain from this wild-type ADE3 gene for use in a color sectoring assay (Bender cross, DOR1-111C, was crossed to DOR1-113 and the resulting and Pringle 1991). The RPS0A gene was excised from plasmid diploids were tested for temperature-sensitive growth. The pJM9 as a 2.7-kb BamHI/SalI fragment (Demianova et al. diploids were temperature sensitive, demonstrating that loci 1996) and inserted into the unique BamHI and SalI sites of involved in the synthetic interactions with the RPS0B deletion plasmid pTSV30A. This plasmid was transformed into the formed a single complementation group (data not shown). strain WYY5-2, which contains a disrupted allele of RPS0B and The synthetic interactions with the RPS0B deletion in the mutant alleles of ade2 and ade3.
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