Functional characterization of the Saccharomyces cerevisiae VHS3 gene: a regulatory subunit of the Ppz1 protein phosphatase with novel, phosphatase-unrelated functions.
The yeast gene VHS3 (YOR054c) has been recently identified as a multicopy suppressor of the G(1)/S cell cycle blockade of a conditional sit4 and hal3 mutant. Vhs3 is structurally related to Hal3, a negative regulatory subunit of the Ser/Thr protein phosphatase Ppz1 important for cell integrity, salt tolerance, and cell cycle control. Phenotypic analyses using vhs3 mutants and overexpressing strains clearly show that Vhs3 has functions reminiscent to those of Hal3 and contrary to those of Ppz1. Mutation of Vhs3 His(459), equivalent to the supposedly functionally relevant His(90) in the plant homolog AtHal3a, did not affect Vhs3 functions mentioned above. Similarly to Hal3, Vhs3 binds in vivo to the C-terminal catalytic moiety of Ppz1 and inhibits in vitro its phosphatase activity. Therefore, our results indicate that Vhs3 plays a role as an inhibitory subunit of Ppz1. We have found that the vhs3 and hal3 mutations are synthetically lethal. Remarkably, lethality is not suppressed by deletion of PPZ1, PPZ2, or both phosphatase genes, indicating that it is not because of an excess of Ppz phosphatase activity. Furthermore, a Vhs3 version carrying the H459A mutation did not rescue the synthetically lethal phenotype. A conditional vhs3 tetO:HAL3 double mutant displays, in the presence of doxycycline, a flocculation phenotype that is dependent on the presence of Flo8 and Flo11. These results indicate that, besides its role as Ppz1 inhibitory subunit, Vhs3 (and probably Hal3) might have important Ppz-independent functions.
Ruiz
Amparo
A
Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain.
Muñoz
Ivan
I
Serrano
Raquel
R
González
Asier
A
Simón
Ernesto
E
Ariño
Joaquín
J
D000595:Amino Acid Sequence; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000235:genetics*; D020134:Catalytic Domain; D003062:Codon; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology; D017353:Gene Deletion; D005838:Genotype; D005982:Glutathione Transferase / Q000378:metabolism; D006579:Heterozygote; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D054645:Protein Phosphatase 1; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012492:Salts / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D001616:beta-Galactosidase / Q000378:metabolism
D003062:Codon; C110690:FLO11 protein, S cerevisiae; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D012492:Salts; D005982:Glutathione Transferase; C092048:GLC7 protein, S cerevisiae; C075119:PPZ1 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C490367:VHS3 protein, S cerevisiae; D001616:beta-Galactosidase; D002262:Carboxy-Lyases; D004318:Doxycycline