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J. Biol. Chem., Vol. 279, Issue 33, 34421-34430, August 13, 2004
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From the Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain
The yeast gene VHS3 (YOR054c) has been recently identified as a multicopy suppressor of the G1/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 His459, equivalent to the supposedly functionally relevant His90 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.
Received for publication, January 20, 2004 , and in revised form, June 7, 2004.
* This work was supported by Grants BMC2002-04011-C05-04 and GEN2001-4707-C08-03 (Ministerio de Ciencia y Tecnología, Spain and Fondo Europeo de Desarrollo Regional), and 2001SGR00193 from the Generalitat de Catalunya (to J. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a fellowship from the DURSI (Generalitat de Catalunya).
Recipients of fellowships from the Ministerio de Educación, Cultura y Deporte, Spain.
¶ Recipient of a fellowship from the Universitat Autònoma de Barcelona.
|| To whom correspondence should be addressed: Dept. Bioquímica i Biologia Molecular, Facultat de Veterinària, Ed. V, Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain. Tel.: 34-93-5812182; Fax: 34-93-5812006; E-mail: Joaquin.Arino{at}uab.es.
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