RESUMO
The regulation of cellular membrane dynamics is crucial for maintaining proper cell growth and division. The Cdc48-Npl4-Ufd1 complex is required for several regulated membrane-associated processes as part of the ubiquitin-proteasome system, including ER-associated degradation and the control of lipid composition in yeast. In this study we report the results of a genetic screen in Saccharomyces cerevisiae for extragenic suppressors of a temperature-sensitive npl4 allele and the subsequent analysis of one suppressor, GET3/ARR4. The GET3 gene encodes an ATPase with homology to the regulatory component of the bacterial arsenic pump. Mutants of GET3 rescue several phenotypes of the npl4 mutant and transcription of GET3 is coregulated with the proteasome, illustrating a functional relationship between GET3 and NPL4 in the ubiquitin-proteasome system. We have further found that Get3 biochemically interacts with the trans-membrane domain proteins Get1/Mdm39 and Get2/Rmd7 and that Deltaget3 is able to suppress phenotypes of get1 and get2 mutants, including sporulation defects. In combination, our characterization of GET3 genetic and biochemical interactions with NPL4, GET1, and GET2 implicates Get3 in multiple membrane-dependent pathways.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Membrana/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Complexos Multiproteicos/metabolismo , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Fenótipo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Esporos Fúngicos/genética , Supressão Genética/fisiologia , Distribuição Tecidual , Proteína com Valosina , Proteínas de Transporte VesicularRESUMO
Spore formation in Saccharomyces cerevisiae involves the sequential deposition of multiple spore wall layers between the prospore membranes that surround each meiotic product. The Smk1p mitogen-activated protein (MAP) kinase plays a critical role in spore formation, but the proteins that interact with Smk1p to regulate spore morphogenesis have not been described. Using mass spectrometry, we identify Gsc2p as a Smk1p-associated protein. Gsc2p is a 1,3-beta-glucan synthase subunit involved in synthesizing an inner spore wall layer. We find that 1,3-beta-glucan synthase activity is elevated in smk1 mutants, suggesting that SMK1 negatively regulates GSC2. Although deposition of the two inner spore wall layers is normal in smk1 mutants, deposition of the outer layers is aberrant. However, eliminating GSC2 activity restores normal deposition of the third spore wall layer in smk1 mutants, indicating that negative regulation of GSC2 by SMK1 is important for spore wall deposition. Our findings suggest a model for the coordination of spore wall layer deposition in which Smk1p facilitates the transition between early and late phases of spore wall deposition by inhibiting a spore wall-synthesizing enzyme important for early phases of spore wall deposition.
