PMID:18165238

Citation	Estrella, LA, Krishnamurthy, S, Timme, CR and Hampsey, M (2008) The Rsp5 E3 ligase mediates turnover of low affinity phosphate transporters in Saccharomyces cerevisiae. J. Biol. Chem. 283:5327-34
Abstract	In an effort to identify novel components of the PHO regulon in Saccharomyces cerevisiae, we have isolated and characterized suppressors of the Pho(-) phenotype associated with deletion of the Pho4 transcriptional activator. Here we report that either a defective form of the Rsp5 E3 ubiquitin ligase or deletion of the End3 component of the endocytic pathway restores growth of the pho4 Delta mutant in the presence of limiting inorganic phosphate (P i). The spa1-1 suppressor allele of RSP5 encodes a phenylalanine-to-valine replacement at position 748 (F748V) within the catalytic HECT domain of Rsp5. Consistent with suppression due to impaired ubiquitin ligase activity, the heat-sensitive growth defect of the spa1-1 mutant is suppressed either by overexpression of ubiquitin or by osmotic stabilization. Western blot analyses revealed that the cellular levels of the Pho87 and Pho91 low affinity P i are markedly increased in the spa1-1 mutant, yet Pho84 high affinity P i transporter levels are unaffected. Furthermore, Pho87 and Pho91 are ubiquitinated in vivo in an Rsp5-dependent manner, and the Pho+ phenotype of the spa1-1 suppressor is dependent upon Pho87 and Pho91. We conclude that turnover of the low affinity P i transporters is initiated by Rsp5-mediated ubiquitination followed by internalization and degradation by the endocytic pathway.
Links	PubMed Online version:10.1074/jbc.M703630200
Keywords	Alleles; Amino Acid Substitution; Cytoskeletal Proteins/genetics; Cytoskeletal Proteins/metabolism; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Endocytosis/physiology; Endosomal Sorting Complexes Required for Transport; Mutation, Missense; Phosphate Transport Proteins/genetics; Phosphate Transport Proteins/metabolism; Saccharomyces cerevisiae/enzymology; Saccharomyces cerevisiae/genetics; Saccharomyces cerevisiae Proteins/genetics; Saccharomyces cerevisiae Proteins/metabolism; Transcription Factors/genetics; Transcription Factors/metabolism; Ubiquitin-Protein Ligase Complexes/genetics; Ubiquitin-Protein Ligase Complexes/metabolism; Ubiquitination/physiology
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