The Rsp5 E3 ligase mediates turnover of low affinity phosphate transporters in Saccharomyces cerevisiae.
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.
Estrella
Luis A
LA
Department of Biochemistry, Division of Nucleic Acids Enzymology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Krishnamurthy
Shankarling
S
Timme
Cindy R
CR
Hampsey
Michael
M
D000483:Alleles; D019943:Amino Acid Substitution; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport; D020125:Mutation, Missense; D028061:Phosphate Transport Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*
D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; C091908:END3 protein, S cerevisiae; D056827:Endosomal Sorting Complexes Required for Transport; C060292:PHO4 protein, S cerevisiae; C525960:Pho91 protein, S cerevisiae; D028061:Phosphate Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D043743:Ubiquitin-Protein Ligase Complexes; C469287:RSP5 protein, S cerevisiae