Isolation and functional analysis of yeast ubiquitin ligase Rsp5 variants that alleviate the toxicity of human α-synuclein

J Biochem. 2015 Apr;157(4):251-60. doi: 10.1093/jb/mvu069. Epub 2014 Nov 14.

Abstract

The essential ubiquitin ligase Rsp5 is a key enzyme involved in the degradation of abnormal or unfavourable proteins in the yeast Saccharomyces cerevisiae. Overexpression of human α-synuclein (α-syn), a small lipid-binding protein implicated in several neurodegenerative diseases, in S. cerevisiae leads to growth inhibition due to many intracellular defects, including accumulation of reactive oxygen species (ROS). Here, to understand the mechanism of Rsp5-mediated detoxification of α-syn, we isolated novel Rsp5 variants (T255A, D295G, P343S and N427D), which conferred α-syn tolerance to yeast cells. Interestingly, these mutants were phenotypically distinguished from our previously identified RSP5(T357A) mutation, which increases ubiquitination of the general amino acid permease Gap1. Among them, the RSP5(P343S) substitution accelerated the degradation of α-syn, suppressed the accumulation of intracellular ROS and enhanced the interaction with α-syn and its ubiquitination. In contrast, the RSP5(T255A) mutation did not contribute to degradation of α-syn, but improved cell growth under acetate stress conditions, possibly leading to alleviation of the α-syn toxicity. Thus, these novel mutations might be useful not only in elucidating the molecular basis by which disused proteins are specifically recognized and effectively removed but also in screening drug candidates for neurodegenerative diseases or in improving ethanol production under acidic fermentation conditions.

Keywords: Rsp5; Saccharomyces cerevisiae; WW domain; ubiquitin; α-synuclein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Endosomal Sorting Complexes Required for Transport / genetics
  • Endosomal Sorting Complexes Required for Transport / isolation & purification
  • Endosomal Sorting Complexes Required for Transport / metabolism*
  • Humans
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / isolation & purification
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Ubiquitin-Protein Ligase Complexes / genetics
  • Ubiquitin-Protein Ligase Complexes / isolation & purification
  • Ubiquitin-Protein Ligase Complexes / metabolism*
  • alpha-Synuclein / metabolism*

Substances

  • Endosomal Sorting Complexes Required for Transport
  • SNCA protein, human
  • Saccharomyces cerevisiae Proteins
  • alpha-Synuclein
  • Ubiquitin-Protein Ligase Complexes
  • RSP5 protein, S cerevisiae