Phosphorylation of the 19S regulatory particle ATPase subunit, Rpt6, modifies susceptibility to proteotoxic stress and protein aggregation

PLoS One. 2017 Jun 29;12(6):e0179893. doi: 10.1371/journal.pone.0179893. eCollection 2017.

Abstract

The ubiquitin proteasome system (UPS) is a highly conserved and tightly regulated biochemical pathway that degrades the majority of proteins in eukaryotic cells. Importantly, the UPS is responsible for counteracting altered protein homeostasis induced by a variety of proteotoxic stresses. We previously reported that Rpt6, the ATPase subunit of the 19S regulatory particle (RP) of the 26S proteasome, is phosphorylated in mammalian neurons at serine 120 in response to neuronal activity. Furthermore, we found that Rpt6 S120 phosphorylation, which regulates the activity and distribution of proteasomes in neurons, is relevant for proteasome-dependent synaptic remodeling and function. To better understand the role of proteasome phosphorylation, we have constructed models of altered Rpt6 phosphorylation in S. cerevisiae by introducing chromosomal point mutations that prevent or mimic phosphorylation at the conserved serine (S119). We find that mutants which prevent Rpt6 phosphorylation at this site (rpt6-S119A), had increased susceptibility to proteotoxic stress, displayed abnormal morphology and had reduced proteasome activity. Since impaired proteasome function has been linked to the aggregation of toxic proteins including the Huntington's disease (HD) related huntingtin (Htt) protein with expanded polyglutamine repeats, we evaluated the extent of Htt aggregation in our phospho-dead (rpt6-S119A) and phospho-mimetic (rpt6-S119D) mutants. We showed Htt103Q aggregate size to be significantly larger in rpt6-S119A mutants compared to wild-type or rpt6-S119D strains. Furthermore, we observed that phosphorylation of endogenous Rpt6 at S119 is increased in response to various stress conditions. Together, these data suggest that Rpt6 phosphorylation at S119 may play an important function in proteasome-dependent relief of proteotoxic stress that can be critical in protein aggregation pathologies.

MeSH terms

  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / metabolism*
  • Amino Acid Sequence
  • Animals
  • Humans
  • Phosphorylation
  • Proteasome Endopeptidase Complex / chemistry
  • Proteasome Endopeptidase Complex / metabolism*
  • Protein Binding
  • Protein Folding
  • Rats
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Sequence Homology, Amino Acid
  • Stress, Physiological*

Substances

  • Saccharomyces cerevisiae Proteins
  • Proteasome Endopeptidase Complex
  • Adenosine Triphosphatases
  • RPT6 protein, S cerevisiae