An investigation of the genetic control of protein mutability: The role of the ubiquitin system in protein based inheritance in Saccharomyces cerevisiae.

摘要

The yeast prion [PSI+] is the self-propagating aggregated isoform of the translational termination factor Sup35 (eRF3). [PSI +] provides a powerful model for amyloidogenic and polyglutamine disorders such as Alzheimer's and Huntington's diseases. While the ubiquitination of amyloids and other cytopathological aggregates have been described in the literature, whether or not the ubiquitin (Ub) system plays a role in aggregate formation or its associated toxicity has not been unknown. Here we show that cellular levels of free Ub modulate the induced formation of [PSI+ ] as well as [PSI+]-mediated nonsense suppression. In both cases, increased Ub levels were seen to assist [PSI+], increasing induced [PSI+] formation and nonsense suppression, whereas Ub depletion had the opposite effects. The “[PSI+]-helper” effects of excess Ub are reminiscent of those previously reported for Hsp70 protein, Ssa. Here we report that, like high Ub levels, excess Ssa also facilitates [PSI+] induction by overproduced Sup35.; Depletion of the ubiquitin-conjugating enzyme, Ubc4, significantly compromises the curing of [PSI+] by excess Hsp104, a chaperone shown to be generally important to prion maintenance in yeast. This strongly suggests a direct role for ubiquitination in Hsp104-mediated [PSI+] curing. Furthermore, ubc4Δ increases the frequency of spontaneous [PSI+] formation in the absence of [RNQ+] (aka [PIN+]) another prion that is usually needed for initial [PSI+] formation. This “protein mutator” effect is stronger than that previously reported for ssb1,2Δ and is unique in its independence of [PIN+]. Hsp70 Ssb, has also been shown to play a role in protein turnover. Like ubc4Δ, we found that ssb1,2Δ compromises curing by excess Hsp104.; We propose that the ubiquitination of Sup35 by Ubc4 normally removes misfolded intermediates that provide a source for new prions and prion recovery after curing. It is possible that ubc4Δ and ssb1,2Δ effects occur by similar mechanisms. Such a role for the ubiquitin system in prion formation and curing has never before been described. Additionally, we observed that overproduction of Ssa or Ssb in combination with excess Sup35 provokes [PSI+] loss. These findings have important implications for understanding the mechanisms that drive aggregate formation and its relationship to toxicity in yeast models of human disease.