The Proteostasis Function of the Saccharomyces Cerevisiae Metacaspase Yca1

Abstract

In addition to apoptosis, metacapases function to regulate various other processes that promote and sustain life. For example, the Saccharomyces cerevisiae metacaspase Yca1 promotes cellular fitness by regulating insoluble protein levels. However, the mechanism(s) that regulate this proteostasis function for Yca1 have remained elusive. Here, using proteomics coupled to protein interaction studies, we describe a role for Yca1 in restraining deposition to the insoluble proteome and further identify a post-translational regulatory mechanism for modulating Yca1 function. Our initial analyses uncovered a role for Yca1 in aggregate assembly where Yca1, in coordination with the Cdc48 chaperone, regulates the composition of the insoluble proteome. Interestingly, loss of Yca1 was correlated with reduced sequestration of proteins related to ribosomal and translational processes in the insoluble protein fraction during heat stress. Subsequent proteomic analyses identified a regulatory mechanism for Yca1 mediated by the ubiquitin system, a feature that was instrumental for limiting insoluble protein content. Specifically, we noted K355 ubiquitination and S346 phosphorylation as key modifications that directed Yca1 function to maintain proteostasis. Loss of function mutations at these sites led to increased retention of insoluble protein and increased vacuolar structures. Surprisingly, loss of Yca1 also affected ubiquitin homeostasis in vivo as observed by reduced levels of low molecular weight free ubiquitin. Upon further analysis, we observed that the ubiquitin precursor protein Rsp31 was cleaved by Yca1 suggesting a possible role for Yca1 in de novo ubiquitin synthesis. Together, these analyses suggest that post-translational modifications of Yca1 are critical regulatory features for this protease, and that this enzyme regulates cell proteostasis in combination with other chaperone and protein degradation machinery.