Abstract
Metacaspase enzymes are critical regulatory factors that paradoxically engage apoptosis and also maintain cell viability. For example, the Saccharomyces cerevisiae metacaspase Yca1 has been shown to be important for maintaining cellular proteostasis during stress, and the loss of this enzyme results in increased retention of aggregated material within the insoluble proteome. However, the molecular mechanism(s) by which Yca1 maintains cellular proteostasis remains unknown. Here, using proteomic analysis coupled with protein interaction studies we identified a direct interplay between Yca1 and the ubiquitin-proteasome system. We noted multiple ubiquitination sites on Yca1 and established Rsp5 as the candidate E3 ligase involved in this process. Further characterization of the ubiquitination sites identified the K355 residue on Yca1 as a critical modification for proteostasis function, managing both insoluble protein content and vacuolar response. We also identified a Yca1 phosphorylation site at S346, which promoted interaction with Rsp5 and the aggregate dispersal function of the metacaspase. Interestingly, proteomic analysis also revealed that Yca1 interacts with the ubiquitin precursor protein Rps31, cleaving the protein to release free ubiquitin. In turn, loss of Yca1 or its catalytic activity reduced the levels of monomeric ubiquitin in vivo, concurrent to increased protein aggregation. The K355 and S346 residues were also observed to influence the abundance of low-molecular weight ubiquitin. Together, these observations suggest that Yca1 maintains proteostasis and limits protein aggregation by ensuring a free flow of monoubiquitin, an essential precursor for ligase-enhanced Yca1 enzymatic activity and general proteasome-mediated protein degradation.
Highlights
Metacaspases are functional orthologs of caspase enzymes and are grouped together into the clan cysteine dependent aspartic specific (C/D) family of proteases[1,2]
Using an unbiased immunoprecipitation screen coupled to LC-MS/MS we identified a series of known and novel protein interactions for the yeast metacaspase Yca[1] (Supplementary Table S1)
We identified both ligase and ubiquitin moieties that regulated Yca[1] activity, and further confirmed the latter to be generated by Yca[1] enzymatic activity. These observations suggest that Yca[1] controls proteostasis by managing the free ubiquitin pool, a biochemical step that (1) feeds back to tag Yca[1] through direct ligase interaction which ensures the metacaspase is active, and (2) provides free ubiquitin moieties which may be deployed for general proteasome targeting and aggregate dispersal
Summary
Metacaspases are functional orthologs of caspase enzymes and are grouped together into the clan cysteine dependent aspartic specific (C/D) family of proteases[1,2]. The yeast Saccharomyces cerevisiae possesses a single type I metacaspase known as Yca[1] which has been shown to mediate apoptosis in yeast under various stresses[3]. The Leishmania metacaspase, LmjMCA, has been shown to promote transit through the cell cycle, suggesting a conserved non-death activity[6]. Yca[1] was shown to mediate key interactions with known members of the proteostasis network such as Cdc[48], Hsp[104], and the Hsp70/40 chaperone systems. Yca[1] has been shown to Shrestha et al Cell Discovery (2019)5:6 limit/prevent protein aggregation in daughter cells during yeast aging studies[8]. Despite compelling observations that link Yca[1] to protein aggregate management, the essential molecular controls that guide this physiologic function remain unknown
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