Murine Sirt3 protein isoforms have variable half-lives

Abstract

Sirt3 is a NAD+-dependent protein deacetylase mainly localized in mitochondria. Recent studies indicate that the murine Sirt3 gene expresses different transcript variants resulting in three possible Sirt3 protein isoforms with variable lengths at the N-terminus: M1 (aa 1-334), M2 (aa 15-334), and M3 (aa 78-334). In this study, we stably expressed these variants in several cell lines. We found that Sirt3 M1 or M2 can be stably expressed with predominant mitochondrial localization. However, stable expression of Sirt3 M3 protein was consistently at very low levels. Fast proteasomal degradation contributes to the low expression of Sirt3 M3 protein, as proteasome inhibitor treatment increased Sirt3 M3 protein levels in these cells. Sirt3 M3 protein is ubiquitinated and the E3 ubiquitin ligase subunit Skp2 is involved in Sirt3 M3 protein degradation. Additionally, we found Sirt3 M3 protein can be produced from Sirt3 transcripts encoding longer M1 and M2 isoforms. To explore the mechanism underlying the instability of Sirt3 M3 protein, we found that Sirt3 M1 and M2 proteins, but not M3, specifically interact with HSP60. This suggests that heat shock proteins might play a role in the maintenance of Sirt3 protein stability.