蛋白酶體19S Rpt5 ATPase受SUMO化修飾之研究

Abstract

Rpt5 (Regulatory particle triple-A ATPase 5) is one of the 19S proteasomal ATPase subunits, which forms the 19S base together with Rpt1, Rpt2, Rpt3, Rpt4 and Rpt6. Rpts modulate the functions of recognizing polyubiquitin chain, substrate unfolding, gate opening of the 20S core particle, and translocation of target proteins. Our previous findings have shown that Rpt3 and Rpt5 were SUMOylated by SUMO1 (small ubiquitin-like modifier 1) and SUMO2 (small ubiquitin-like modifier 2). In this study, by expressing truncated Rpt5 fragments in the Escherichia coli SUMOylation system, the results reveal that there are two lysine residues located in the Rpt51-242 and Rpt5243-355 regions are the potential SUMOylation sites. Notably, K56 located in the SUMOylation consensus motif of Rpt5 is not the SUMOylated site. By using site-directed mutagenesis at nine lysine residues within Rpt5243-355, the data showed that K294 might be the lysine residue for SUMO conjugation. Rpt5 contains six putative SUMO interacting motifs (SIM), herein named SIM1-6. The SUMOylation level of Rpt5 is markedly reduced as substituting the hydrophobic residues of SIMs to alanine. Among six SIMs, SIM5 seems to play the most significant role in mediating Rpt5 SUMOylation. In addition to Rpt3 and Rpt5, this study also showed that Rpt2 was SUMOylated by SUMO1 in the E. coli SUMOylation system. However, it remains unclear that whether Rpt1, Rpt4 and Rpt6 are SUMOylated or not, since they cannot be expressed properly in the E. coli SUMOylation system.