蛋白酶體 19S Rpt3 ATPase 之 SUMO 化修飾研究

Abstract

Rpt3 (regulatory particle triple-A ATPase3) is a subunit of the proteasome 19S regulatory particle (RP). The primary functions of 19S RP include recognition of ubiquitinated target proteins, unfolding and translocation of target proteins into the catalytic cavity of the 20S core particle. In our previous study, Rpt3 was found to be SUMOylated by SUMO2 in COS7 cells. In this study, the strong SUMOylation of Rpt3 by SUMO1 and SUMO2 was clearly observed in HEK293T cells. However, the exact SUMOylation site of Rpt3 was not determined by expression of truncated Rpt3 and Rpt3 mutants containing K62R, K69R and K408R mutations within the Ψ-K-x-D/E SUMOylation consensus motifs. Nonetheless, the SUMO-interacting motif SIM3 on Rpt3 may be involved in modulating Rpt3 SUMOylation, as mutation of SIM3 reduced the SUMOylation level of Rpt3 in HEK293T cells. By using the live cell imaging to analyze the expression of GFP-Rpt3 fusion protein in HEK293T cells, the data showed that GFP-Rpt3 was primarily distributed in the cytoplasm, and a small population of cells containing GFP-Rpt3 in the nucleus. However, the cell population containing nuclear GFP-Rpt3 only slightly increased after treatment of proteasome inhibitor MG132. This study suggests that Rpt3 is SUMOylated by SUMO1 and SUMO2 in HEK293T cells. However, the detailed SUMOylation mechanism and the SUMOylation sites of Rpt3 remain elusive.