Study of the Proteasome 19S Rpt5 ATPase Activity Affected by SUMO Modification
Date Issued
2014
Date
2014
Author(s)
Huang, Pei-Tzu
Abstract
The 26S proteasome is composed of 20S core particle (CP) capped with 19S regulatory particle (RP). Regulatory particle triple-A ATPase 5 (Rpt5) is one of the subunits of the 19S RP, which forms the base of 19S RP together with Rpt1, Rpt2, Rpt3, Rpt4 and Rpt6. The 19S base performs several functions, such as polyubiquitin chain recognition, substrate unfolding, gate opening and also translocation of target proteins into 20S CP. Our previous study has revealed that Rpt5 was modified by small ubiquitin-like modifier 2 (SUMO2) in COS7 cells, and modified by SUMO1 and SUMO2 in the E. coli sumoylation system. In the present study, the in vitro sumoylation assay further demonstrated that recombinant Rpt5 can be modified by SUMO1.
Because ATP binding and hydrolysis play critical roles in the regulation of proteasome function, this study was aimed to examine whether SUMO modification may affect the ATPase activity of Rpt5. The result showed that the ATPase activity of Rpt5 was reduced by SUMO2 modification. Furthermore, Rpt5 was found to contain several putative SUMO interacting motifs (SIMs). Although previous in vitro experimental results showed that sumoylation of Rpt5 by SUMO1 was markedly reduced while SIM3 was mutated, the level of Rpt5 sumoylation was not lowered when SIM3 was mutated in HEK293T cells. To rule out the possibility that endogenous Rpt5 might interfere with the observation of sumoylation pattern, shRNAs were applied to knockdown the expression level of endogenous Rpt5. However, the cells with inhibited Rpt5 expression were not viable. Therefore, whether SIM3 is involved in Rpt5 sumoylation remain elusive.
Subjects
Proteasome
Rpt5
SUMO
sumoylation
SIM
Type
thesis
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