Study of the Function of the SUMO-Interacting Motif in 19S Proteasomal Rpt5 ATPase
Date Issued
2012
Date
2012
Author(s)
Kao, Yi-Hsuan
Abstract
Regulatory particle triple-A ATPase 5 (Rpt5) is one of six AAA ATPases of 19S regulatory particle of 26S proteasome. Rpt5 performs several functions, such as unfolding target proteins and translocating these substrates to the 20S core particle, in the regulation of the proteasome catalytic activity. Furthermore, Rpt5 can also function independently from proteasome and usually participate in part of a protein complex which involved in transcription regulation.
In the previous study, we found that Rpt5 was modified by small ubiquitin-like modifier (SUMO) in an in vitro E. coli SUMOylation assay system even though its SUMOylation consensus motif MKSE was mutated.
In the present study, we found that Rpt5 contains six putative SUMO interacting motifs (SIMs), herein named SIM1-6, suggesting that Rpt5 may interact with SUMO protein in a non-covalent way. Therefore, we hypothesized that the SIMs of Rpt5 may play an important role in its SUMOylation process. By using Rpt5 mutants, whose hydrophobic residues of the putative SIMs were substituted to alanine residues, as the substrates, we demonstrated that SUMOylation of Rpt5 by SUMO1 was markedly inhibited while SIM3, SIM4, or SIM5 was mutated, respectively. In addition, SUMOylation of Rpt5 by SUMO2 was also inhibited while SIM3 was mutated. Furthermore, the non-covalent interaction between Rpt5 and SUMO1 was observed by using anti-HA binding agarose pull-down assay. Mutation of SIM3 in Rpt5 also abolished its non-covalent interaction with SUMO1. However, the SIM4, SIM5, or SIM6 Rpt5 mutant displayed greater interaction with SUMO1. These findings suggest that different SIMs of Rpt5 may play various roles in regulation of its SUMOylation and non-covalent interaction with SUMO1. The non-covalent interaction between Rpt5 and SUMO2 was not detected in this study.
Subjects
Rpt5
SUMO
SIM
non-covalent interaction
Type
thesis
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