Selective chemical inactivation of AAA proteins reveals distinct functions of proteasomal ATPases

Russell, S. J., Gonzalez, F., Joshua-Tor, L., Johnston, S. A. (2001) Selective chemical inactivation of AAA proteins reveals distinct functions of proteasomal ATPases. Chemistry & Biology, 8 (10). pp. 941-950. ISSN 1074-5521

URL: https://www.ncbi.nlm.nih.gov/pubmed/11590019
DOI: 10.1016/S1074-5521(01)00060-6

Abstract

Background: The 26S proteasome contains six highly related ATPases of the AAA family. We have developed a strategy that allows selective inhibition of individual proteasomal ATPases in the intact proteasome. Mutation of a threonine in the active site of Sug1/Rpt6 or Sug2/Rpt4 to a cysteine sensitizes these proteins to inactivation through alkylation by the sulfhydryl modifying agent NEM. Using this technique the individual contributions of Sug1 and Sug2 to proteasome function can be assessed. Results: We show that both Sug1 and Sug2 can be selectively alkylated by NEM in the context of the intact 26S complex and as predicted by structural modeling, this inactivates the ATPase function. Using this technique we demonstrate that both Sug 1 and 2 are required for full peptidase activity of the proteasome and that their functions are not redundant. Kinetic analysis suggests that Sug2 may have an important role in maintaining the interaction between the 19S regulatory complex and the 20S proteasome. In contrast, inhibition of Sug1 apparently decreases peptidase activity of the 26S proteasome by another mechanism. Conclusions: These results describe a useful technique for the selective inactivation of AAA proteins. In addition, they also demonstrate that the functions of two related proteasomal AAA proteins are not redundant, suggesting differential roles of proteasomal AAA proteins in protein degradation. (C) 2001 Elsevier Science Ltd. All rights reserved.

Item Type: Paper
Uncontrolled Keywords: chemical inactivation AAA proteins proteasomal ATPases SENSITIVE FUSION PROTEIN sensitive fusion protein REGULATORY PARTICLE regulatory particle CRYSTAL-STRUCTURE crystal structure 20S PROTEASOME proteasome YEAST yeast DOMAIN domain ATP ACTIVATION activatin MOLSCRIPT molscript TRANSPORT transport
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein structure rendering
Investigative techniques and equipment > x ray crystallography
CSHL Authors:
Communities: CSHL labs > Joshua-Tor lab
Depositing User: CSHL Librarian
Date Deposited: 21 Mar 2012 20:07
Last Modified: 17 Jan 2017 16:00
Related URLs:
URI: http://repository.cshl.edu/id/eprint/25492

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