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Molecular and Cellular Biology, May 2005, p. 3842-3853, Vol. 25, No. 9
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.9.3842-3853.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Proteasomal ATPase-Associated Factor 1 Negatively Regulates Proteasome Activity by Interacting with Proteasomal ATPases

Yoon Park,^1, Yong-Pil Hwang,^1, Jong-Sik Lee,¹ Sang-Hyun Seo,¹ Sungjoo Kim Yoon,² and Jong-Bok Yoon^1*

Department of Biochemistry and Protein Network Research Center, Yonsei University, Seoul 120-749, Korea,¹ Department of Biomedical Sciences, Catholic University of Korea, Seoul 137-040, Korea²

Received 8 October 2004/ Returned for modification 23 November 2004/ Accepted 25 January 2005

The 26S proteasome, composed of the 20S core and the 19S regulatory complex, plays a central role in ubiquitin-dependent proteolysis by catalyzing degradation of polyubiquitinated proteins. In a search for proteins involved in regulation of the proteasome, we affinity purified the 19S regulatory complex from HeLa cells and identified a novel protein of 43 kDa in size as an associated protein. Immunoprecipitation analyses suggested that this protein specifically interacted with the proteasomal ATPases. Hence the protein was named proteasomal ATPase-associated factor 1 (PAAF1). Immunoaffinity purification of PAAF1 confirmed its interaction with the 19S regulatory complex and further showed that the 19S regulatory complex bound with PAAF1 was not stably associated with the 20S core. Overexpression of PAAF1 in HeLa cells decreased the level of the 20S core associated with the 19S complex in a dose-dependent fashion, suggesting that PAAF1 binding to proteasomal ATPases inhibited the assembly of the 26S proteasome. Proteasomal degradation assays using reporters based on green fluorescent protein revealed that overexpression of PAAF1 inhibited the proteasome activity in vivo. Furthermore, the suppression of PAAF1 expression that is mediated by small inhibitory RNA enhanced the proteasome activity. These results suggest that PAAF1 functions as a negative regulator of the proteasome by controlling the assembly/disassembly of the proteasome.

These two authors contributed equally to this work.

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