Proteasomal ATPase-associated factor 1 negatively regulates proteasome activity by interacting with proteasomal ATPases.
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.
Park
Yoon
Y
Department of Biochemistry, College of Science, Yonsei University, 134 Shinchon-Dong, Seodaemoon-Gu, Seoul 120-749, Korea.
Hwang
Yong-Pil
YP
Lee
Jong-Sik
JS
Seo
Sang-Hyun
SH
Yoon
Sungjoo Kim
SK
Yoon
Jong-Bok
JB
D048868:Adaptor Proteins, Signal Transducing; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D015536:Down-Regulation; D010450:Endopeptidases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D016415:Sequence Alignment; D015533:Transcriptional Activation
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C501074:PAAF1 protein, human; D061988:Proteasome Inhibitors; D010450:Endopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; C119011:26S proteasome non-ATPase regulatory subunit 13; D000251:Adenosine Triphosphatases