Subcomplexes of PA700, the 19 S regulator of the 26 S proteasome, reveal relative roles of AAA subunits in 26 S proteasome assembly and activation and ATPase activity.
We have identified, purified, and characterized three subcomplexes of PA700, the 19 S regulatory complex of the 26 S proteasome. These subcomplexes (denoted PS-1, PS-2, and PS-3) collectively account for all subunits present in purified PA700 but contain no overlapping components or significant levels of non-PA700 proteins. Each subcomplex contained two of the six AAA subunits (Rpt1-6) that form the binding interface of PA700 with the 20 S proteasome, the protease component of the 26 S proteasome. Unlike intact PA700, no individual PA700 subcomplex displayed ATPase activity or proteasome activating activity. However, both activities were manifested by ATP-dependent in vitro reconstitution of PA700 from the subcomplexes. We exploited functional reconstitution to define and distinguish roles of different PA700 subunits in PA700 function by selective alteration of subunits within individual subcomplexes prior to reconstitution. Carboxypeptidase treatment of either PS-2 or PS-3, subcomplexes containing specific Rpt subunits previously shown to have important roles in 26 S proteasome assembly and activation, inhibited these processes but did not affect PA700 reconstitution or ATPase activity. Thus, the intact C termini of both subunits are required for 26 S proteasome assembly and activation but not for PA700 reconstitution. Surprisingly, carboxypeptidase treatment of PS-1 also inhibited 26 S proteasome assembly and activation upon reconstitution with untreated PS-2 and PS-3. These results suggest a previously unidentified role for other PA700 subunits in 26 S proteasome assembly and activation. Our results reveal relative structural and functional relationships among the AAA subunits of PA700 and new insights about mechanisms of 26 S proteasome assembly and activation.
Thompson
David
D
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9040, USA.
Hakala
Kevin
K
DeMartino
George N
GN
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism; D005990:Glycerol / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008954:Models, Biological; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
C085834:PA700 proteasome activator; D000255:Adenosine Triphosphate; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D000251:Adenosine Triphosphatases; D005990:Glycerol