Structural and functional effects of PA700 and modulator protein on proteasomes.
Control and targeting of the proteolytic activity of the major intracellular protease, the proteasome, is accomplished by various regulatory protein complexes that may form higher-order assemblies with the proteasome. An activator of proteolytic activity, PA700, has been shown to have an ATP-dependent stimulatory effect on the peptidase activities of the proteasome, and another protein factor, the modulator, further enhances the effect of PA700. Here we show that the addition of PA700 endows the proteasome with the ability to cleave ubiquitinated proteins, a property associated with the previously isolated 26 S form of the proteasome. The modulator further stimulates this specific activity, without having any such effect on the proteasome alone. Using electron microscopy, we show that addition of PA700 causes the appearance of protein "caps" at one or both ends of proteasomes, forming structures that are indistinguishable from 26 S proteasomes. Quantitation of the numbers of uncapped, singly capped and doubly capped complexes indicates cooperativity in the association of PA700 with the two ends of the proteasome. Addition of modulator protein makes no further structural modification that is detectable by electron microscopy, but does cause an increase in the number of capped complexes visible at subsaturating concentrations of PA700. Hence PA700 converts the proteasome both functionally and structurally to the 26 S form, and the modulator promotes this transformation, apparently without stable association with the resulting complex.
Adams
G M
GM
School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110, USA.
Falke
S
S
Goldberg
A L
AL
Slaughter
C A
CA
DeMartino
G N
GN
Gogol
E P
EP
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000502:physiology* / Q000648:ultrastructure; D004789:Enzyme Activation; D007091:Image Processing, Computer-Assisted; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000502:physiology* / Q000648:ultrastructure; D009113:Muramidase / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000502:physiology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014452:Ubiquitins / Q000378:metabolism
D009097:Multienzyme Complexes; C085834:PA700 proteasome activator; D011506:Proteins; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D009113:Muramidase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex