Abstract
The proteasome-associated deubiquitinating enzyme Usp14/Ubp6 inhibits protein degradation by catalyzing substrate deubiquitination and by poorly understood allosteric actions. However, upon binding a ubiquitin chain, Usp14 enhances proteasomal degradation by stimulating ATP and peptide degradation. These studies were undertaken to clarify these seemingly opposite regulatory roles of Usp14 and their importance. To learn how the presence of Usp14 on 26S proteasomes influences its different activities, we compared enzymatic and regulatory properties of 26S proteasomes purified from wild-type mouse embryonic fibroblast cells and those lacking Usp14. The proteasomes lacking Usp14 had higher basal peptidase activity than WT 26S, and this activity was stimulated to a greater extent by adenosine 5'-O-(thiotriphosphate) (ATPγS) than with WT particles. These differences were clear even though Usp14 is present on only a minor fraction (30-40%) of the 26S in WT mouse embryonic fibroblast cells. Addition of purified Usp14 to the WT and Usp14-defficient proteasomes reduced both their basal peptidase activity and the stimulation by ATPγS. Usp14 inhibits these processes allosterically because a catalytically inactive Usp14 mutant also inhibited them. Proteasomes lacking Usp14 also exhibited greater deubiquitinating activity by Rpn11 and greater basal ATPase activity than WT particles. ATP hydrolysis by WT proteasomes is activated if they bind a ubiquitinated protein, which is loosely folded. Surprisingly, proteasomes lacking Usp14 could be activated by such proteins even without a ubiquitin chain present. Furthermore, proteasomes lacking Usp14 are much more active in degrading non-ubiquitinated proteins (e.g. Sic1) than WT particles. Thus, without a ubiquitinated substrate present, Usp14 suppresses multiple proteasomal activities, especially basal ATP consumption and degradation of non-ubiquitinated proteins. These allosteric effects thus reduce ATP hydrolysis by inactive proteasomes and nonspecific proteolysis and enhance proteasomal specificity for ubiquitinated proteins.
Highlights
In eukaryotic cells, 26S proteasomes are the major site for protein degradation
To understand the regulatory role of Usp14, we compared subunit composition and various enzymatic activities of 26S proteasomes purified from WT mouse embryonic fibroblast (MEF) cells and MEFs lacking Usp14 (Usp14KO)
In the WT MEF cells, we determined that Usp14 was present on ϳ40% of the 26S proteasomes based on Western blotting using increasing amounts of purified Usp14 as a standard and a proteasome with molecular mass of 2.5 MDa (Fig. 1A)
Summary
Ubiquitin; MEF, mouse embryonic fibroblast; UBL, ubiquitin-like; DUB, deubiquitinating enzyme; UIM, ubiquitin-interacting motif; Ub-al, ubiquitin aldehyde; ATP␥S, adenosine 5Ј-O-(thiotriphosphate); USP, ubiquitin-specific protease; Ni-NTA, nickelnitrilotriacetic acid; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; amc, 7-amino-4-methylcoumarin; Boc, t-butoxycarbonyl; Suc, succinyl; Nle/nL, norleucine; Ub-VS, ubiquitin vinyl sulfone; PAN, proteasome-activating nucleotidase. Studies of Ubp6-deficient yeast mutants had shown that Ubp reduces proteolysis both by acting as a DUB and an allosteric mechanism, which remains unexplained [25, 26] This deubiquitinating activity of Usp is dramatically stimulated upon its association with the 19S particle, which requires the UBL domain [24, 27]. The role of Usp as a central regulator of 26S function has been further supported by the recent cryoelectron microscopy (cryo-EM) studies by Baumeister and co-workers [30], Martin and co-workers [31], and Shi and co-workers [32] that revealed that the association of Ub-al with Usp14/Ubp induces marked changes in the structure of the 19S complex After binding of this substrate analog, the central channel in the ATPase ring is enlarged, and the catalytic domain of Ubp becomes located closer to the ATPase ring and Rpn. These findings imply that, in the absence of a substrate, Usp normally maintains the 26S proteasome in a quiescent state, but upon binding a Ub conjugate, it allosterically activates several enzymatic processes essential for efficient destruction of Ub conjugates
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