Revised Manuscript Received April 16, 1991 abstract: Pathways of ubiquitin-dependent protein degradation have in common two requirements for ATP. Ubiquitin activation by the enzyme El is accompanied by ATP hydrolysis to yield AMP and PPj, and during conjugate breakdown, the ubiquitin-dependent protease hydrolyzes ATP to ADP and Pj. We show here that either of two/9, 7-nonhydrolyzable ATP analogues, 5'-adenylyl imidodiphosphate or 5'-adenylyl methylenediphosphate, can support ubiquitin-protein conjugation. With the ubiquitin-dependent protease, however, neither analogue could substitute for ATP. Thus, the substitution of a/3, 7-nonhydrolyzable analogue for ATP offers a simple method to uncouple ubiquitin conjugation from proteolysis in crude systems. On the basis of pyrophosphate exchange kinetics, El has apparent Km and Fmax values that are similar for ATP and the analogues, but substrate inhibition by 5'-adenylyl methylenediphosphate made use of the/3, 7-imido analogue preferable. In one application,/3, 7-imido-ATP was used in combinationwith ubiquitin aldehyde (an inhibitor of ubiquitin-protein isopeptidases) to establish that several unfolded RNase A derivatives are recognized equally as ubiquitination substrates. This result extends an earlier study [Dunten, R. L., & Cohen, R. E.(1989) J. Biol. Chem. 264, 16739-16747] to show that conjugateyields, upon which relative ubiquitination rates were based, were not influenced by differential ubiquitin-dependentproteolysis. In a second application, ATP and/8, 7-imido-ATP were compared in a pulse-chase experiment to investigate the con-tributions of ATP-dependent proteolysis and isopeptidase activities to conjugate stability. liapid protein degradation within normally growing euka-ryotic cells has been characterized as predominantly nonly-sosomal and energy-dependent (Goldberg & St. John, 1976; Hershko & Ciechanover, 1982). A major system for intracellular proteolysis involves the attachment of ubiquitin, a 76-residue polypeptide, as an intermediate step in the selection and subsequent degradation of protein substrates [for reviews, see Rechsteiner (1987), Hershko (1988), and Jentsch et al.(1990)]. Thebasic reactions of ubiquitin-mediated proteolysis are activation of the ubiquitin C-terminal carboxylate by the enzyme El, transfer of the activated ubiquitin to a carrier/conjugating enzyme (E2), and subsequent covalent conjugation (“ubiquitination”) of a protein target in a process that, for some substrates, requires E3, a ubiquitin-protein ligase (Hershko et al., 1983). Multiple ubiquitination of a protein usually precedes, and may be obligatory for, its ultimate degradation by the ubiquitin-dependent 26S protease [Hershko et al., 1984; Hough et al., 1987; Chau et al., 1989; Gregori et al., 1990; but see also Hershko and Heller (1985) and Haas et al.(1990)]. Both ubiquitin activation by El (Ciechanoveret al., 1981, 1982) and proteolysis by the 26S protease (Hough et al., 1987; Ganoth et al., 1988; Armon et al., 1990) require ATP hydrolysis, andthese reactions may account for much of the energy requirement of intracellular protein turnover. Studies of ubiquitin-protein conjugation and proteolysis in vitro predominantly have employed the ubiquitin-depleted reticulocyte lysate (“fraction II”) described by Hershko and his co-workers (Ciechanover et al., 1978; Hershko et al., 1983). The considerable complexity of this system is evident from the multitude of ubiquitin conjugation components (Hershko et al., 1983; Pickart & Rose, 1985; Pickart & Vella, 1988; Haas tThis work was supported by USPHS Grant GM37666 from the National Institutes of Health.