AN ESSENTIAL YEAST GENE ENCODING A HOMOLOG OF UBIQUITIN-ACTIVATING ENZYME

Ubiquitin (Ub) activation by the Ub-activating (E1) enzyme is the init ial and essential step common to all of the known processes that invol ve post-translational conjugation of Ub to itself or other proteins. T he ''activated'' Ub, linked via a thioester bond to a specific cystein e residue of E1 enzyme, can be transferred to a cysteine residue in on e of several Ub-conjugating (E2) enzymes, which catalyze the formation of isopeptide bonds between the C-terminal glycine of Ub and lysine r esidues of acceptor proteins. In the yeast Saccharomyces cerevisiae, a 114-kDa E1 enzyme is encoded by an essential gene termed UBA1 (McGrat h, J. P., Jentsch, S., and Varshavsky, A. (1991) EMBO J. 10, 227-236). We describe the isolation and analysis of another essential gene, ter med UBA2, that encodes a 71-kDa protein with extensive sequence simila rities to both the UBA1-encoded yeast E1 and E1 enzymes of other organ isms. The regions of similarities between Uba1p and Uba2p encompass a putative ATP-binding site as well as a sequence that is highly conserv ed between the known E1 enzymes and contains the active-site cysteine of E1. This cysteine is shown to be required for an essential function of Uba2p, suggesting that Uba2p-catalyzed reactions involve a transie nt thioester bond between Uba2p and either Ub or another protein. Uba2 p is located largely in the nucleus. The putative nuclear localization signal of Uba2p is near its C terminus. The Uba1p (E1 enzyme) and Uba 2p cannot complement each others essential functions even if their sub cellular localization is altered by mutagenesis. Uba2p appears to inte ract with itself and several other S. cerevisiae proteins with apparen t molecular masses of 52, 63, 87, and 120 kDa. Uba2p is multiubiquitin ated in vivo, suggesting that at least a fraction of Uba2p is metaboli cally unstable. Uba2p is likely to be a component of the Ub system tha t functions as either an E2 or E1/E2 enzyme.