An Essential Yeast Gene Encoding a Homolog of Ubiquitin-activating Enzyme

• Published in: The Journal of biological chemistry 1995-07, Vol.270 (30), p.18099-18109
• Main Authors: Dohmen, R J, Stappen, R, McGrath, J P, Forrová, H, Kolarov, J, Goffeau, A, Varshavsky, A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; DNA, Fungal; Genes, Fungal; Humans; Ligases - genetics; Ligases - metabolism; Molecular Sequence Data; Nuclear Proteins - genetics; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Sequence Homology, Amino Acid; Ubiquitin-Activating Enzymes; Ubiquitin-Protein Ligases; Ubiquitins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.30.18099

Ubiquitin (Ub) activation by the Ub-activating ( E 1) enzyme is the initial and essential step common to all of the known processes that involve post-translational conjugation of Ub to itself or other proteins. The “activated” Ub, linked via a thioester bond to a specific cysteine residue of E 1 enzyme, can be transferred to a cysteine residue in one of several Ub-conjugating ( E 2) enzymes, which catalyze the formation of isopeptide bonds between the C-terminal glycine of Ub and lysine residues of acceptor proteins. In the yeast Saccharomyces cerevisiae , a 114-kDa E 1 enzyme is encoded by an essential gene termed UBA1 (McGrath, J. P., Jentsch, S., and Varshavsky, A.(1991) EMBO J. 10, 227-236). We describe the isolation and analysis of another essential gene, termed UBA2 , that encodes a 71-kDa protein with extensive sequence similarities to both the UBA1 -encoded yeast E 1 and E 1 enzymes of other organisms. The regions of similarities between Uba1p and Uba2p encompass a putative ATP-binding site as well as a sequence that is highly conserved between the known E 1 enzymes and contains the active-site cysteine of E 1. This cysteine is shown to be required for an essential function of Uba2p, suggesting that Uba2p-catalyzed reactions involve a transient thioester bond between Uba2p and either Ub or another protein. Uba2p is located largely in the nucleus. The putative nuclear localization signal of Uba2p is near its C terminus. The Uba1p ( E 1 enzyme) and Uba2p cannot complement each others essential functions even if their subcellular localization is altered by mutagenesis. Uba2p appears to interact with itself and several other S. cerevisiae proteins with apparent molecular masses of 52, 63, 87, and 120 kDa. Uba2p is multiubiquitinated in vivo , suggesting that at least a fraction of Uba2p is metabolically unstable. Uba2p is likely to be a component of the Ub system that functions as either an E 2 or E 1/ E 2 enzyme.