Volume 270,
Number 30,
Issue of July 28, pp. 18099-18109, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An
Essential Yeast Gene Encoding a Homolog of Ubiquitin-activating Enzyme
(Received for publication, March 30, 1995; and in revised form, May 18,
1995)
R. Jrgen
Dohmen
,
Reiner
Stappen
,
John
P.
McGrath
,
Helena
Forrov
,
Jordan
Kolarov
,
Andr
Goffeau
,
Alexander
Varshavsky
From the
(1)Institut fr
Mikrobiologie, Heinrich-Heine-Universitt
Dsseldorf,
Universittsstra
e 1, Geb. 26.12, D-40225,
Dsseldorf, Germany,
(2)Alkermes Inc., Cambridge, Massachusetts 02139,
the
(3)Department of Biochemistry, Comenius University,
Mlynska dolina CH-1, 842-15 Bratislava, Slovakia, the
(4)Unit de Biochemie
Physiologique, Universit Catholique de Louvain,
Place Croix du Sud, 2-20, B-1348, Louvain-la-Neuve, Belgium, and the
(5)Division of Biology, California Institute of
Technology, Pasadena, California 91125
Ubiquitin (Ub) activation by the Ub-activating (E1)
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 E1 enzyme,
can be transferred to a cysteine residue in one of several
Ub-conjugating (E2) 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 E1 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 E1 and E1 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 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 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 (E1 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 E2 or E1/E2 enzyme.
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