Ubc9p Is the Conjugating Enzyme for the Ubiquitin-like Protein Smt3p*
- From the Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021
Abstract
At least one essential function of Smt3p, aSaccharomyces cerevisiae ubiquitin-like protein similar to the mammalian protein SUMO-1, involves its posttranslational covalent attachment to other proteins. Using Smt3p affinity chromatography, we have isolated the second enzyme of the Smt3p conjugation pathway and have found that it is identical to Ubc9p, a previously identified protein that has extensive sequence similarity to the ubiquitin-conjugating enzymes (E2s) and that is required for yeast to progress through mitosis. A hallmark of E2s is the ability to form a thioester bond-containing covalent intermediate with ubiquitin (Ub). While we were unable to detect formation of a Ub∼Ubc9p thioester, Ubc9p was found to form a thioester with Smt3p, indicating that Ubc9p is the functional analog of E2s in the Smt3p pathway and that this step is distinct from the ubiquitin pathway. Ubc9p is required for attachment of Smt3p to other proteins in vitro, suggesting that it is the only such enzyme in S. cerevisiae. These results suggest that, like ubiquitination, Smt3p conjugation may be a critical modification in cell cycle regulation.
Footnotes
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↵* This work was supported by the Howard Hughes Medical Institute (to G. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Postdoctoral fellow of the American Cancer Society (PF-4114). To whom correspondence should be addressed: Rockefeller University, Box 168, 1230 York Ave., New York, NY 10021. Tel.: 212-327-8181; Fax: 212-327-7880; E-mail: johnsoe{at}rockvax.rockefeller.edu.
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↵1 The abbreviations used are: RanGAP1, Ran-GTPase-activating protein; Ub, ubiquitin; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; HF-Smt3p, His6- and FLAG-epitope-tagged mature Smt3p; DTT, dithiothreitol; NTA, nitrilotriacetic acid; EF1α, elongation factor 1α; PAGE, polyacrylamide gel electrophoresis; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
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↵2 R. Beckmann, personal communication.
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- Received August 19, 1997.
- Revision received August 26, 1997.
