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Molecular and Cellular Biology, August 2003, p. 5388-5400, Vol. 23, No. 15
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.15.5388-5400.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Cdc34 Self-Association Is Facilitated by Ubiquitin Thiolester Formation and Is Required for Its Catalytic Activity

Xaralabos Varelas,1 Christopher Ptak,1* and Michael J. Ellison1,2

Department of Biochemistry,1 Institute for Biomolecular Design, University of Alberta, Edmonton, Alberta T6G 2H7, Canada2

Received 17 October 2002/ Returned for modification 10 January 2003/ Accepted 8 April 2003

Using a coimmunoprecipitation strategy, we showed that the Cdc34 ubiquitin (Ub)-conjugating enzyme from Saccharomyces cerevisiae self-associates in cell lysates, thereby indicating an in vivo interaction. The ability of Cdc34 to interact with itself is not dependent on its association with the ubiquitin ligase Skp1-Cdc53/Cul1-Hrt1-F-box complex. Rather, this interaction depends upon the integrity of the Cdc34~Ub thiolester. Furthermore, several principal determinants within the Cdc34 catalytic domain, including the active-site cysteine, amino acid residues S73 and S97, and its catalytic domain insertion, also play a role in self-association. Mutational studies have shown that these determinants are functionally important in vivo and operate at the levels of both Cdc34~Ub thiolester formation and Cdc34-mediated multi-Ub chain assembly. These determinants are spatially situated in a region that is close to the active site, corresponding closely to the previously identified E2-Ub interface. These observations indicate that the formation of the Cdc34~Ub thiolester is important for Cdc34 self-association and that the interaction of Cdc34~Ub thiolesters is in turn a prerequisite for both multi-Ub chain assembly and Cdc34's essential function(s). A conclusion from these findings is that the placement of ubiquitin on the Cdc34 surface is a structurally important feature of Cdc34's function.

* Corresponding author. Mailing address: 5-76 Medical Science Building, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Phone: (780) 492-6352. Fax: (780) 492-0886. E-mail for Michael J. Ellison: mike.ellison{at}ualberta.ca. E-mail for Christopher Ptak: cptak{at}biochem.ualberta.ca.

Molecular and Cellular Biology, August 2003, p. 5388-5400, Vol. 23, No. 15
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.15.5388-5400.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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