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The bacterially expressed yeast CDC34 gene product can undergo autoubiquitination to form a multiubiquitin
1Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201.
Abstract:
The CDC34 gene of Saccharomyces cerevisiae encodes a 295-residue ubiquitin-conjugating enzyme (E2). The function of this ubiquitin-conjugating activity remains to be defined as its in vivo substrates are presently unknown. The bacterially expressed and purified Cdc34 protein is shown here to catalyze its own ubiquitination via an intramolecular transfer of its thiol ester-linked ubiquitin to a lysine. In this process, multiple ubiquitin groups are added to Cdc34, and these ubiquitin groups were shown to be arranged predominantly in the form of a single Lys48-specific multiubiquitin chain. Analysis of the hydroxylamine-dependent cleavage of ubiquitin-Cdc34 conjugates at the single Asn-Gly sequence in Cdc34 placed the major ubiquitin linkage site within the C-terminal 215-295 residues of Cdc34. The 4 Lys residues (Lys273, Lys277, Lys293, and Lys294) in this region of CDC34 were substituted by arginine either singly or in combination to produce a set of Cdc34 mutants. Analysis of these Cdc34 mutants for autoubiquitination revealed that the multiubiquitin chain can be formed on any one of these 4 lysines although most Cdc34 conjugates contain a single multiubiquitin chain. Since the presence of a Lys48-specific multiubiquitin chain in protein conjugates is known to target acceptor proteins for degradation in the ubiquitin-mediated proteolytic pathway, the present result raises the possibility that one function of the ubiquitin-conjugating activity in CDC34 may be used to target its own degradation.
Insights
The CDC34 gene product, a ubiquitin-conjugating enzyme (E2), can ubiquitinate itself. This autoubiquitination, forming Lys48-linked chains, suggests CDC34 may target itself for degradation.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- The Saccharomyces cerevisiae CDC34 gene encodes a ubiquitin-conjugating enzyme (E2) whose precise function is not fully understood.
- In vivo substrates for the Cdc34 protein are currently unknown, limiting the definition of its role.
Purpose of the Study:
- To investigate the in vitro activity of bacterially expressed Cdc34 protein.
- To determine the mechanism and potential function of Cdc34 autoubiquitination.
Main Methods:
- Bacterial expression and purification of Cdc34 protein.
- In vitro autoubiquitination assays.
- Site-directed mutagenesis of lysine residues in Cdc34.
- Hydroxylamine cleavage to map ubiquitin linkage sites.
Main Results:
- Cdc34 catalyzes its own ubiquitination, forming multiubiquitin chains predominantly linked via Lys48.
- Autoubiquitination occurs on one of four specific lysine residues (Lys273, Lys277, Lys293, Lys294) in the C-terminal region.
- The formation of Lys48-linked chains suggests a role in targeting Cdc34 for proteasomal degradation.
Conclusions:
- The ubiquitin-conjugating activity of Cdc34 can target itself for degradation.
- Autoubiquitination via Lys48-specific chains may be a regulatory mechanism for Cdc34 protein levels.