The bacterially expressed yeast CDC34 gene product can undergo autoubiquitination to form a multiubiquitin chain-linked protein.
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.
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201.
A
A
Banerjee
L
L
Gregori
Y
Y
Xu
V
V
Chau
D064173:Anaphase-Promoting Complex-Cyclosome; D001483:Base Sequence; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded; D004926:Escherichia coli; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013438:Sulfhydryl Compounds / Q000737:chemistry; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
D004277:DNA, Single-Stranded; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D013438:Sulfhydryl Compounds; D014452:Ubiquitins; C577449:CDC34 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D008239:Lysine