The yeast UBC4 ubiquitin conjugating enzyme monoubiquitinates itself in vivo: evidence for an E2-E2 homointeraction.
Here we report that the stress-related conjugating enzyme UBC4 from Saccharomyces cerevisiae is monoubiquintinated in vivo. The UBC4-ubiquitin conjugate was detected by the coexpression in yeast of epitope-tagged ubiquitin in combination with either untagged or epitope-tagged versions of UBC4. Under these conditions the UBC4 conjugate proved to be the most abundant conjugate detected. Using chemical mapping and site-directed mutation, the site of ubiquitination was localized to a single lysine (K144) near the carboxy terminus of UBC4. A second lysine within UBC4 (K64) was also identified whose mutation resulted in the loss of ubiquitination at K144. The mutation of either K64 or K144 had no obvious effect on the known in vivo functions associated with UBC4. In another experiment, a nonfunctional UBC4 derivative with a mutation at the active site was also found to be monoubiquitinated in a manner that depended on the expression of active UBC4. This result indicated that ubiquitin was transferred in an intermolecular reaction from one UBC4 monomer to another. Cross-linking analysis demonstrated that UBC4 monomers directly and specifically interact with one another in vitro. Both the in vivo and in vitro observations reported here, in combination with previous findings, support the view that interactions between ubiquitin conjugating enzymes represent a general phenomenon.
Gwozd
C S
CS
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Arnason
T G
TG
Cook
W J
WJ
Chau
V
V
Ellison
M J
MJ
D000595:Amino Acid Sequence; D005656:Fungal Proteins / Q000378:metabolism; D008025:Ligases / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism*
D005656:Fungal Proteins; D014452:Ubiquitins; D044763:Ubiquitin-Conjugating Enzymes; C063109:ubiquitin-conjugating enzyme UBC4; D008025:Ligases; D008239:Lysine