Expression of a ubiquitin derivative that conjugates to protein irreversibly produces phenotypes consistent with a ubiquitin deficiency.
Ubiquitin (Ub) exists in a dynamic equilibrium between the free form and the conjugated form. This equilibrium is maintained and regulated through the antagonistic actions of the conjugation system and a class of enzymes referred to collectively as the Ub-protein hydrolases. Using a previously described epitope-tagged Ub approach (Ellison, M., and Hochstrasser, M. (1991) J. Biol. Chem. 266, 21150-21157) we show here that a single amino acid substitution at the carboxyl terminus of Ub (Gly-76 to Ala-76) results in a derivative of Ub (UbA-76) that becomes irreversibly conjugated to protein when expressed in the yeast Saccharomyces cerevisiae, producing a profound effect on the Ub-conjugate equilibrium. The major target of UbA-76 conjugation is itself (and presumably wild-type Ub) producing unanchored chains at the expense of the free form. Unsurprisingly, the expression of UbA-76 results in yeast phenotypes that would be expected in situations of Ub deprivation. Such cells show slow growth characteristics and sensitivity to various forms of environmental stress and to ultraviolet light. In view of these findings, the expression of UbA-76 in higher organisms may represent a convenient epigenetic strategy for examining the physiological consequences of Ub deprivation or Ub-protein hydrolase disfunction in living cells without the need for gene disruption or replacement. The observation that UbA-76 couples to itself irreversibly also provides an effective tool for elucidating the role of Ub as the proteolytic signal.
Hodgins
R R
RR
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Ellison
K S
KS
Ellison
M J
MJ
D000596:Amino Acids / Q000235:genetics; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000378:metabolism*; D015870:Gene Expression; D006867:Hydrolases / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014452:Ubiquitins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D005656:Fungal Proteins; D014452:Ubiquitins; D006867:Hydrolases