INCREASED UBIQUITIN EXPRESSION SUPPRESSES THE CELL-CYCLE DEFECT ASSOCIATED WITH THE YEAST UBIQUITIN-CONJUGATING ENZYME, CDC34 (UBC3) - EVIDENCE FOR A NONCOVALENT INTERACTION BETWEEN CDC34 AND UBIQUITIN
Ja. Prendergast et al., INCREASED UBIQUITIN EXPRESSION SUPPRESSES THE CELL-CYCLE DEFECT ASSOCIATED WITH THE YEAST UBIQUITIN-CONJUGATING ENZYME, CDC34 (UBC3) - EVIDENCE FOR A NONCOVALENT INTERACTION BETWEEN CDC34 AND UBIQUITIN, The Journal of biological chemistry, 270(16), 1995, pp. 9347-9352
The yeast ubiquitin (Uh) conjugating enzyme CDC34 plays a crucial role
in the progression of the cell cycle from the G(1) to S phase, In an
effort to identify proteins that interact with CDC34 we undertook a ge
netic screen to isolate genes whose increased expression suppressed th
e cell cycle defect associated with the cdc34-2 temperature-sensitive
allele, From this screen, the poly-Ub gene UBI4 was identified as a mo
derately strong suppressor, The fact that the overexpression of a gene
encoding a single Ub protein could also suppress the cdc34-2 allele i
ndicated that suppression was related to the increased abundance of Ub
, Ub overexpression was found to suppress two other structurally unrel
ated cdc34 mutations, in addition to the cdc34-2 allele, In all three
cases, suppression depended on the expression of Ub with an intact car
boxyl terminus, Only the cdc34-2 allele, however, could be suppressed
by Ub with an amino acid substitution at lysine 48 which is known to b
e involved in multi-Ub chain assembly, Genetic results showing allele
specific suppression of cdc34 mutations by various Ub derivatives sugg
ested a specific noncovalent interaction between Ub and CDC34, Consist
ent with this prediction, we have shown by chemical crosslinking the e
xistence of a specific noncovalent Ub binding site on CDC34, Together,
these genetic and biochemical experiments indicate that Ub suppressio
n of these cdc34 mutations results from the combined contributions of
Ub-CDC34 thiol ester formation and a noncovalent interaction between U
b and CDC34 and therefore suggest that the correct positioning of Uh o
n a surface of the ubiquitin conjugating enzyme is a requirement of en
zyme function.