NOVEL CDC34 (UBC3) UBIQUITIN-CONJUGATING ENZYME MUTANTS OBTAINED BY CHARGE-TO-ALANINE SCANNING MUTAGENESIS

CDC34 (UBC3) encodes a ubiquitin-conjugating (E2) enzyme required for transition from the G(1) phase to the S phase of the budding yeast cel l cycle. CDC34 consists of a 170-residue catalytic N-terminal domain o nto which is appended an acidic C-terminal domain. A portable determin ant of cell cycle function resides in the C-terminal domain, but deter minants for specific function must reside in the N-terminal domain as well. We have explored the utility of ''charge-to-alanine'' scanning m utagenesis to identify novel N-terminal domain mutants of CDC34 that a re enzymatically competent with respect to unfacilitated (ES-independe nt) ubiquitination but that nevertheless are defective with respect to its cell cycle function. Such mutants may reveal determinants of spec ific in vivo function, such as those required for interaction with sub strates or trans-acting regulators of activity and substrate selectivi ty, Three of 18 ''single-scan'' mutants (in which small clusters of ch arged residues were mutated to alanine) were compromised with respect to in vivo function. One mutant (cdc34-109, 111, 113A) targeted a 12-r esidue segment of the Cdc34 protein not found in most other E2s and wa s unable to complement a cdc34 null mutant at low copy numbers but cou ld complement a null mutant when overexpressed from an induced GALI pr omoter. Combining adjacent pairs of single-scan mutants to produce ''d ouble-scan'' mutants yielded four additional mutants, two of which sho wed heat and cold sensitivity conditional defects, Most of the mutant proteins expressed in Escherichia coli displayed unfacilitated (E3-ind ependent) ubiquitin-conjugating activity, but two mutants differed fro m wild-type and other mutant Cdc34 proteins in the extent of multiubiq uitination they catalyzed during an autoubiquitination reaction. Our r esults validate the use of clustered charge-to-alanine scanning mutage nesis for exploring ubiquitin-conjugating enzyme function and have ide ntified additional mutant alleles of CDC34 that will be valuable in fu rther genetic and biochemical studies of Cd34-dependent ubiquitination .