Novel CDC34 (UBC3) ubiquitin-conjugating enzyme mutants obtained by charge-to-alanine scanning mutagenesis

Abstract

CDC34 (UBC3) encodes a ubiquitin-conjugating (E2) enzyme required for transition from the G 1 phase to the S phase of the budding yeast cell cycle. CDC34 consists of a 170-residue catalytic N-terminal domain onto which is appended an acidic C-terminal domain. A portable determinant of cell cycle function resides in the C-terminal domain, but determinants for specific function must reside in the N-terminal domain as well. We have explored the utility of ''charge-to-alanine'' scanning mutagenesis to identify novel N-terminal domain mutants of CDC34 that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that nevertheless are defective with respect to its cell cycle function. Such mutants may reveal determinants of specific in vivo function, such as those required for interaction with substrates or trans-acting regulators of activity and substrate selectivity. Three of 18 ''single-scan'' mutants (in which small clusters of charged residues were mutated to alanine) were compromised with respect to in vivo function. One mutant (cdc34-109, 111, 113A) targeted a 12-residue segment of the Cdc34 protein not found in most other E2s and was unable to complement a cdc34 null mutant at low copy numbers but could complement a null mutant when overexpressed from an induced GAL1 promoter. Combining adjacent pairs of single-scan mutants to produce ''double-scan'' mutants yielded four additional mutants, two of which showed heat and cold sensitivity conditional defects. Most of the mutant proteins expressed in Escherichia coli displayed unfacilitated (E3-independent) ubiquitin-conjugating activity, but two mutants differed from wild-type and other mutant Cdc34 proteins in the extent of multiubiquitination they catalyzed during an autoubiquitination reaction. Our results validate the use of clustered charge-to-alanine scanning mutagenesis for exploring ubiquitin-conjugating enzyme function and have identified additional mutant alleles of CDC34 that will be valuable in further genetic and biochemical studies of Cdc34-dependent ubiquitination.Covalent attachment of the highly conserved protein ubiquitin to other eucaryotic proteins is required for many cellular functions, including stress resistance, selective proteolysis of most normal and abnormal short-lived proteins, cell cycle progression, and DNA repair (14,27,31). Ubiquitination of proteins is catalyzed by an elaborate multienzyme conjugation pathway whose components are conserved in eucaryotes (31). The first step in the ubiquitin ligation pathway is the ATPdependent activation of ubiquitin via the formation of a thiol ester between the ubiquitin C terminus and a cysteine residue in the ubiquitin-activating (E1) enzyme. Ubiquitin is then transferred from the E1 to one of several ubiquitin-conjugating (E2) enzymes, also via a thiol ester formed with a conserved Cys residue. Finally, ubiquitin is transferred from the E2 to a protein substrate in a reaction that often requires a third factor called a ubiquitin protein ligase or E3 (14,...

Role of UEV-1, an Inactive Variant of the E2 UbiquitinConjugating Enzymes, in In Vitro Differentiation and Cell Cycle Behavior of HT-29-M6 Intestinal Mucosecretory Cells

“…In E2 enzymes, the core Ubc domain appears to be sufficient for their enzymatic activity, while the extensions at either end could serve as modules for protein-protein interactions that direct the enzyme to specific substrates or subcellular locations (2,20,27,38,48). The unique amino termini of the different isoforms of HsUEV-1 might be involved in specific protein-protein interactions which may affect their localization or activity.…”

Human Cdc34 Employs Distinct Sites To Coordinate Attachment of Ubiquitin to a Substrate and Assembly of Polyubiquitin Chains

“…In addition, conserved ScCdc34 S97 (S95 in human Cdc34) is essential for cell viability (14) and is required for supporting E2 monomer interactions or polyubiquitin chain assembly on the E2 protein (32). Other critical ScCdc34 sites include a conserved acidic insertion loop (residues 103 to 114 [residues 102 to 113 in humans]) (14,23) and the charged cluster (residues D144, D148, R150, K151, and E154 [residues D143, R149, K150, and E153 in humans]) (23).…”

Role of the Ubiquitin-proteasome System in Methylmercury Toxicity in Saccharomyces cerevisiae

“…To identify the functional domain of Cdc34 that is essential for acquisition of resistance to MeHg, we constructed several Cdc34 mutants 4,5) that had no ubiquitin-conjugating activity and examined their MeHg sensitivity, thereafter. Our results showed that the MeHg sensitivity of yeast cells that overexpressed Cdc34 mutants did not significantly differ from that of the control yeast cells.…”