title: The acidic tail of the Cdc34 ubiquitin-conjugating enzyme functions in both binding to and catalysis with ubiquitin ligase SCFCdc4.
abstract: Ubiquitin ligases, together with their cognate ubiquitin-conjugating enzymes, are responsible for the ubiquitylation of proteins, a process that regulates a myriad of eukaryotic cellular functions. The first cullin-RING ligase discovered, yeast SCF(Cdc4), functions with the conjugating enzyme Cdc34 to regulate the cell cycle. Cdc34 orthologs are notable for their highly acidic C-terminal extension. Here we confirm that the Cdc34 acidic C-terminal tail has a role in Cdc34 binding to SCF(Cdc4) and makes a major contribution to the submicromolar K(m) of Cdc34 for SCF(Cdc4). Moreover, we demonstrate that a key functional property of the tail is its acidity. Our analysis also uncovers an unexpected new function for the acidic tail in promoting catalysis. We demonstrate that SCF is functional when Cdc34 is fused to the C terminus of Cul1 and that this fusion retains partial function even when the acidic tail has been deleted. The Cdc34-SCF fusion proteins that lack the acidic tail must interact in a fundamentally different manner than unfused SCF and wild type Cdc34, demonstrating that distinct mechanisms of E2 recruitment to E3, as is seen in nature, can sustain substrate ubiquitylation. Finally, a search of the yeast proteome uncovered scores of proteins containing highly acidic stretches of amino acids, hinting that electrostatic interactions may be a common mechanism for facilitating protein assembly.
authors.affiliation: Howard Hughes Medical Institute and the Division of Biology, California Institute of Technology, Pasadena, California 91125.
authors.forename: Gary
authors.initials: G
authors.lastname: Kleiger
authors.affiliation: Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, Connecticut 06030.
authors.forename: Bing
authors.initials: B
authors.lastname: Hao
authors.affiliation: Howard Hughes Medical Institute and the Division of Biology, California Institute of Technology, Pasadena, California 91125.
authors.forename: Dane A
authors.initials: DA
authors.lastname: Mohl
authors.affiliation: Howard Hughes Medical Institute and the Division of Biology, California Institute of Technology, Pasadena, California 91125. Electronic address: deshaies@caltech.edu.
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Deshaies
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome; D002453:Cell Cycle / Q000502:physiology; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017434:Protein Structure, Tertiary / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D044843:SKP Cullin F-Box Protein Ligases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*
chemical_list: C100318:Cullin 1; D044842:Cullin Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C577449:CDC34 protein, S cerevisiae; C577447:CDC34 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044843:SKP Cullin F-Box Protein Ligases