title: E2 conjugating enzyme selectivity and requirements for function of the E3 ubiquitin ligase CHIP.
abstract: The transfer of ubiquitin (Ub) to a substrate protein requires a cascade of E1 activating, E2 conjugating, and E3 ligating enzymes. E3 Ub ligases containing U-box and RING domains bind both E2∼Ub conjugates and substrates to facilitate transfer of the Ub molecule. Although the overall mode of action of E3 ligases is well established, many of the mechanistic details that determine the outcome of ubiquitination are poorly understood. CHIP (carboxyl terminus of Hsc70-interacting protein) is a U-box E3 ligase that serves as a co-chaperone to heat shock proteins and is critical for the regulation of unfolded proteins in the cytosol. We have performed a systematic analysis of the interactions of CHIP with E2 conjugating enzymes and found that only a subset bind and function. Moreover, some E2 enzymes function in pairs to create products that neither create individually. Characterization of the products of these reactions showed that different E2 enzymes produce different ubiquitination products, i.e. that E2 determines the outcome of Ub transfer. Site-directed mutagenesis on the E2 enzymes Ube2D1 and Ube2L3 (UbcH5a and UbcH7) established that an SPA motif in loop 7 of E2 is required for binding to CHIP but is not sufficient for activation of the E2∼Ub conjugate and consequent ubiquitination activity. These data support the proposal that the E2 SPA motif provides specificity for binding to CHIP, whereas activation of the E2∼Ub conjugate is derived from other molecular determinants.
authors.affiliation: Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232, USA.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Soss
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Yue
authors.forename: Sirano
authors.initials: S
authors.lastname: Dhe-Paganon
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Chazin
mesh_terms: D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D025941:Protein Interaction Mapping / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D014908:Triticum / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry*
chemical_list: D011994:Recombinant Proteins; D025801:Ubiquitin; D004247:DNA; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases