title: Pex10p functions as an E3 ligase for the Ubc4p-dependent ubiquitination of Pex5p.
abstract: The Saccharomyces cerevisiae (Sc) PTS1 import receptor Pex5p is modified by ubiquitin, both in an Ubc4p-dependent and a Pex4p (Ubc10p)-dependent manner. Both of these modifications require the RING domain-containing protein Pex10p in vivo, but the actual role this protein plays in the ubiquitination of Pex5p has so far, remained enigmatic. Here, we report that the RING domain of Pex10p exhibits E(3) ligase activity in vitro, in combination with the human E(2) enzyme UbcH5a, a homologue of ScUbc4p, but not when ScPex4p was used as an E(2) enzyme in the reaction. We have further characterised Pex10p's E(3) ligase activity using mutants designed to disturb this activity and show that Pex10p acts as the E(3) ligase for Ubc4p-dependent ubiquitination of Pex5p but not Pex4p-dependent ubiquitination in vivo. These data imply that the two distinct Pex5p modifications require different E(3) ligases, as well as different E(2) enzymes.
authors.affiliation: Department of Medical Biochemistry, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
authors.forename: Chris
authors.initials: C
authors.lastname: Williams
authors.forename: Marlene
authors.initials: M
authors.lastname: van den Berg
authors.forename: Erica
authors.initials: E
authors.lastname: Geers
authors.forename: Ben
authors.initials: B
authors.lastname: Distel
mesh_terms: D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D006801:Humans; D007930:Leucine / Q000235:genetics; D008565:Membrane Proteins; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009154:Mutation; D000074401:Peroxins; D000074435:Peroxisome-Targeting Signal 1 Receptor; D054829:RING Finger Domains / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination / Q000235:genetics
chemical_list: D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C492057:PEX10 protein, S cerevisiae; C499509:PEX5 protein, S cerevisiae; D000074401:Peroxins; D000074435:Peroxisome-Targeting Signal 1 Receptor; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C097308:UBE2D1 protein, human; C501306:Ubc4 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D007930:Leucine; D000409:Alanine