title: KCMF1 (potassium channel modulatory factor 1) Links RAD6 to UBR4 (ubiquitin N-recognin domain-containing E3 ligase 4) and lysosome-mediated degradation.
abstract: RAD6 is a ubiquitin E2 protein with roles in a number of different biological processes. Here, using affinity purification coupled with mass spectrometry, we identify a number of new RAD6 binding partners, including the poorly characterized ubiquitin E3 ligases KCMF1 (potassium channel modulatory factor 1) and UBR4 (ubiquitin N-recognin domain-containing E3 ligase 4), a protein that can bind N-end rule substrates, and which was recently linked to lysosome-mediated degradation and autophagy. NMR, combined with in vivo and in vitro interaction mapping, demonstrate that the KCMF1 C terminus binds directly to RAD6, whereas N-terminal domains interact with UBR4 and other intracellular vesicle- and mitochondria-associated proteins. KCMF1 and RAD6 colocalize at late endosomes and lysosomes, and cells disrupted for KCMF1 or RAD6 function display defects in late endosome vesicle dynamics. Notably, we also find that two different RAD6A point mutants (R7W and R11Q) found in X-linked intellectual disability (XLID) patients specifically lose the interaction with KCMF1 and UBR4, but not with other previously identified RAD6 interactors. We propose that RAD6-KCMF1-UBR4 represents a unique new E2-E3 complex that targets unknown N-end rule substrates for lysosome-mediated degradation, and that disruption of this complex via RAD6A mutations could negatively affect neuronal function in XLID patients.
authors.affiliation: From the ‡Princess Margaret Cancer Centre, University Health Network and Department of Medical Biophysics, University of Toronto;
authors.forename: Jenny H
authors.initials: JH
authors.lastname: Hong
authors.affiliation: §Structural Genomics Consortium, Toronto, Ontario Canada.
authors.forename: Lilia
authors.initials: L
authors.lastname: Kaustov
authors.affiliation: From the ‡Princess Margaret Cancer Centre, University Health Network and Department of Medical Biophysics, University of Toronto;
authors.forename: Etienne
authors.initials: E
authors.lastname: Coyaud
authors.affiliation: From the ‡Princess Margaret Cancer Centre, University Health Network and Department of Medical Biophysics, University of Toronto;
authors.forename: Tharan
authors.initials: T
authors.lastname: Srikumar
authors.affiliation: From the ‡Princess Margaret Cancer Centre, University Health Network and Department of Medical Biophysics, University of Toronto;
authors.forename: Janet
authors.initials: J
authors.lastname: Wan
authors.affiliation: From the ‡Princess Margaret Cancer Centre, University Health Network and Department of Medical Biophysics, University of Toronto; §Structural Genomics Consortium, Toronto, Ontario Canada.
authors.forename: Cheryl
authors.initials: C
authors.lastname: Arrowsmith
authors.affiliation: From the ‡Princess Margaret Cancer Centre, University Health Network and Department of Medical Biophysics, University of Toronto; brian.raught@uhnres.utoronto.ca.
authors.forename: Brian
authors.initials: B
authors.lastname: Raught
mesh_terms: D001343:Autophagy; D001665:Binding Sites; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D013058:Mass Spectrometry; D038901:X-Linked Intellectual Disability / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D017354:Point Mutation; D040901:Proteomics / Q000379:methods*; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
chemical_list: D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; C471545:UBE2A protein, human; D044763:Ubiquitin-Conjugating Enzymes; C496223:KCMF1 protein, human; C505833:UBR4 protein, human; D044767:Ubiquitin-Protein Ligases