title: HUWE1 and TRIP12 collaborate in degradation of ubiquitin-fusion proteins and misframed ubiquitin.
abstract: In eukaryotic cells an uncleavable ubiquitin moiety conjugated to the N-terminus of a protein signals the degradation of the fusion protein via the proteasome-dependent ubiquitin fusion degradation (UFD) pathway. In yeast the molecular mechanism of the UFD pathway has been well characterized. Recently the human E3 ubiquitin-protein ligase TRIP12 was connected with the UFD pathway, but little is otherwise known about this system in mammalian cells. In the present work, we utilized high-throughput imaging on cells transfected with a targeted siRNA library to identify components involved in degradation of the UFD substrate Ub(G76V)-YFP. The most significant hits from the screen were the E3 ubiquitin-protein ligase HUWE1, as well as PSMD7 and PSMD14 that encode proteasome subunits. Accordingly, knock down of HUWE1 led to an increase in the steady state level and a retarded degradation of the UFD substrate. Knock down of HUWE1 also led to a stabilization of the physiological UFD substrate UBB(+1). Precipitation experiments revealed that HUWE1 is associated with both the Ub(G76V)-YFP substrate and the 26S proteasome, indicating that it functions late in the UFD pathway. Double knock down of HUWE1 and TRIP12 resulted in an additive stabilization of the substrate, suggesting that HUWE1 and TRIP12 function in parallel during UFD. However, even when both HUWE1 and TRIP12 are downregulated, ubiquitylation of the UFD substrate was still apparent, revealing functional redundancy between HUWE1, TRIP12 and yet other ubiquitin-protein ligases.
authors.affiliation: Department of Biology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Esben G
authors.initials: EG
authors.lastname: Poulsen
authors.forename: Cornelia
authors.initials: C
authors.lastname: Steinhauer
authors.forename: Michael
authors.initials: M
authors.lastname: Lees
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Lauridsen
authors.forename: Lars
authors.initials: L
authors.lastname: Ellgaard
authors.forename: Rasmus
authors.initials: R
authors.lastname: Hartmann-Petersen
mesh_terms: D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004586:Electrophoresis; D006801:Humans; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D025521:Tumor Suppressor Proteins; C479419:UBB protein, human; D025801:Ubiquitin; C502830:HUWE1 protein, human; C530813:TRIP12 protein, human; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease