huwe1 and trip12 collaborate in degradation of ubiquitin-fusion proteins and misframed ubiquitinhuwe1 trip12 ubiquitin-fusion蛋白质降解的协作和misframed泛素.pdfVIP

HUWE1 and TRIP12 Collaborate in Degradation of Ubiquitin-Fusion Proteins and Misframed Ubiquitin 1 2 2 1 1 Esben G. Poulsen , Cornelia Steinhauer , Michael Lees , Anne-Marie Lauridsen , Lars Ellgaard , Rasmus Hartmann-Petersen1* 1 Department of Biology, University of Copenhagen, Copenhagen, Denmark, 2 Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark 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 UbG76V-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 UbG76V-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 HUWE