HUWE1 and TRIP12 Collaborate in Degradation of Ubiquitin-Fusion Proteins and Misframed Ubiquitin

Esben G Poulsen,Cornelia Steinhauer,Michael Lees,Lars Ellgaard,Rasmus Hartmann-Petersen,Anne-Marie Lauridsen,Alexander F Palazzo

doi:10.1371/journal.pone.0050548

Esben G Poulsen, Cornelia Steinhauer + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0050548

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Journal: PloS one	Publication Date: Nov 27, 2012
Citations: 61	License type: CC BY 4.0

Affiliation: University of Copenhagen

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 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.

Highlights

The ubiquitin-proteasome system (UPS) is responsible for the majority of intracellular protein degradation, and plays an important regulatory role in many cellular processes, including metabolism, cell division, DNA repair, antigen presentation, signal transduction, and development [1].With few exceptions, proteins must be conjugated to a chain of ubiquitin before they become substrates for the 26S proteasome
At the 26S proteasome, the ubiquitin chains can be further elongated or released, while the substrate is degraded into shorter peptides that can be displayed on the cell surface for immuno surveillance or further degraded to free amino acids by various amino peptidases [6]
It was first described in yeast [7] [2], the ubiquitin fusion degradation (UFD) pathway is conserved in all eukaryotic cells [8]

Summary

Introduction

The ubiquitin-proteasome system (UPS) is responsible for the majority of intracellular protein degradation, and plays an important regulatory role in many cellular processes, including metabolism, cell division, DNA repair, antigen presentation, signal transduction, and development [1]. At the 26S proteasome, the ubiquitin chains can be further elongated or released, while the substrate is degraded into shorter peptides that can be displayed on the cell surface for immuno surveillance or further degraded to free amino acids by various amino peptidases [6]. It was first described in yeast [7] [2], the ubiquitin fusion degradation (UFD) pathway is conserved in all eukaryotic cells [8]. Since we could co-precipitate HUWE1 with the substrate and the 26S proteasome, we propose that HUWE1 directly ubiquitylates UFD substrates at a late step during the degradation pathway

Materials and Methods

Results

Findings

Discussion