Enzyme reversal to explore the function of yeast E3 ubiquitin ‐ligases

In this study we describe tools that may help achieve both of these goals. We describe a strategy whereby the activity of a ubiquitin ligase has been enzymatically reversed, accomplished by fusing it to a catalytic domain of an exogenous deubiquitinating enzyme. We present a library of 72 ‘anti‐ligases’ that appear to work in a dominant‐negative fashion to stabilize their cognate substrates against ubiquitin‐dependent proteasomal and lysosomal degradation. We then used the ligase‐DUb library to screen for E3 ligases involved in post‐Golgi / endosomal trafficking. We identify ligases previously implicated in these pathways (Rsp5 and Tul1), in addition to ligases previously localized to endosomes (Pib1 and Vps8). We also document an optimized workflow for isolating and analyzing the ‘ubiquitome’ of yeast, which can be used with mass‐spectrometry to identify substrates perturbed by expression of particular ligase‐DUb fusions. Graphical abstract MacDonald et al. describe a library of ‘anti‐ligase’ reagents that antagonize the activity of endogenous E3 ubiquitin ligases, perturbing their function and stabilizing endogenous substrate levels. The library was used to identify E3 ligases required for vacuolar sorting from the biosynthetic and endocytic pathways.
Source: Traffic - Category: Research Authors: Tags: TOOLBOX Source Type: research