Methods for studying the regulation of membrane traffic by ubiquitin and the ESCRT pathway

Abstract

Covalent modification of proteins with ubiquitin dynamically regulates their function and fate. The ubiquitination of most plasma membrane proteins initiates endocytosis and ESCRT-mediated sorting to the lysosomal lumen for degradation. Powerful genetic approaches in the budding yeast Saccharomyces cerevisiae have been particularly instrumental in the discovery and elucidation of these molecular mechanisms, which are conserved in all eukaryotes. Here we provide two detailed protocols and tools for studying ubiquitination-dependent membrane trafficking mechanisms in yeast. The first utilizes fusions between a protein of interest and an auxotrophic marker to screen for mutants that affect ubiquitin-mediated endocytosis. The second method artificially ubiquitinates a protein of interest, allowing downstream trafficking steps to be studied independently from the regulatory signals that initiate endocytosis.

Keywords: Auxotrophic marker; Endocytosis; Genetic screen; Rapamycin; Ubiquitin; Yeast.