Genetic and Biochemical Analyses of Yeast ESCRT

Sudeep Banjade; Shaogeng Tang; Scott D Emr

doi:10.1007/978-1-4939-9492-2_8

Genetic and Biochemical Analyses of Yeast ESCRT

Methods Mol Biol. 2019:1998:105-116. doi: 10.1007/978-1-4939-9492-2_8.

Authors

Sudeep Banjade¹, Shaogeng Tang^{1

2}, Scott D Emr³

Affiliations

¹ Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA.
² Department of Biochemistry, Stanford University, Stanford, CA, USA.
³ Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA. sde26@cornell.edu.

PMID: 31250297
DOI: 10.1007/978-1-4939-9492-2_8

Abstract

Budding yeast Saccharomyces cerevisiae is an ideal model organism to study membrane trafficking pathways. The ESCRT (endosomal sorting complexes required for transport) pathway was first identified in this organism. Upon recognition of endocytosed ubiquitinated membrane proteins at endosomes, ESCRTs assemble at these organelles to catalyze the biogenesis of multivesicular bodies (MVBs). Formation of MVBs leads to the trafficking of these membrane proteins to vacuoles for degradation. Here, we describe genetic and biochemical approaches to study ESCRT function. We outline in vivo endocytosis assays using two model cargoes in Saccharomyces cerevisiae and also describe an in vitro approach to analyze ESCRT-III polymerization on lipid monolayers.

Keywords: ESCRT; ESCRT-III polymerization; Endocytosis; Lipid monolayer; Multivesicular bodies (MVB); Saccharomyces cerevisiae; Snf7 spiral.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Transport Systems, Basic / genetics
Amino Acid Transport Systems, Basic / metabolism*
Endocytosis / genetics
Endosomal Sorting Complexes Required for Transport / genetics
Endosomal Sorting Complexes Required for Transport / isolation & purification
Endosomal Sorting Complexes Required for Transport / metabolism*
Green Fluorescent Proteins / chemistry
Membrane Lipids / chemistry
Membrane Lipids / metabolism
Membranes, Artificial
Microscopy, Electron / methods
Microscopy, Fluorescence / methods
Multivesicular Bodies / metabolism*
Mutation
Protein Multimerization / genetics
Recombinant Proteins / genetics
Recombinant Proteins / isolation & purification
Recombinant Proteins / metabolism
Saccharomyces cerevisiae / genetics
Saccharomyces cerevisiae / metabolism*
Saccharomyces cerevisiae Proteins / genetics
Saccharomyces cerevisiae Proteins / isolation & purification
Saccharomyces cerevisiae Proteins / metabolism*
Vacuoles / metabolism

Substances

Amino Acid Transport Systems, Basic
CAN1 protein, S cerevisiae
Endosomal Sorting Complexes Required for Transport
Membrane Lipids
Membranes, Artificial
Recombinant Proteins
SNF7 protein, S cerevisiae
Saccharomyces cerevisiae Proteins
Green Fluorescent Proteins