Protein aggregation induced during glass bead lysis of yeast.

Yeast
Irene PapanayotouNicholas G Davis

Abstract

Yeast cell lysates produced by mechanical glass bead disruption are widely used in a variety of applications, including for the analysis of native function, e.g. protein-protein interaction, enzyme assays and membrane fractionations. Below, we report a striking case of protein denaturation and aggregation that is induced by this lysis protocol. Most of this analysis focuses on the type 1 casein kinase Yck2, which normally tethers to the plasma membrane through C-terminal palmitoylation. Surprisingly, when cells are subjected to glass bead disruption, non-palmitoylated, cytosolic forms of the kinase denature and aggregate, while membrane-associated forms, whether attached through their native palmitoyl tethers or through a variety of artificial membrane-tethering sequences, are wholly protected from denaturation and aggregation. A wider look at the yeast proteome finds that, while the majority of proteins resist glass bead-induced aggregation, a significant subset does, in fact, succumb to such denaturation. Thus, yeast researchers should be aware of this potential artifact when embarking on biochemical analyses that employ glass bead lysates to look at native protein function. Finally, we demonstrate an experimental utility for...Continue Reading

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Citations

Jun 10, 2011·Molecular Biology of the Cell·Amy F RothNicholas G Davis
Nov 16, 2010·Biochimie·Le-Le HuKuo-Chen Chou
Aug 9, 2016·Journal of Microbiological Methods·Frédérique GourietFlorence Fenollar
Apr 6, 2019·Applied Microbiology and Biotechnology·Lisa Van RenterghemWim Soetaert
Aug 13, 2017·Analytical and Bioanalytical Chemistry·Anna M DonnellAnne P Vonderheide
Aug 26, 2020·Environmental Science and Pollution Research International·Lorena da Graça Pedrosa de MacenaMarize Pereira Miagostovich
Dec 29, 2020·Applied and Environmental Microbiology·Masashi YoshidaShingo Izawa
Sep 6, 2013·Journal of Agricultural and Food Chemistry·Elsa VieiraIsabel M P L V O Ferreira

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