Protein aggregation induced during glass bead lysis of yeast.
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 glass bead-induced aggregation, using its fine discrimination of membrane-associated from non-associated Yck2 forms to discern fractional palmitoylation states of Yck2 mutants that are partially defective for palmitoylation.
Papanayotou
Irene
I
Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Sun
Beimeng
B
Roth
Amy F
AF
Davis
Nicholas G
NG
D047389:Casein Kinase I / Q000737:chemistry / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D005898:Glass; D054878:Lipoylation; D008863:Microspheres; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism
D029701:Saccharomyces cerevisiae Proteins; D047389:Casein Kinase I; C479666:YCK2 protein, S cerevisiae