Palmitoylation and the yeast casein kinase yck2

PALMITOYLATION AND THE YEAST CASEIN KINASE YCK2byIRENE PAPANAYOTOUMay 2011Advisor: Dr. Nicholas G. DavisMajor: PharmacologyDegree: Doctor of Philosophy ! The Saccharomyces cerevisiae type I casein kinase Yck2 is tethered to thecytosolic face of the plasma membrane through palmitoylation of its two C-terminalcysteines. Yck2 palmitoylation is mediated by the Golgi-localized palmitoyl transferaseAkr1. Yck2 is a cytosolic protein and is presumably synthesized on cytosolic ribosomes.While the trafficking events following palmitoylation of Yck2 at the Golgi are wellcharacterized,the initial trafficking events after synthesis remain unknown. We sought toelucidate these early events of how Yck2 might travel to the Golgi-localized Akr1 after itis synthesized.! Our initial experimental analysis of Yck2 utilized glass bead cell lysates. The useof glass beads to mechanically disrupt yeast cells is used in a variety of applications,including for the analysis of native protein function (e.g. protein-protein interaction,enzyme assays, membrane fractionations). We describe a surprising case of proteindenaturation and aggregation that is induced by this lysis protocol. Interestingly, whenyeast cells are subjected to glass bead disruption, non-palmitoylated, cytosolic forms of118