Plasma Membrane Targeting of the Yck2 Casein Kinase 1 Isoform

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The Yck1 and Yck2 protein kinases are together essential for viability in Saccharomyces cerevisiae. The two proteins are members of an abundant protein kinase family that is highly conserved in eukaryotic cells, whose properties have been well-studied but whose functions are not yet clear in most organisms. The plasma membrane-associated Yck protein kinases are required for cellular morphogenesis and cytokinesis in yeast, and are enriched at specific sites within the membrane during the cell cycle. Studies with a green fluorescent protein fusion to Yck2p allowed determination that the route of delivery of Yck2p to the plasma membrane requires a functioning secretory pathway. This was unexpected because the Yck proteins carry a C-terminal sequence necessary for membrane localization that could direct modification by a prenyltransferase that heretofore was only known to prenylate members of the Rab family of ras-like GTPases. This research is directed toward understanding the targeting mechanisms of the Yck2 protein by determining the cis-acting elements on Yck2p and the trans-acting factors that are required for modification and localization. Also, the question of whether prenylation has functional consequences, either to the targeting mechanism or to the protein's interaction with targets, will be tested. Finally, evidence that the Yck2 kinase cycles on and off the plasma membrane in association with vesicular structures will be explored, to characterize the apparent cycling and to determine whether this is an integral part of the essential function of Yck in yeast. The results of these studies will be important not only in understanding the localization mechanism of an essential protein kinase, but will address important questions in the area of protein prenylation -- especially the question of whether prenylation can determine targeting of these proteins to particular membranes and the functional consequences of this modification. The project addresses a fundamental question in cell biology, namely, how do specific proteins, that need to be located in specific places inside the cell in order to perform their function, get to where they need to be? The protein under investigation in this project, Yck, is a protein kinase, a type of protein that functions in intracellular signal transduction events by activating or inactivating other specific proteins by phosphorylating them. The project will explore the role of protein prenylation in subcellular localization of proteins. The biochemistry of prenylation (an important post-translational modification of a protein that results from the covalent addition of a specific fatty acyl group to the protein; such a modification allows a protein, that would otherwise not interact with membranes, to interact with membranes) is reasonably well understood. However, the specific cell biological consequences of prenylation are not so well understood. The study will be done in a model organism, the yeast Saccharomyces cerevisiae, that historically has richly provided cell biologists with fundamental information about cellular processes that are applicable to a broad range of other types of organisms, including plants and mammals.