New Tools and Approaches for Studying the Role of the Saccharomyces
cerevisiae phosphatidylinositol 4-kinase Pik1 in the Yeast Nucleus
Evguenia S. Klimenko
Doctor of Philosophy in Molecular and Cell Biology
University of California, Berkeley
Professor Jeremy W. Thorner, Chair
Phosphoinositides are a specialized type of glycerophospholipids found in all eukaryotes and their levels undergo dynamic temporal and spatial changes mediated by the actions of dedicated phosphatidylinositol kinases, phosphatases, and lipases. The Saccharomyces cerevisiae genome encodes two distinct Type III PtdIns 4-kinases that share sequence homology to each other: PIK1 and STT4. Together, Pik1 and Stt4 account for synthesis of more than 90% of the PtdIns4P detectable in yeast extracts, each is essential for viability, and the inactivation of either Pik1 or Stt4 via conditional alleles results in distinct non-overlapping phenotypes. It is thought that the independent functions of Pik1 and Stt4 result from their localization to specific cellular compartments. In the budding yeast, Stt4 localizes exclusively to the plasma membrane, whereas Pik1 localizes primarily to the Golgi body, but is also found in the cytoplasm and in the nucleus.
For the first part of my dissertation research (Chapter 3), I have characterized a novel potential specific inhibitor of Pik1. Compound ST016598, dubbed optimistically "pikostatin", specifically inhibits the growth of cells with reduced dosage of PIK1, without affecting Pik1 stability, in vivo protein-protein interactions, or localization of fluorescently-tagged Pik1. Although ST016598 (pikostatin) is only a weak inhibitor of the lipid kinase activity of Pik1 in vitro, pikostatin treatment nevertheless results in a specific, rapid, and reversible depletion of the Pik1-dependent Golgi body-specific pool of PtdIns4P, without affecting the separate pool of PtdIns4P created at the plasma membrane by Stt4. I also found that a subset of pik1ts alleles exhibit hypersensitivity to pikostatin even under permissive conditions, with subsequent implications for the mechanism by which pikostatin might inhibit Pik1 activity at the Golgi body.
Previous work from this lab used two differentially-localized Pik1 constructs to demonstrate that localization of Pik1 both to the Golgi and to the nucleus is required for viability. However, so far, no attempts have been made to separate the function of Pik1 in the nucleus from its now well-characterized essential function in the Golgi compartment. For the second part of my dissertation research (Chapter 4), I have tested directly whether Pik1 is responsible for generating nuclear PtdIns4P that might serve as the precursor for production of a soluble cofactor (InsP6) that is necessary for mRNA export. I also attempted to apply an unbiased genetic selection for dosage suppressors of the lack of nuclear Pik1 as a means to identify potential phosphoinositide- or inositol-polyphosphate-binding effectors in the yeast nucleus.