Saccharomyces cerevisiae contains a Type II phosphoinositide 4-kinase

Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-9041, USA.
Biochemical Journal (Impact Factor: 4.4). 05/2003; 371(Pt 2):533-40. DOI: 10.1042/BJ20021407
Source: PubMed


The yeast Saccharomyces cerevisiae contains two known phosphoinositide 4-kinases (PI 4-kinases), which are encoded by PIK1 and STT4; both are essential. Pik1p is important for exocytic transport from the Golgi, whereas Stt4p plays a role in cell-wall integrity and cytoskeletal rearrangements. In the present study, we report that cells have a third PI 4-kinase activity encoded by LSB6, a protein identified previously in a two-hybrid screen as interacting with LAS17p. Although Pik1p and Stt4p are closely related members of the Type III class of PI 4-kinases, Lsb6p belongs to the distinct Type II class, based on its amino acid sequence, its sensitivity to inhibition by adenosine and its insensitivity to wortmannin. Lsb6p is the first fungal Type II enzyme cloned. The protein was expressed and purified from Sf9 cells and used to define kinetic parameters. As commonly observed for surface-active enzymes, activities varied both with substrate concentration and lipid/detergent molar ratios. Maximal activities of approx. 100 min(-1) were obtained at the PI/Triton X-100 ratio of 1:5. The K (m) value for ATP was 266 microM, intermediate between the values reported for mammalian Type II and III kinases. Epitope-tagged protein, expressed in yeast, was entirely particulate, and about half of it could be extracted with non-ionic detergent. Lsb6p-green fluorescent protein was found both on vacuolar membranes and on the plasma membrane, suggesting a role in endocytic or exocytic pathways.

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Available from: Joel M Goodman, Feb 24, 2014
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    • "Phosphoinositide kinases, such as PI 4-kinases or PI4P 5-kinases, can each be represented by different subfamilies of enzymes, that can have non-redundant functionality. For instance, in the yeast Saccharomyces cerevisiae three PI 4-kinases with non-redundant functions exist (Table 1) which represent two distinct enzyme classes (Shelton et al. 2003; Demmel et al. 2008). In A. thaliana, four ubiquitously expressed homologs of the yeast enzymes "
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    ABSTRACT: Phosphoinositides (PIs) are minor, but essential phospholipid constituents of eukaryotic membranes, and are involved in the regulation of various physiological processes. Recent genetic and cell biological advances indicate that PIs play important roles in the control of polar tip growth in plant cells. In root hairs and pollen tubes, PIs control directional membrane trafficking required for the delivery of cell wall material and membrane area to the growing tip. So far, the exact mechanisms by which PIs control polarity and tip growth are unresolved. However, data gained from the analysis of plant, fungal and animal systems implicate PIs in the control of cytoskeletal dynamics, ion channel activity as well as vesicle trafficking. The present review aims at giving an overview of PI roles in eukaryotic cells with a special focus on functions pertaining to the control of cell polarity. Comparative screening of plant and fungal genomes suggests diversification of the PI system with increasing organismic complexity. The evolutionary conservation of the PI system among eukaryotic cells suggests a role for PIs in tip growing cells in models where PIs so far have not been a focus of attention, such as fungal hyphae.
    Protoplasma 04/2010; 240(1-4):13-31. DOI:10.1007/s00709-009-0093-0 · 2.65 Impact Factor
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    • "The third yeast PI4K, Lsp6p, also termed Pik2p, is a g-group member. Loss of Lsp6p is not lethal and leads to only moderate alterations in PtdIns4P levels (Han et al. 2002; Shelton et al. 2003). Two mammalian a-group members are isoforms of a single gene, PI4KIIIa, with isoform 1 containing just the c-terminal portion of the protein. "
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    ABSTRACT: Polyphosphoinositides (PPIs) are important signaling molecules for a variety of cellular processes including intracellular membrane trafficking. In plants, recent studies have led to an increased understanding of the role of PPIs in a number of cellular processes. Here, we discuss data concerning phosphatidylinositol 4-phosphate (PtdIns4P) function in tip growing cells. Plants lacking members of phosphatidylinositol 4-OH kinase (PI4K) or PtdIns4P phosphatase families display defects in growth and morphology of root hairs and pollen tubes. Imaging of PtdIns4P localization in root hairs and pollen tubes revealed that PtdIns4P is present on internal compartments and is enriched at the plasma membrane at the tips of growing cells. These data indicate that regulation of PtdIns4P formation and turnover is important for tip growth. Once viewed as an intermediate for synthesis of other PPIs and as a substrate for production of signaling molecules, PtdIns4P is emerging as an important PPI in its own right.
    12/2009: pages 65-77;
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    • "Before our study, Lsb6, the sole type II PI 4-kinase of yeast, had been characterized biochemically (Han et al., 2002; Shelton et al., 2003). However, its function was unknown because lsb6⌬ mutants did not exhibit growth defects (Han et al., 2002; Shelton et al., 2003), in contrast to the lethal growth deficits of mutants defective in the type III PI 4-kinases Stt4 or Pik1. However, we show herein that Lsb6 is required for the motility of endosomes containing Ste2, a novel function for type II PI 4-kinases. "
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    ABSTRACT: Endosomes in yeast have been hypothesized to move through the cytoplasm by the momentum gained after actin polymerization has driven endosome abscision from the plasma membrane. Alternatively, after abscission, ongoing actin polymerization on endosomes could power transport. Here, we tested these hypotheses by showing that the Arp2/3 complex activation domain (WCA) of Las17 (Wiskott-Aldrich syndrome protein [WASp] homologue) fused to an endocytic cargo protein (Ste2) rescued endosome motility in las17DeltaWCA mutants, and that capping actin filament barbed ends inhibited endosome motility but not endocytic internalization. Motility therefore requires continual actin polymerization on endosomes. We also explored how Las17 is regulated. Endosome motility required the Las17-binding protein Lsb6, a type II phosphatidylinositol 4-kinase. Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility. Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis. Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility.
    The Journal of Cell Biology 11/2005; 171(1):133-42. DOI:10.1083/jcb.200501086 · 9.83 Impact Factor
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