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. 100min−1 were obtained at the PI/Triton X-100 ratio of 1:5. The Km value for ATP was 266μM, 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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April 2003
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Research Article|
April 15 2003
Saccharomyces cerevisiae contains a Type II phosphoinositide 4-kinase
Shary N. SHELTON;
Shary N. SHELTON
∗Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-9041, U.S.A.
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Barbara BARYLKO;
Barbara BARYLKO
∗Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-9041, U.S.A.
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Derk D. BINNS;
Derk D. BINNS
∗Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-9041, U.S.A.
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Bruce F. HORAZDOVSKY;
Bruce F. HORAZDOVSKY
†Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-9041, U.S.A.
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Joseph P. ALBANESI;
Joseph P. ALBANESI
∗Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-9041, U.S.A.
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Joel M. GOODMAN
Joel M. GOODMAN
1
∗Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-9041, U.S.A.
1To whom correspondence should be addressed (e-mail Joel.Goodman@UTSouthwestern.edu).
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Publisher: Portland Press Ltd
Received:
September 05 2002
Revision Received:
January 08 2003
Accepted:
January 10 2003
Accepted Manuscript online:
January 10 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 371 (2): 533–540.
Article history
Received:
September 05 2002
Revision Received:
January 08 2003
Accepted:
January 10 2003
Accepted Manuscript online:
January 10 2003
Citation
Shary N. SHELTON, Barbara BARYLKO, Derk D. BINNS, Bruce F. HORAZDOVSKY, Joseph P. ALBANESI, Joel M. GOODMAN; Saccharomyces cerevisiae contains a Type II phosphoinositide 4-kinase. Biochem J 15 April 2003; 371 (2): 533–540. doi: https://doi.org/10.1042/bj20021407
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