Saccharomyces cerevisiae contains a Type II phosphoinositide 4-kinase

Shary N. Shelton, Barbara Barylko, Derk D. Binns, Bruce F. Horazdovsky, Joseph P. Albanesi, Joel M. Goodman

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

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 Km value for ATP was 266 μM, 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.

Original languageEnglish (US)
Pages (from-to)533-540
Number of pages8
JournalBiochemical Journal
Volume371
Issue number2
DOIs
StatePublished - Apr 15 2003

Fingerprint

1-Phosphatidylinositol 4-Kinase
Phosphatidylinositols
Yeast
Saccharomyces cerevisiae
Phosphotransferases
Detergents
Yeasts
Sf9 Cells
Proteins
Octoxynol
Enzymes
Green Fluorescent Proteins
Adenosine
Cell Wall
Epitopes
Amino Acid Sequence
Enzyme activity
Cell membranes
Adenosine Triphosphate
Kinetic parameters

Keywords

  • LSB6
  • Phosphoinositide
  • Protein trafficking
  • Yeast

ASJC Scopus subject areas

  • Biochemistry

Cite this

Saccharomyces cerevisiae contains a Type II phosphoinositide 4-kinase. / Shelton, Shary N.; Barylko, Barbara; Binns, Derk D.; Horazdovsky, Bruce F.; Albanesi, Joseph P.; Goodman, Joel M.

In: Biochemical Journal, Vol. 371, No. 2, 15.04.2003, p. 533-540.

Research output: Contribution to journalArticle

Shelton, Shary N. ; Barylko, Barbara ; Binns, Derk D. ; Horazdovsky, Bruce F. ; Albanesi, Joseph P. ; Goodman, Joel M. / Saccharomyces cerevisiae contains a Type II phosphoinositide 4-kinase. In: Biochemical Journal. 2003 ; Vol. 371, No. 2. pp. 533-540.
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