Molecular interactions of yeast frequenin (Frq1) with the phosphatidylinositol 4-kinase isoform, Pik1

J Biol Chem. 2003 Feb 14;278(7):4862-74. doi: 10.1074/jbc.M207920200. Epub 2002 Dec 10.

Abstract

Frq1, a 190-residue N-myristoylated calcium-binding protein, associates tightly with the N terminus of Pik1, a 1066-residue phosphatidylinositol 4-kinase. Deletion analysis of an Frq1-binding fragment, Pik1-(10-192), showed that residues within 80-192 are necessary and sufficient for Frq1 association in vitro. A synthetic peptide (residues 151-199) competed for binding of [(35)S]Pik1-(10-192) to bead-immobilized Frq1, whereas shorter peptides (164-199 and 174-199) did not. Correspondingly, a deletion mutant, Pik1(delta152-191), did not co-immunoprecipitate efficiently with Frq1 and did not support growth at elevated temperature. Site-directed mutagenesis of Pik1-(10-192) suggested that recognition determinants lie over an extended region. Titration calorimetry demonstrated that binding of an 83-residue fragment, Pik1-(110-192), or the 151-199 peptide to Frq1 shows high affinity (K(d) approximately 100 nm) and is largely entropic, consistent with hydrophobic interaction. Stoichiometry of Pik1-(110-192) binding to Frq1 was 1:1, as judged by titration calorimetry, by changes in NMR spectrum and intrinsic tryptophan fluorescence, and by light scattering. In cell extracts, Pik1 and Frq1 exist mainly in a heterodimeric complex, as shown by size exclusion chromatography. Cys-15 in Frq1 is not S-palmitoylated, as assessed by mass spectrometry; a Frq1(C15A) mutant and even a non-myristoylated Frq1(G2A,C15A) double mutant rescued the inviability of frq1Delta cells. This study defines the segment of Pik1 required for high affinity binding of Frq1.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 1-Phosphatidylinositol 4-Kinase / chemistry
  • 1-Phosphatidylinositol 4-Kinase / metabolism*
  • Binding Sites / genetics
  • Calcium-Binding Proteins / chemistry
  • Calcium-Binding Proteins / metabolism*
  • Mass Spectrometry
  • Protein Binding
  • Protein Conformation
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Sequence Deletion
  • Structure-Activity Relationship

Substances

  • Calcium-Binding Proteins
  • FRQ1 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • 1-Phosphatidylinositol 4-Kinase
  • PIK1 protein, S cerevisiae