Details

Autor(en) / Beteiligte

• Huttner, Inken G
• Strahl, Thomas
• Osawa, Masanori
• King, David S
• Ames, James B
• Thorner, Jeremy

Titel

Molecular Interactions of Yeast Frequenin (Frq1) with the Phosphatidylinositol 4-Kinase Isoform, Pik1

Ist Teil von

• The Journal of biological chemistry, 2003-02, Vol.278 (7), p.4862-4874

Ort / Verlag

United States: American Society for Biochemistry and Molecular Biology

Erscheinungsjahr

2003

Quelle

EZB-FREE-00999 freely available EZB journals

Beschreibungen/Notizen

• 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(Δ152–191), 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 ∼100 n m ) 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 frq1 Δ cells. This study defines the segment of Pik1 required for high affinity binding of Frq1.