Abstract
Frequenin, also known as neuronal calcium sensor-1 (NCS-1), is an N-myristoylated Ca2+-binding protein that has been conserved in both sequence and three-dimensional fold during evolution. We demonstrate using both genetic and biochemical approaches that the observed structural conservation between Saccharomyces cerevisiae frequenin (Frq1) and human NCS-1 is also reflected at the functional level. In yeast, the sole essential target of Frq1 is the phosphatidylinositol 4-kinase isoform, Pik1; both FRQ1 and PIK1 are indispensable for cell viability. Expression of human NCS-1 in yeast, but not a close relative (human KChIP2), rescues the inviability of frq1 cells. Furthermore, in vitro, Frq1 and NCS-1 (either N-myristoylated or unmyristoylated) compete for binding to a small 28-residue motif near the N terminus of Pik1. Site-directed mutagenesis indicates that the binding determinant in Pik1 is a hydrophobic alpha-helix and that frequenins bind to one side of this alpha-helix. We propose, therefore, that the function of NCS-1 in mammals may closely resemble that of Frq1 in S. cerevisiae and, hence, that frequenins in general may serve as regulators of certain isoforms of phosphatidylinositol 4-kinase.
Highlights
Frequenin, known as neuronal calcium sensor-1 (NCS-1), is an N-myristoylated Ca2؉-binding protein that has been conserved in both sequence and threedimensional fold during evolution
Inviability of S. cerevisiae frq1⌬ Cells Is Rescued by Human NCS-1—Frequenins seem to be conserved at the level of both primary and tertiary structure from yeast to humans
As one approach to address this question, we examined whether NCS-1 is able to functionally substitute for Frq1 in S. cerevisiae cells
Summary
Plasmid Construction—Plasmids were constructed using standard methods for the manipulation of recombinant DNA [21, 22]. In Vitro Pull-down Assays—NCS-1 (0.7 g), expressed in and purified from bacteria [2], was incubated at 4 °C either alone or with 7 g of the different Pik1-His derivatives in a final volume of 0.5 ml of YLB (50 mM Tris/HCl, pH 7.2, 100 mM NaCl, 1 M CaCl2, 1 mM DTT) on a roller drum for 1 h, and 20 l of a 50% slurry of Ni2ϩ-saturated NTAagarose beads (Qiagen) that had been preblocked in YLB containing 1% (w/v) bovine serum albumin were added. Construction of Yeast Strains—Heterozygous diploid strain YKBH1 (frq1::HIS3/FRQ1) was transformed with 2-m DNA-based (high copy) plasmids carrying either wild-type or mutant versions of human NCS-1 or KChIP2 under the control of the strong inducible GAL1 promoter. Values were determined assuming that all proteins present were in their native and functional state and not corrected for the possible presence of inactive species
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