Fission yeast homolog of neuronal calcium sensor-1 (Ncs1p) regulates sporulation and confers calcium tolerance.
The neuronal calcium sensor (NCS) proteins (e.g. recoverin, neurocalcins, and frequenin) are expressed at highest levels in excitable cells, and some of them regulate desensitization of G protein-coupled receptors. Here we present NMR analysis and genetic functional studies of an NCS homolog in fission yeast (Ncs1p). Ncs1p binds three Ca2+ ions at saturation with an apparent affinity of 2 microm and Hill coefficient of 1.9. Analysis of NMR and fluorescence spectra of Ncs1p revealed significant Ca2+-induced protein conformational changes indicative of a Ca2+-myristoyl switch. The amino-terminal myristoyl group is sequestered inside a hydrophobic cavity of the Ca2+-free protein and becomes solvent-exposed in the Ca2+-bound protein. Subcellular fractionation experiments showed that myristoylation and Ca2+ binding by Ncs1p are essential for its translocation from cytoplasm to membranes. The ncs1 deletion mutant (ncs1Delta) showed two distinct phenotypes: nutrition-insensitive sexual development and a growth defect at high levels of extracellular Ca2+ (0.1 m CaCl(2)). Analysis of Ncs1p mutants lacking myristoylation (Ncs1p(G2A)) or deficient in Ca2+ binding (Ncs1p(E84Q/E120Q/E168Q)) revealed that Ca2+ binding was essential for both phenotypes, while myristoylation was less critical. Exogenous cAMP, a key regulator for sexual development, suppressed conjugation and sporulation of ncs1Delta, suggesting involvement of Ncs1p in the adenylate cyclase pathway turned on by the glucose-sensing G protein-coupled receptor Git3p. Starvation-independent sexual development of ncs1Delta was also complemented by retinal recoverin, which controls Ca2+-regulated desensitization of rhodopsin. In contrast, the Ca2+-intolerance of ncs1Delta was not affected by cAMP or recoverin, suggesting that the two ncs1Delta phenotypes are mechanistically independent. We propose that Schizosaccharomyces pombe Ncs1p negatively regulates sporulation perhaps by controlling Ca2+-dependent desensitization of Git3p.
Hamasaki-Katagiri
Nobuko
N
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD 20850, USA.
Molchanova
Tatiana
T
Takeda
Kazuyo
K
Ames
James B
JB
D000483:Alleles; D000595:Amino Acid Sequence; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D002462:Cell Membrane; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005136:Eye Proteins; D017353:Gene Deletion; D005947:Glucose / Q000378:metabolism; D049452:Green Fluorescent Proteins; D051597:Hippocalcin; D008074:Lipoproteins; D008164:Luminescent Proteins / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D019814:Myristic Acid / Q000378:metabolism; D009419:Nerve Tissue Proteins; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D051603:Recoverin; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism / Q000502:physiology*; D012568:Schizosaccharomyces / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013347:Subcellular Fractions / Q000378:metabolism
D002135:Calcium-Binding Proteins; D005136:Eye Proteins; C400903:FRQ1 protein, S cerevisiae; D008074:Lipoproteins; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D012997:Solvents; C416142:git3 protein, S pombe; D019814:Myristic Acid; D051603:Recoverin; D049452:Green Fluorescent Proteins; D051597:Hippocalcin; D000242:Cyclic AMP; D005947:Glucose; D002118:Calcium