Structure and Function Analysis of the Calcium-Related Gene spray in Neurospora crassa

The spray gene was cloned, and wildtype and mutant alleles were sequenced. Spray+ has a 3452-bp open reading frame plus seven introns. The spray mutant had a T → G transversion close to the carboxyl end, creating a stop codon (TGA). The sequence shows no match to genes of known function, but the carboxyl end shows seven transmembrane domains and matches putative membrane proteins of yeast. The most abundant transcript detected was 4.4 kb in size. Repeat-induced point mutagenesis produced the mutant spray phenotype. Electrophysiological analysis showed that ion fluxes in the spray plasma membrane are normal; furthermore, whereas the spray mutant was known to have no organelle-based calcium fluorescence, the cytosol shows a tip-high calcium gradient. The spray mutant is sensitive to calcineurin inhibitors. The results suggest that the SPRAY protein is located in an organellar membrane, regulating the distribution of Ca2+ via calcineurin.