Structure and function analysis of the calcium-related gene spray in Neurospora crassa

Bok, J.-W., Sone, T., Silverman-Gavrila, L. B., Lew, R. R., Bowring, F. J., Catcheside, D. E. A., and Griffiths, A. J. F. 2001. Structure and function analysis of the calcium-related gene spray in Neurospora crassa, Fungal Ge netics and Biology 32, 145-158. The spray gene was cloned, and wildtype and mutant alleles were sequenced. Spray(+) has a 3452-bp open reading frame p lus 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 dom ains and matches putative membrane proteins of yeast. The most abundant tra nscript detected was 4.4 kb in size. Repeat-induced point mutagenesis produ ced the mutant spray phenotype. Electrophysiological analysis showed that i on fluxes in the spray plasma membrane are normal; furthermore, whereas the spray mutant was known to have no organelle-based calcium fluorescence, th e cytosol shows a tip-high calcium gradient. The spray mutant is sensitive to calcineurin inhibitors. The results suggest that the SPRAY protein is lo cated in an organellar membrane, regulating the distribution of Ca2+ via ca lcineurin. (C) 2001 Academic Press.