CA2-POMBE AS REVEALED BY MUTAGENESIS AND NMR-SPECTROSCOPY( BINDING TOCALMODULIN AND ITS ROLE IN SCHIZOSACCHAROMYCES)

As a first step toward identifying the important structural elements o f calmodulin from Schizosaccharomyces pombe, we examined the ability o f heterologous calmodulins and Ca2+-binding site mutant S. pombe calmo dulins to replace the essential cam1(+) gene, A cDNA encoding vertebra te calmodulin allows growth of S. pombe. However, calmodulin from Sacc haromyces cerevisiae does not support growth even though the protein i s produced at high levels. With one exception, all mutant S. pombe cal modulins with one or more intact Ca2+ binding sites allow growth at 21 degrees C. A mutant containing only an intact Ca2+-binding site 3 fai ls to support growth, as does S. pombe calmodulin with all four Ca2+ b inding sites mutated. Several of the mutant proteins confer a temperat ure-sensitive phenotype. Analysis of the degree of temperature sensiti vity allows the Ca2+ binding sites to be ranked by their ability to su pport fission yeast proliferation. Site 2 is more important than site 1, which is more important than site 4, which is more important than s ite 3. A visual colony color screen based on the fission yeast ade1(+) gene was developed to perform these genetic analyses. To compare the Ca2+ binding properties of individual sites to their functional import ance for viability, Ca2+ binding to calmodulin from S. pombe was studi ed by H-1 NMR spectroscopy. NMR analysis indicates a Ca2+-binding prof ile that differs from those previously determined for vertebrate and S . cerevisiae calmodulins. Ca2+-binding site 3 has the highest relative affinity for Ca2+, while the affinities of sites 1, 2, and 4 are indi stinguishable. A combination of an in vivo functional assay and an in vitro physical assay reveals that the relative affinity of a site for Ca2+ does not predict its functional importance.