Identification and characterization of nuclear calmodulin-binding proteins of Saccharomyces cerevisiae.
Nuclear calmodulin-binding proteins of the yeast Saccharomyces cerevisiae were investigated. The soluble fractions after serial treatments of the isolated nuclei with buffers containing the nonionic detergent NP-40 (F1), 0.5 M KCl (F2) and 2.0 M KCl (F3) in this order, and the residual proteins (F4) were obtained. The calmodulin-binding proteins of the nucleus and nuclear subfractions were identified using the gel overlay method using 125I-calmodulin. Each subnuclear fraction contained a large number of components that bound calmodulin in a Ca(2+)-dependent or -independent manners. The calmodulin-binding proteins were isolated from F1 and F2 subnuclear fractions by affinity chromatography. The affinity-purified proteins bound calmodulin in a Ca(2+)-dependent manner when analyzed using the gel overlay method. The major calmodulin-binding components of F1 were 44, 42, 36, 32 and 29 kDa proteins, and those of F2 were 200, 100, 40, 42, 36, 34 and 32 kDa proteins. The isolated proteins also contained several Coomassie-blue stained proteins that did not bind calmodulin and, therefore, may represent the proteins associated with the calmodulin-binding proteins. Antisera raised against the affinity-purified preparation of F1 and F2 recognized almost all of the calmodulin-binding proteins present in the fraction and several other proteins of the nucleus. The presence of Ca(2+)-dependent protein phosphatase (type 2B) in the nucleus was demonstrated by Western blotting. The enzyme was localized predominantly in F1 and F4.
Hiraga
K
K
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Japan.
Suzuki
K
K
Tsuchiya
E
E
Miyakawa
T
T
D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002467:Cell Nucleus / Q000737:chemistry* / Q000648:ultrastructure; D002846:Chromatography, Affinity; D003902:Detergents; D008954:Models, Biological; D012441:Saccharomyces cerevisiae / Q000737:chemistry*
D002148:Calmodulin-Binding Proteins; D003902:Detergents