Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone.
By using an assay specific for detection of calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase, this enzyme was purified approximately 5,000-fold from extracts of the yeast Saccharomyces cerevisiae. Cna1p and Cna2p, the products of two yeast genes encoding the catalytic (A) subunits of calcineurin, were major constituents of the purified fraction. A third prominent component of apparent molecular mass 16 kDa displayed several properties, including ability to bind 45Ca2+, that are characteristic of the regulatory (B) subunit of mammalian calcineurin and was recognized by an antiserum raised against bovine calcineurin. These antibodies were used to isolate the structural gene (CNB1) encoding this protein from a yeast expression library in the vector lambda gt11. The nucleotide sequence of CNB1 predicted a polypeptide similar in length and highly related in amino acid sequence (56% identity) to the mammalian calcineurin B subunit. Like its counterpart in higher cells, yeast Cnb1p was myristoylated at its N terminus. Mutants lacking Cnb1p, or all three calcineurin subunits (Cna1p, Cna2p, and Cnb1p), were viable. Extracts of cnb1 delta mutants contained no detectable calcineurin activity, even though Cna1p and Cna2p were present at normal levels, suggesting that the B subunit is required for full enzymatic activity in vitro. As was observed previously for MATa cna1 cna2 double mutants, MATa cnb1 mutants were defective in their ability to recover from alpha-factor-induced growth arrest. Thus, the B subunit also is required for the function of calcineurin in promoting adaptation of haploid yeast cells to pheromone in vivo.
Cyert
M S
MS
Department of Molecular and Cell Biology, University of California 94720.
Thorner
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D005814:Genes, Viral; D006801:Humans; D007106:Immune Sera; D007700:Kinetics; D046911:Macromolecular Substances; D000072235:Mating Factor; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology / Q000494:pharmacology*; D010675:Pheromones / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D015678:Viral Structural Proteins / Q000235:genetics*
D002148:Calmodulin-Binding Proteins; D004271:DNA, Fungal; D007106:Immune Sera; D046911:Macromolecular Substances; D010455:Peptides; D010675:Pheromones; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D000072235:Mating Factor; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases