Myristoylation of calcineurin B is not required for function or interaction with immunophilin-immunosuppressant complexes in the yeast Saccharomyces cerevisiae.
Calcineurin is a heterodimeric Ca2+/calmodulin-dependent protein phosphatase that regulates signal transduction and is the target of immunophilin-immunosuppressive drug complexes in T-lymphocytes and in yeast. Calcineurin is composed of a catalytic A subunit and a regulatory B subunit that is myristoylated at its amino terminus. We employed genetic and biochemical approaches to investigate the functional roles of myristoylation of calcineurin B (CNB1) in Saccharomyces cerevisiae. A calcineurin B mutant in which glycine 2 was substituted by alanine (CNB1-G2A) did not incorporate [3H]myristate when expressed in yeast. Both wild-type calcineurin B and the CNB1-G2A mutant protein are partially associated with membranes and cytoskeletal structures; hence, myristoylation is not required for these associations. In several independent genetic assays of calcineurin functions (recovery from alpha-factor arrest, survival during cation stress, and viability of a calcineurin-dependent strain), the nonmyristoylated CNB1-G2A mutant protein exhibited full biological activity. In vitro, both wild-type and CNB1-G2A mutant proteins formed complexes with both cyclophilin A-cyclosporin A (CsA) and FKBP12-FK506 that contained calcineurin A. Interestingly, expression of the nonmyristoylated CNB1-G2A mutant protein rendered yeast cells partially resistant to the immunosuppressant CsA, but not to FK506. This study demonstrates that calcineurin B myristoylation is not required for function, but may participate in inhibition by the cyclophilin A-CsA complex.
Zhu
D
D
Department of Genetics, Duke University Medical Center, Durham, North Carolina 27710, USA.
Cardenas
M E
ME
Heitman
J
J
D000591:Amino Acid Isomerases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D019703:Calcineurin; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D016572:Cyclosporine / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D007166:Immunosuppressive Agents / Q000378:metabolism*; D008969:Molecular Sequence Data; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013329:Structure-Activity Relationship; D016559:Tacrolimus / Q000378:metabolism; D022021:Tacrolimus Binding Proteins
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D007166:Immunosuppressive Agents; D009227:Myristic Acids; D019814:Myristic Acid; D016572:Cyclosporine; D019703:Calcineurin; D000591:Amino Acid Isomerases; D022021:Tacrolimus Binding Proteins; D019696:Peptidylprolyl Isomerase; D002118:Calcium; D016559:Tacrolimus