A mutation in an ATP-binding loop of Saccharomyces cerevisiae actin (S14A) causes a temperature-sensitive phenotype in vivo and in vitro.
The Ser14 hydroxyl group of actin is one of six groups that potentially form hydrogen bonds with the gamma-phosphate of the ATP bound in the cleft separating the two domains of the protein. To understand the importance of this group in actin function, we mutated Ser14 of Saccharomyces cerevisiae actin and studied the effects of these mutations in vivo and in vitro. Substitution of Cys of Gly resulted in cell death. Substitution of Thr for Ser resulted in an actin with wild-type properties in vivo and in vitro. Cells carrying the Ser14-->Ala (S14A) mutation were viable but displayed a temperature sensitive lethality at 37 degrees C preceded by delocalization of actin patches, the appearance of bar-like structures, and finally the disappearance of identifiable actin structures. The mutation caused no effect on the critical concentration of polymerization but resulted in an actin with an increased rate of polymerization, an altered protease susceptibility, and a decreased filament ATPase activity. At 37 degrees C, Mg-, but not Ca-S14A-actin irreversibly lost the ability to polymerize. These results demonstrate the importance of the ATP-Ser14 hydroxyl hydrogen bond in regulating actin function in vivo and in vitro and the magnification of the effects of the mutation when Mg2+ is substituted for Ca2+ in the protein.
Chen
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Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242-1104, USA.
Rubenstein
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PA
D000199:Actins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000409:Alanine; D001483:Base Sequence; D001665:Binding Sites; D005838:Genotype; D007700:Kinetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D010641:Phenotype; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D012694:Serine; D013696:Temperature; D013997:Time Factors; D014783:Viscosity
D000199:Actins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012694:Serine; D000255:Adenosine Triphosphate; D009218:Myosins; D000409:Alanine