A mutation in an ATP-binding loop of Saccharomyces cerevisiae actin (S14A) causes a temperature-sensitive phenotype in vivo and in vitro

Xin Chen; Peter A Rubenstein

doi:10.1074/jbc.270.19.11406

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A mutation in an ATP-binding loop of Saccharomyces cerevisiae actin (S14A) causes a temperature-sensitive phenotype in vivo and in vitro

Journal article

Open access

Peer reviewed

A mutation in an ATP-binding loop of Saccharomyces cerevisiae actin (S14A) causes a temperature-sensitive phenotype in vivo and in vitro

Xin Chen and Peter A Rubenstein

The Journal of biological chemistry, Vol.270(19), pp.11406-11414

05/12/1995

DOI: 10.1074/jbc.270.19.11406

PMID: 7744777

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Abstract

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.

Phenotype

Viscosity

Temperature

Saccharomyces cerevisiae - genetics

Serine

Actins - metabolism

Actins - ultrastructure

Molecular Sequence Data

Saccharomyces cerevisiae - metabolism

Time Factors

Adenosine Triphosphate - metabolism

Base Sequence

Actins - chemistry

Myosins - metabolism

Binding Sites

Recombinant Proteins - metabolism

Alanine

Oligodeoxyribonucleotides

Mutagenesis, Site-Directed

Recombinant Proteins - ultrastructure

Recombinant Proteins - chemistry

Genotype

Microscopy, Electron

Point Mutation

Kinetics

Saccharomyces cerevisiae - growth & development

Details

Title: Subtitle: A mutation in an ATP-binding loop of Saccharomyces cerevisiae actin (S14A) causes a temperature-sensitive phenotype in vivo and in vitro
Creators: Xin Chen - Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242-1104, USA
Peter A Rubenstein
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.270(19), pp.11406-11414
DOI: 10.1074/jbc.270.19.11406
PMID: 7744777
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: GM-33689 / NIGMS NIH HHS
Language: English
Date published: 05/12/1995
Academic Unit: Stead Family Department of Pediatrics; Biochemistry and Molecular Biology; Internal Medicine
Record Identifier: 9984025256402771

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