Functional studies of yeast actin mutants corresponding to human cardiomyopathy mutations.
The molecular mechanisms by which different mutations in actin lead to distinct cardiomyopathies are unknown. Here, actin mutants corresponding to alpha-cardiac actin mutations causing hypertrophic cardiomyopathy [(HCM) P164A and A331P] and dilated cardiomyopathy [(DCM) R312H and E361G] were expressed in yeast and purified for in vitro functional studies. While P164A appeared unaltered compared to wild-type (WT) actin, A331P function was impaired. A331P showed reduced stability in circular dichroism melting experiments; its monomer unfolding transition was 10 degrees C lower compared to WT actin. Additionally, in vitro filament formation was hampered, and yeast cell cultures were temperature sensitive, implying perturbations in actin-actin interactions. Filament instability of the A331P mutant actin could lead to actomyosin dysfunction observed in HCM. Yeast strains harboring the R312H mutation did not grow well in culture, suggesting that cell viability is compromised. The E361G substitution is located at an alpha-actinin binding region where the actin filament is anchored. The mutant actin, though unaltered in the in vitro motility and standard actomyosin functions, had a threefold reduction in alpha-actinin binding. This could result in impairment of force-transduction in muscle fibers, and a DCM phenotype.
Wong
W W
WW
Department of Chemistry and Biochemistry, Molecular Biology Institute, University of California, Los Angeles 90095, USA.
Doyle
T C
TC
Cheung
P
P
Olson
T M
TM
Reisler
E
E
D000185:Actinin / Q000378:metabolism; D000199:Actins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D009202:Cardiomyopathies / Q000235:genetics*; D006801:Humans; D016296:Mutagenesis / Q000502:physiology; D020125:Mutation, Missense / Q000502:physiology; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism; D010641:Phenotype; D011108:Polymers / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D000199:Actins; D015879:Myosin Subfragments; D011108:Polymers; D000185:Actinin; D000251:Adenosine Triphosphatases