Charge-reversion mutagenesis of Dictyostelium actin to map the surface recognized by myosin during ATP-driven sliding motion

Masaaki Johara; Yoko Yano Toyoshima; Akihiko Ishijima; Hiroaki Kojima; Toshio Yanagida; Kazuo Sutoh

doi:10.1073/pnas.90.6.2127

Charge-reversion mutagenesis of Dictyostelium actin to map the surface recognized by myosin during ATP-driven sliding motion

Masaaki Johara, Yoko Yano Toyoshima, Akihiko Ishijima, Hiroaki Kojima, Toshio Yanagida, Kazuo Sutoh

Research output: Contribution to journal › Article › peer-review

70 Citations (Scopus)

Abstract

Amino acid residues D24/D25, E99/E100, E360/E361, and D363/E364 in subdomain 1 of Dictyostelium actin were replaced with histidine residues by site-directed mutagenesis. Mutant actins were expressed in Dictyostelium cells and purified to homogeneity. The sliding movement of mutant actin filaments on heavy meromyosin attached to a glass surface was measured to assess the effect of the mutation on the motility of actin. For two C-terminal mutants, force generated by a single actin filament and myosin was also measured. These measurements indicated that both D24/D25 and E99/E100 are involved in ATP-driven sliding, whereas E360/E361/ D363/E364 are not essential for ATP-driven sliding and force generation.

Original language	English
Pages (from-to)	2127-2131
Number of pages	5
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	90
Issue number	6
DOIs	https://doi.org/10.1073/pnas.90.6.2127
Publication status	Published - 1993 Mar 15
Externally published	Yes

Keywords

Actin-activated myosin ATPase
Force generation
In vitro motility assay
Site-directed mutagenesis

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Charge-reversion mutagenesis of Dictyostelium actin to map the surface recognized by myosin during ATP-driven sliding motion. / Johara, Masaaki; Toyoshima, Yoko Yano; Ishijima, Akihiko et al.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 90, No. 6, 15.03.1993, p. 2127-2131.

Research output: Contribution to journal › Article › peer-review

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abstract = "Amino acid residues D24/D25, E99/E100, E360/E361, and D363/E364 in subdomain 1 of Dictyostelium actin were replaced with histidine residues by site-directed mutagenesis. Mutant actins were expressed in Dictyostelium cells and purified to homogeneity. The sliding movement of mutant actin filaments on heavy meromyosin attached to a glass surface was measured to assess the effect of the mutation on the motility of actin. For two C-terminal mutants, force generated by a single actin filament and myosin was also measured. These measurements indicated that both D24/D25 and E99/E100 are involved in ATP-driven sliding, whereas E360/E361/ D363/E364 are not essential for ATP-driven sliding and force generation.",

keywords = "Actin-activated myosin ATPase, Force generation, In vitro motility assay, Site-directed mutagenesis",

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AU - Johara, Masaaki

AU - Toyoshima, Yoko Yano

AU - Ishijima, Akihiko

AU - Kojima, Hiroaki

AU - Yanagida, Toshio

AU - Sutoh, Kazuo

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N2 - Amino acid residues D24/D25, E99/E100, E360/E361, and D363/E364 in subdomain 1 of Dictyostelium actin were replaced with histidine residues by site-directed mutagenesis. Mutant actins were expressed in Dictyostelium cells and purified to homogeneity. The sliding movement of mutant actin filaments on heavy meromyosin attached to a glass surface was measured to assess the effect of the mutation on the motility of actin. For two C-terminal mutants, force generated by a single actin filament and myosin was also measured. These measurements indicated that both D24/D25 and E99/E100 are involved in ATP-driven sliding, whereas E360/E361/ D363/E364 are not essential for ATP-driven sliding and force generation.

AB - Amino acid residues D24/D25, E99/E100, E360/E361, and D363/E364 in subdomain 1 of Dictyostelium actin were replaced with histidine residues by site-directed mutagenesis. Mutant actins were expressed in Dictyostelium cells and purified to homogeneity. The sliding movement of mutant actin filaments on heavy meromyosin attached to a glass surface was measured to assess the effect of the mutation on the motility of actin. For two C-terminal mutants, force generated by a single actin filament and myosin was also measured. These measurements indicated that both D24/D25 and E99/E100 are involved in ATP-driven sliding, whereas E360/E361/ D363/E364 are not essential for ATP-driven sliding and force generation.

KW - Actin-activated myosin ATPase

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KW - In vitro motility assay

KW - Site-directed mutagenesis

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Charge-reversion mutagenesis of Dictyostelium actin to map the surface recognized by myosin during ATP-driven sliding motion

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Keywords

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