Charge-reversion mutagenesis of Dictyostelium actin to map the surface recognized by myosin during ATP-driven sliding motion.
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.
Johara
M
M
Department of Pure and Applied Sciences, College of Arts and Sciences, University of Tokyo, Japan.
Toyoshima
Y Y
YY
Ishijima
A
A
Kojima
H
H
Yanagida
T
T
Sutoh
K
K
D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism*; D006639:Histidine; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015879:Myosin Subfragments / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000199:Actins; D015879:Myosin Subfragments; D011994:Recombinant Proteins; D006639:Histidine; D000255:Adenosine Triphosphate; D009218:Myosins