Myosin VIIB from Drosophila is a high duty ratio motor.
Myosin VII is an unconventional myosin widely expressed in organisms ranging from amoebae to mammals that has been shown to play vital roles in cell adhesion and phagocytosis. Here we present the first study of the mechanism of action of a myosin VII isoform. We have expressed a truncated single-headed Drosophila myosin VIIB construct in the baculovirus-Sf9 system that bound calmodulin light chains. By using steady-state and transient kinetic methods, we showed that myosin VIIB exhibits a fast release of phosphate and a slower, rate-limiting ADP release from actomyosin. As a result, myosin VIIB will be predominantly strongly bound to actin during steady-state ATP hydrolysis (its duty ratio will be at least 80%). This kinetic pattern is in many respects similar to that of the single-molecule vesicle transporters myosin V and VI. The enzymatic properties of myosin VIIB provide a kinetic basis for processivity upon possible dimerization via the C-terminal domains of the heavy chain. Our experiments also revealed conformational heterogeneity of the actomyosin VIIB complex in the absence of nucleotide.
Yang
Yi
Y
Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1762, USA.
Kovács
Mihály
M
Xu
Qian
Q
Anderson
John B
JB
Sellers
James R
JR
D000199:Actins / Q000737:chemistry; D000205:Actomyosin / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D002448:Cell Adhesion; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D029721:Drosophila Proteins / Q000737:chemistry* / Q000502:physiology*; D004331:Drosophila melanogaster; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007313:Insecta; D007700:Kinetics; D046911:Macromolecular Substances / Q000737:chemistry; D008956:Models, Chemical; D018995:Myosin Heavy Chains / Q000737:chemistry* / Q000502:physiology*; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism; D010587:Phagocytosis; D010710:Phosphates / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D013997:Time Factors
D000199:Actins; D018076:DNA, Complementary; D029721:Drosophila Proteins; D046911:Macromolecular Substances; D010710:Phosphates; D020033:Protein Isoforms; C504724:myosin VIIB, Drosophila; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000205:Actomyosin; D000251:Adenosine Triphosphatases; D018995:Myosin Heavy Chains; D009218:Myosins