Recombinant motor domain constructs of Chara corallina myosin display fast motility and high ATPase activity.
The mechanism and structural features that are responsible for the fast motility of Chara corallina myosin (CCM) have not been elucidated, so far. The low yields of native CCM that can be purified to homogeneity were the major reason for this. Here, we describe the expression of recombinant CCM motor domains, which support the fast movement of actin filaments in an in vitro motility assay. A CCM motor domain without light chain binding site moved actin filaments at a velocity of 8.8 microm/s at 30 degrees C and a CCM motor domain with an artificial lever arm consisting of two alpha-actinin repeats moved actin filaments at 16.2 microm/s. Both constructs displayed high actin-activated ATPase activities ( approximately 500 Pi/s/head), which is indicative of a very fast hydrolysis step. Our results provide an excellent system to dissect the specific structural and functional features that distinguish the myosin responsible for fast cytoplasmic streaming.
Ito
Kohji
K
Department of Biology, Chiba University, Inage-ku, 263-8522, Chiba, Japan. k-ito@faculty.chiba-u.jp
Kashiyama
Taku
T
Shimada
Kiyo
K
Yamaguchi
Akira
A
Awata
Jun ya
Jy
Hachikubo
You
Y
Manstein
Dietmar J
DJ
Yamamoto
Keiichi
K
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000502:physiology; D044784:Chara / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D004789:Enzyme Activation; D007700:Kinetics; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D009038:Motion; D009068:Movement / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000737:chemistry* / Q000502:physiology; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D020409:Molecular Motor Proteins; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; D009218:Myosins