Myosin XI-I is Mechanically and Enzymatically Unique Among Class-XI Myosins in Arabidopsis.
Arabidopsis possesses 13 genes encoding class-XI myosins. Among these, myosin XI-I is phylogenetically distant. To examine the molecular properties of Arabidopsis thaliana myosin XI-I (At myosin XI-I), we performed in vitro mechanical and enzymatic analyses using recombinant constructs of At myosin XI-I. Unlike other biochemically studied class-XI myosins, At myosin XI-I showed extremely low actin-activated ATPase activity (Vmax = 3.7 Pi s(-1) head(-1)). The actin-sliding velocity of At myosin XI-I was 0.25 µm s(-1), >10 times lower than those of other class-XI myosins. The ADP dissociation rate from acto-At myosin XI-I was 17 s(-1), accounting for the low actin-sliding velocity. In contrast, the apparent affinity for actin in the presence of ATP, estimated from Kapp (0.61 µM) of actin-activated ATPase, was extremely high. The equilibrium dissociation constant for actin was very low in both the presence and absence of ATP, indicating a high affinity for actin. To examine At myosin XI-I motility in vivo, green fluorescent protein-fused full-length At myosin XI-I was expressed in cultured Arabidopsis cells. At myosin XI-I localized not only on the nuclear envelope but also on small dots moving slowly (0.23 µm s(-1)) along actin filaments. Our results show that the properties of At myosin XI-I differ from those of other Arabidopsis class-XI myosins. The data suggest that At myosin XI-I does not function as a driving force for cytoplasmic streaming but regulates the organelle velocity, supports processive organelle movement or acts as a tension generator.
Department of Biology, Graduate School of Science, Chiba University, Inage-ku, Chiba, 263-8522 Japan These authors contributed equally to this work. k-ito@faculty.chiba-u.jp motominaga@waseda.jp.
Takeshi
T
Haraguchi
Faculty of Education and Integrated Arts and Sciences, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo, 162-8480 Japan These authors contributed equally to this work. k-ito@faculty.chiba-u.jp motominaga@waseda.jp.
Motoki
M
Tominaga
Live Cell Super-Resolution Imaging Research Team, Extreme Photonics Research Group, RIKEN Center for Advanced Photonics, Wako, Saitama, 351-0198 Japan Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan.
Akihiko
A
Nakano
Department of Biology, Graduate School of Science, Chiba University, Inage-ku, Chiba, 263-8522 Japan.
Keiichi
K
Yamamoto
Department of Biology, Graduate School of Science, Chiba University, Inage-ku, Chiba, 263-8522 Japan k-ito@faculty.chiba-u.jp motominaga@waseda.jp.
Kohji
K
Ito
D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D017360:Arabidopsis / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003595:Cytoplasmic Streaming; D017930:Genes, Reporter; D020409:Molecular Motor Proteins / Q000235:genetics / Q000378:metabolism*; D015388:Organelles / Q000378:metabolism; D021381:Protein Transport
D000199:Actins; D029681:Arabidopsis Proteins; D020409:Molecular Motor Proteins; C000588851:myosin XI-i protein, Arabidopsis; D000255:Adenosine Triphosphate
ATPase; Actin; Arabidopsis; Kinetics; Molecular motor; Myosin