Subcellular localization of myosin I in A10 smooth muscle cell.
We isolated and identified a 110-kDa myosin I from porcine aorta media smooth muscle [Y. Hasegawa et al. (1996) J. Biochem. 120, 971-976]. Partial peptide sequences of the 110-kDa myosin I fragments were homologous to amino acid sequences deduced from myosin Ibeta of bovine brain and adrenal gland. We investigated biochemically the distribution of the 110-kDa myosin I by cell fractionation methods. About 10% of myosin I present in whole cells could be extracted by treatment with 0.02% saponin, which does not liberate organelles, indicating that at least 10% of myosin I present in A10 cells is associated with neither organelles nor cytoskeleton. After treatment of A10 cells with 0.5% Triton X-100, the insoluble cytoskeleton contained 45% of myosin I present in whole cells. Treatment with MgATP extracted most of myosin I from the cytoskeleton, indicating that the distribution of myosin I is maintained by binding of the myosin I head to an actin filament. On the other hand, when the cell homogenate was fractionated on sucrose density step gradients, about 80% of myosin I was associated with membranes of various densities. An attempt to dissociate the myosin I from the membranes in the presence of MgATP was not successful. These results show that about 80% of total myosin I is associated directly with membranes, not through F-actin. The amounts of myosin I associated with membranes or cytoskeleton provide evidence that myosin I in A10 cells is associated in part with only membrane and in part with both cytoskeleton and membranes. Our results lead to conclusion that myosin I exist in several states: membrane-and-cytoskeleton-associated, membrane-associated, and membrane-and-cytoskeleton-free. These states may be in dynamic equilibrium, allowing myosin I to respond to the cellular requirements.
Department of Applied Chemistry, Muroran Institute of Technology, Muroran, Hokkaido, 050, Japan.
Y
Y
Hasegawa
S
S
Tsuwaki
N
N
Yamada
T
T
Araki
S
S
Kimura
J
J
Sugawara
K
K
Yamamoto
Y
Y
Okamoto
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D002460:Cell Line; D003599:Cytoskeleton / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D019084:Fluorescent Antibody Technique, Indirect; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000737:chemistry; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000737:chemistry* / Q000166:cytology; D009218:Myosins / Q000302:isolation & purification*; D051381:Rats; D017421:Sequence Analysis; D013347:Subcellular Fractions / Q000737:chemistry
D009218:Myosins