Dictyostelium myosin 25-50K loop substitutions specifically affect ADP release rates.
While most of the sequence of myosin's motor domain is highly conserved among various organisms and tissue types, the junctions between the 25 and 50 kDa domains and the 50 and 20 kDa domains are strikingly divergent. The 50-20K loop is positioned to interact with actin, while the 25-50K loop is situated nearer the ATP binding site [Rayment, I., et al. (1993) Science 261, 50-58]. Chimeric studies of the 50-20K loop [Uyeda, T. Q.-P., et al. (1994) Nature 368, 567-569; Rovner, A. S., et al. (1995) J. Biol. Chem. 270 (51), 30260-30263] have shown that this loop affects actin activation of ATPase activity. Given the function of myosin as a molecular motor, it was proposed that the 25-50K loop might specifically alter ADP release [Spudich, J. A. (1994) Nature 374, 515-518]. Here we study the role of this loop by engineering chimeras containing the Dictyostelium myosin heavy chain with loops from two enzymatically diverse myosins, rabbit skeletal and Acanthamoeba. The chimeric myosins complement the myosin null phenotype in vivo, bind nucleotide normally, interact normally with actin, and display wild-type levels of actin-activated ATPase activity. However, the rate of ADP release from the myosins, normally the slowest step involved in motility, was changed in a manner that reflects the activity of the donor myosin. In summary, studies of Dictyostelium myosin heavy chain chimeras have shown that the 50-20K sequence specifically affects the actin-activated ATPase activity [Uyeda, T. Q.-P., et al. (1994)] while the 25-50K sequence helps determine the rate of ADP release.
Murphy
C T
CT
Departments of Biochemistry and Developmental Biology, Stanford University School of Medicine, California 94305, USA.
Spudich
J A
JA
D000048:Acanthamoeba / Q000235:genetics; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000345:Affinity Labels; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D002465:Cell Movement / Q000235:genetics / Q000502:physiology; D002645:Chickens; D004023:Dictyostelium / Q000737:chemistry* / Q000235:genetics; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D009218:Myosins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011485:Protein Binding / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000502:physiology; D062367:ortho-Aminobenzoates / Q000378:metabolism
D000345:Affinity Labels; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D062367:ortho-Aminobenzoates; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate; D009218:Myosins