Biochemical and motile properties of Myo1b splice isoforms.
Myo1b is a widely expressed myosin-I isoform that concentrates on endosomal and ruffling membranes and is thought to play roles in membrane trafficking and dynamics. Myo1b is alternatively spliced within the regulatory domain of the molecule, yielding isoforms with six (myo1b(a)), five (myo1b(b)), or four (myo1b(c)) non-identical IQ motifs. The calmodulin binding properties of the myo1b IQ motifs have not been investigated, and the mechanical and cell biological consequences of alternative splicing are not known. Therefore, we expressed the alternatively spliced myo1b isoforms truncated after the final IQ motif and included a sequence at their C termini that is a substrate for bacterial biotin ligase. Site-specific biotinylation allows us to specifically attach the myosin to motility surfaces via a biotin-streptavidin linkage. We measured the ATPase and motile properties of the recombinant myo1b splice isoforms, and we correlated these properties with calmodulin binding. We confirmed that calcium-dependent changes in the ATPase activity are due to calcium binding to the calmodulin closest to the motor. We found that calmodulin binds tightly to some of the IQ motifs (Kd < 0.2 microM) and very weakly to the others (Kd > 5 microM), suggesting that a subset of the IQ motifs are not calmodulin bound under physiological conditions. Finally, we found the in vitro motility rate to be dependent on the myo1b isoform and the calmodulin concentration and that the myo1b regulatory domain acts as a rigid lever arm upon calmodulin binding to the high affinity and low affinity IQ motifs.
Lin
Tianming
T
Department of Physiology and The Pennsylvania Muscle Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Tang
Nanyun
N
Ostap
E Michael
EM
D000199:Actins / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D002147:Calmodulin / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008969:Molecular Sequence Data; D024461:Myosin Type I / Q000737:chemistry* / Q000378:metabolism; D009218:Myosins / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013997:Time Factors
D000199:Actins; D002147:Calmodulin; C104297:Myo1b protein, rat; D020033:Protein Isoforms; D000244:Adenosine Diphosphate; D001710:Biotin; D000251:Adenosine Triphosphatases; D024461:Myosin Type I; D009218:Myosins