Calcium, nucleotide, and actin affect the interaction of mammalian Myo1c with its light chain calmodulin.
To investigate the interaction of mammalian class I myosin, Myo1c, with its light chain calmodulin, we expressed (with calmodulin) truncation mutants consisting of the Myo1c motor domain followed by 0-4 presumed calmodulin-binding (IQ) domains (Myo1c (0IQ)-Myo1c (4IQ)). The amount of calmodulin associating with the Myo1c heavy chain increased with increasing number of IQ domains from Myo1c (0IQ) to Myo1c (3IQ). No calmodulin beyond that associated with Myo1c (3IQ) was found with Myo1c (4IQ) despite its availability, showing that Myo1c binds three molecules of calmodulin with no evidence of a fourth IQ domain. Unlike Myo1c (0IQ), the basal ATPase activity of Myo1c (1IQ) was >10-fold higher in Ca (2+) vs EGTA +/- exogenous calmodulin, showing that regulation is by Ca (2+) binding to calmodulin on the first IQ domain. The K m and V max of the actin-activated Mg (2+)-ATPase activity were largely independent of the number of IQ domains present and moderately affected by Ca (2+). In binding assays, some calmodulin pelleted with Myo1c heavy chain when actin was present, but a considerable fraction remained in the supernatant, suggesting that calmodulin is displaced most likely from the second IQ domain. The Myo1c heavy chain associated with actin in a nucleotide-dependent fashion. In ATP a smaller proportion of calmodulin pelleted with the heavy chain, suggesting that Myo1c undergoes nucleotide-dependent conformational changes that affect the affinity of calmodulin for the heavy chain. The studies support a model in which Myo1c in the inner ear is regulated by both Ca (2+) and nucleotide, which exert their effects on motor activity through the light-chain-binding region.
Lieto-Trivedi
Alena
A
Boston Biomedical Research Institute, 64 Grove Street, Watertown, Massachusetts 02472, USA.
Coluccio
Lynne M
LM
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D005973:Glutamine; D007532:Isoleucine; D008969:Molecular Sequence Data; D018994:Myosin Light Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024461:Myosin Type I / Q000235:genetics / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011817:Rabbits; D051381:Rats
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; C088340:Myo1c protein, rat; D018994:Myosin Light Chains; D009711:Nucleotides; D007532:Isoleucine; D005973:Glutamine; D024461:Myosin Type I; D002118:Calcium