The two IQ‐motifs and Ca²⁺/calmodulin regulate the rat myosin 1d ATPase activity

Abstract

The light chain binding domain of rat myosin 1d consists of two IQ‐motifs, both of which bind the light chain calmodulin (CaM). To analyze the Myo1d ATPase activity as a function of the IQ‐motifs and Ca2+/CaM binding, we expressed and affinity purified the Myo1d constructs Myo1d‐head, Myo1d‐IQ1, Myo1d‐IQ1.2, Myo1d‐IQ2 and Myo1dΔLV‐IQ2. IQ1 exhibited a high affinity for CaM both in the absence and presence of free Ca2+. IQ2 had a lower affinity for CaM in the absence of Ca2+ than in the presence of Ca2+. The actin‐activated ATPase activity of Myo1d was ∼75% inhibited by Ca2+‐binding to CaM. This inhibition was observed irrespective of whether IQ1, IQ2 or both IQ1 and IQ2 were fused to the head. Based on the measured Ca2+‐dependence, we propose that Ca2+‐binding to the C‐terminal pair of high affinity sites in CaM inhibits the Myo1d actin‐activated ATPase activity. This inhibition was due to a conformational change of the C‐terminal lobe of CaM remaining bound to the IQ‐motif(s). Interestingly, a similar but Ca2+‐independent inhibition of Myo1d actin‐activated ATPase activity was observed when IQ2, fused directly to the Myo1d‐head, was rotated through 200° by the deletion of two amino acids in the lever arm α‐helix N‐terminal to the IQ‐motif.

Two‐dimensional electrophoresis with cationic detergents: A powerful tool for the proteomic analysis of myelin proteins. Part 2: Analytical aspects

“…Myo1D (rat: myr4) is an unconventional myosin protein (Bahler et al, 1994;Kohler et al, 2005) and is linked to membrane trafficking along the recycling pathway (Huber et al, 2000). In developmental analysis of myelin fraction, Myo1D was detected from 3 weeks of age and increased with age in the same way as major myelin proteins (PLP, DM-20, and CNP; Fig.…”

Myosin I: From yeast to human

“…Myo1d has two calmodulin (light chain) binding sites in the neck region and an additional site in the tail domain, with different Ca 2+ dependence on binding [103]. Binding of Ca 2+ to calmodulin can inhibit ATPase activity of Myo1d [104]. Fusion of early endosomes with recycling endosomes in vitro was specifically inhibited by an antibody against the tail region of rat Myo1d, suggesting that Myo1d is involved in the membrane recycling pathway [105].…”

Leveraging the membrane – cytoskeleton interface with myosin-1

“…These results may indicate that CaM binding to the first IQ motif plays a role as a clutch, to control the transmission of conformational changes in the motor domain to the C-terminus of the molecule. It is important to note that myosin-1d appears to be divergent from the other characterized class 1 myosins, as it is not expected to be load-sensitive (it lacks a two-step working stroke)[24] and shows a different response to Ca 2+ (ATPase is inhibited by Ca 2+ ) [82]. …”