Abstract
All class 2 myosins contain an N-terminal extension of approximately 80 residues that includes an Src homology 3 (SH3)-like subdomain. To explore the functional importance of this region, which is also present in most other myosin classes, we generated truncated constructs of Dictyostelium discoideum myosin-2. Truncation at position 80 resulted in the complete loss of myosin-2 function in vivo. Actin affinity was more than 80-fold, and the rate of ADP release approximately 40-fold decreased in this mutant. In contrast, a myosin construct that lacks only the SH3-like subdomain, corresponding to residues 33-79, displayed much smaller functional defects. In complementation experiments with myosin-2 null cells, this construct rescued myosin-2-dependent processes such as cytokinesis, fruiting body formation, and sporogenesis. An 8-fold reduction in motile activity and changes of similar extent in the affinity for ADP and filamentous actin indicate the importance of the SH3-like subdomain for correct communication between the functional regions within the myosin motor domain and suggest that local perturbations in this region can play a role in modulating myosin-2 motor activity.
Highlights
Members of the myosin superfamily of actin-based motors act in a variety of cellular functions such as muscle contraction, cell and organelle movement, membrane trafficking, and signal transduction
Myosin motor domains show a high degree of sequence conservation, the individual myosin classes are clearly defined by differences in the head structure [1]
In the presence of saturating concentrations of ATP and in the absence of F-actin, the basal ATPase activity of ⌬N1-M761 was 0.014 sϪ1 and ϳ2-fold slower than the value determined for M761
Summary
The experimental conditions were 25 mM HEPES, pH 7.4, 25 mM KCl, 4 mM MgCl2 at 25 °C. Protein Expression and Purification—Dictyostelium AX3ORFϩ cells producing the motor domain constructs and ⌬N2myosin myosins were grown as described previously [15]. The results shown here were obtained with ⌬N1-M761 from five different preparations, and all measurements were performed within 24 h after elution of the protein from the Ni2ϩ-NTA column. Following purification by Ni2ϩ-NTA affinity chromatography [15], yields of 0.5, 1.5, and 4.0 mg of purified protein were obtained for ⌬N1-M761, ⌬N2-M761, and M761 per gram of cells. The reduced yields that were obtained with ⌬N1-M761 and ⌬N2-M761 result from increased losses during the initial steps of the purification including the wash step prior to ATP extraction of the recombinant motor domains. The concentrations of the recombinant Dictyostelium myosin motor domains were determined using the Bradford assay. The conditions were: 25 mM imidazole, 25 mM KCl, 4 mM MgCl2, 0.5 mM DTT, 0.5 mM ATP, 0.2
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