Abstract
Abstract: :
Purpose: This study examines the actin-binding properties of myosin IIIA. This protein localizes specifically to the core actin filament bundles of photoreceptor calycal processes. Myo3A has an N-terminal kinase domain and a highly conserved motor domain, followed by neck and tail domains. The neck and tail contain nine putative calmodulin binding sites followed by two conserved domains: a ~50 amino acid tail homology domain I (3THDI), seen in all class III myosins to date, and a 22 amino acid tail homology domain II (3THDII) specific to myosin IIIAs. Methods: We began by examining the ability of native Myo3A and a 3A-tail fusion protein to co-sediment with the detergent extracted photoreceptor cytoskeleton. We next used heterologous expression studies in Hela cells and actin-spindown assays to examine the actin binding properties of the Myo3A. Results: Native Myo3A co-sediments with the photoreceptor cytoskeleton, and can be released by the addition of ATP. In Hela cells, expressed Myo3A localizes to actin filaments and concentrates at the tips of filopodia. A point mutation critical to motor activity abolishes this filapodaial localization, as does the deletion of the C-terminal tail-tip (3THDII), the conserved domain at the extreme C-terminus. A GST fusion protein containing the C-terminal 202 amino acids of the myoIIIA tail co-sedimented with the cytoskeletal fraction of rod photoreceptors. In Hela cell transfections, GFP-tail and tail-tip (3THDII) fusion proteins localize to actin filaments, whereas the GFP-tail minus 3THDII was cytosolic. The 202-amino acid tail fusion protein (containing 3THDI and 3THDII) and a 22-amino acid tail-tip fusion protein (containing only 3THDII) both co-sedimented with F-actin, but a tail fusion protein missing the 3THDII remained in the supernatant. Within 3THDII, we identified an actin-binding motif DXRXXL, first identified as a novel actin-binding motif in myosin light chain kinase (MLCK, Smith et al., 1999). An Arg to Ala point mutation in this motif eliminated the ability of the 3THDII to localize to actin filaments in Hela cells and similarly the GFP-3THDII(R to A) did not bind to actin in sedimentation assays. Conclusion: Myo3A associates with the cytoskeleton in an ATP-dependent manner in vitro and localizes to actin filaments in vivo. Actin association is dependent on a functional motor domain and an intact actin-binding motif at the tip of the tail domain. (Supported by NIH grant EY03575)
Keywords: cytoskeleton • photoreceptors