FIGURE 2.

Overproduction of M. musculus myosin-18A. A, purification of myosin-18A-S1 and -HMM constructs. Lanes are marked as either molecular weight markers (M) or purified fractions of each motor construct. The dashed line indicates that the image is a composite of two separate gel scans. Note that previous studies of nonmuscle myosin 2 and smooth muscle isoforms have revealed that the ELC binds Coomassie dye poorly and always appears lighter in intensity than does the RLC (16, 48). B, confirmation of phosphorylation of the bound RLC using a gel shift Phos-tag assay. Each of the myosin fragments described at the top was either untreated (−) or treated (+) with MLCK to phosphorylate the RLC as described under “Experimental Procedures.” C, alignments of the neck region of representative myosin-18 and myosin-2 heavy chain sequences. Genome sources are M. musculus (Mm), Homo sapiens (Hs), Xenopus tropicalis (Xt), and D. melanogaster (Dm). Myosin-2 H. sapiens genes are MYH9 (nm myo-2A), MYH7 (card myo-2), and MYH4 (sk myo-2). Blue highlighting shows (from left to right) the consensus first IQ motif; the second, more divergent motif that forms the binding site for RLC in the aligned motors; the bend region; and the Pro that marks the end of the neck. The heptad register for the beginning of the coiled-coil motif is shown at the bottom.