Human deafness mutation E385D disrupts the mechanochemical coupling and subcellular targeting of myosin-1a.
Missense mutations in the membrane-binding actin-based motor protein, myosin-1a (Myo1a), have recently been linked to sensorineural deafness in humans. One of these mutations, E385D, impacts a residue in the switch II region of the motor domain that is present in virtually all members of the myosin superfamily. We sought to examine the impact of E385D on the function of Myo1a, both in terms of mechanochemical activity and ability to target to actin-rich microvilli in polarized epithelial cells. While E385D-Myo1a demonstrated actin-activated ATPase activity, the V(MAX) was reduced threefold relative to wild-type. Despite maintaining an active mechanochemical cycle, E385D-Myo1a was unable to move actin in the sliding filament assay. Intriguingly, when an enhanced-green-fluorescent-protein-tagged form of E385D-Myo1a was stably expressed in polarized epithelial cells, this mutation abolished the microvillar targeting normally demonstrated by wild-type Myo1a. Notably, these data are the first to suggest that mechanical activity is essential for proper localization of Myo1a in microvilli. These studies also provide a unique example of how even the most mild substitution of invariant switch II residues can effectively uncouple enzymatic and mechanical activity of the myosin motor domain.
Yengo
Christopher M
CM
Ananthanarayanan
Shobana K
SK
Brosey
Chris A
CA
Mao
Suli
S
Tyska
Matthew J
MJ
D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics*; D003638:Deafness / Q000235:genetics*; D004023:Dictyostelium; D018698:Glutamic Acid / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D040542:Mechanotransduction, Cellular; D009154:Mutation / Q000235:genetics*; D018995:Myosin Heavy Chains; D024461:Myosin Type I; D024462:Myosin Type II / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
D000199:Actins; C062291:MYO1A protein, human; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D024461:Myosin Type I; D024462:Myosin Type II; D018995:Myosin Heavy Chains