Human deafness mutation E385D disrupts the mechanochemical coupling and subcellular targeting of myosin-1a
Christopher M. Yengo 1, Shobana K. Ananthanarayanan 1, Chris A. Brosey 2, Suli Mao 2 and Matthew J. Tyska 2*
1 Department of Biology, University of North Carolina, Charlotte, North Carolina
2 Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN
* To whom correspondence should be addressed. E-mail: matthew.tyska{at}vanderbilt.edu.
Submitted on September 23, 2007
Revised on October 8, 2007
Accepted on 25 October 2007
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Abstract |
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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 VMAX was reduced three-fold 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 EGFP-tagged form of E385D-Myo1a was stably expressed in polarized epithelial cells, this mutation abolished the microvillar targeting normally demonstrated by wildtype 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.
Key Words:
actin, membrane, microvilli, motility, motor, mutation
