Chaperone-enhanced purification of unconventional myosin 15, a molecular motor specialized for stereocilia protein trafficking

• DOI: 10.1073/pnas.1409459111
• Publication Date: 2014-08-12T06:59:19Z
• Authors: Jonathan E. Bird; Yasuharu Takagi; Neil Billington; Marie-Paule Strub; James R. Sellers; Thomas B. Friedman
• Keywords: Molecular motor; Meromyosin; Chaperone (clinical)

Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of cause profound hearing loss humans and mice; however, the properties this its regulation stereocilia organelle are unknown. To address these questions, we subfragment 1-like (S1) truncation mouse 15, comprising predicted domain plus three light-chain binding sites. Following unsuccessful attempts to express functional 15-S1 using Spodoptera frugiperda (Sf9)-baculovirus system, discovered coexpression muscle-myosin-specific chaperone UNC45B, addition heat-shock 90 (HSP90) significantly increased yield protein. Surprisingly, did not bind calmodulin with high affinity. Instead, IQ domains bound essential regulatory light chains normally associated class II myosins. We show barbed-end-directed moves actin filaments gliding assay (∼ 430 nm · s(-1) at 30 °C), power stroke 7.9 nm. The maximum ATPase rate (k(cat) ∼ 6 s(-1)) was similar actin-detachment (k(det) = 6.2 determined single molecule optical trapping experiments, indicating limited by transit through strongly actin-bound states, other processive motors. Our data further indicate folding muscle myosin, UNC45B facilitates maturation an unconventional myosin. speculate may be simple method optimize purification motors from Sf9 insect cells.