title: Chaperone-enhanced purification of unconventional myosin 15, a molecular motor specialized for stereocilia protein trafficking.
abstract: Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin-specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end-directed motor that moves actin filaments in a gliding assay (∼ 430 nm · s(-1) at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (k(cat) ∼ 6 s(-1)) was similar to the actin-detachment rate (k(det) = 6.2 s(-1)) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells.
authors.affiliation: Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, and.
authors.forename: Jonathan E
authors.identifier: http://orcid.org/0000-0001-5531-8794
authors.initials: JE
authors.lastname: Bird
authors.affiliation: Laboratory of Molecular Physiology and.
authors.forename: Yasuharu
authors.initials: Y
authors.lastname: Takagi
authors.affiliation: Laboratory of Molecular Physiology and.
authors.forename: Neil
authors.initials: N
authors.lastname: Billington
authors.affiliation: Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Marie-Paule
authors.initials: MP
authors.lastname: Strub
authors.affiliation: Laboratory of Molecular Physiology and friedman@nidcd.nih.gov sellersj@nhlbi.nih.gov.
authors.forename: James R
authors.initials: JR
authors.lastname: Sellers
authors.affiliation: Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, and friedman@nidcd.nih.gov sellersj@nhlbi.nih.gov.
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Friedman
mesh_terms: D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D018994:Myosin Light Chains / Q000378:metabolism; D015879:Myosin Subfragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009218:Myosins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D052898:Optical Tweezers; D017510:Protein Folding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D061987:Sf9 Cells; D018411:Spodoptera; D059547:Stereocilia / Q000378:metabolism*
chemical_list: D000199:Actins; D002147:Calmodulin; D018841:HSP90 Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; C114013:Myo15 protein, mouse; D018994:Myosin Light Chains; D015879:Myosin Subfragments; D011993:Recombinant Fusion Proteins; C529478:Unc45 protein, mouse; D009218:Myosins
keywords: DFNB3; UNC-45; deafness; myosin XV
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