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.

Keywords: DFNB3; UNC-45; deafness; myosin XV.

Fig. 1.

Purification of mouse myosin 15 from Sf9 cells by chaperone coexpression. (A) Schematic domain structure of mouse myosin 15 isoforms. (B) Motor domain constructs used in this study. All expressed proteins have a C-terminal FLAG epitope for affinity purification. (C) ClustalW alignments of the second IQ of myosin 15 show similarity to the ELC binding site (first IQ) of class II myosins. (D) Coexpression with UNC45B/HSP90AA1 increases the solubility of EGFP-Myo15-3IQ in Sf9 insect cells (abbreviated to 3IQ for D–F). Western blot probed with antibody against FLAG (green) and alpha-tubulin (red). Total and soluble fractions were prepared from equal volumes of Sf9 cell cultures expressing EGFP-Myo15-3IQ, plus combinations of chaperones and light chains as shown. EGFP-Myo15-3IQ sedimented when coexpressed with calmodulin (CALM) alone, or with CALM + ELC + RLC. Expression of UNC45B + HSP90AA1 increased EGFP-Myo15-3IQ in the supernatant (compare arrowheads), but did not change heavy chain expression overall (compare stars). (E) Supernatants from D were bound to FLAG-M2 affinity resin, eluted with FLAG peptide (0.5-mL fractions), and assayed for EGFP fluorescence (representative of three experiments). Individual images (outlined in white) for each condition were captured with identical settings and consolidated. Samples coexpressing RLC + ELC in addition to UNC45B + HSP90AA1 eluted in earlier fractions (third row), compared with coexpressing UNC45B/HSP90AA1 and CALM (second row). (F) SDS/PAGE of FLAG-purified proteins from Sf9 cells expressing UNC45B + HSP90AA1 and combinations of Myo15 IQ mutants and lights chains as indicated. Note increased mobility of Myo15-1IQ (arrowhead) without an EGFP fusion.

Fig. 2.

Chaperone-purified myosin 15 is correctly folded and the lever arm fully occupied by ELC and RLC. (A) Representative Coomassie-stained SDS/PAGE gel of EGFP-Myo15-2IQ and Myo15-2IQ-EGFP after large-scale expression and purification by ion exchange and size exclusion chromatography. The identity of the myosin 15 heavy chain (HC), RLC, and ELC were confirmed by LC-MS/MS sequencing. (B) Representative raw TEM micrograph of negative-stained Myo15-2IQ-EGFP. (Scale bar, 50 nm.) (C) Class averages of Myo15-2IQ-EGFP with no-nucleotide (apo) or with ADP·AlF₄ bound shows postpower stroke- (rigor) and prepower stroke-like states, respectively. The globular EGFP moiety is visible (arrowhead) at the C terminus. (Scale bars, 20 nm.) (D) Class average of EGFP-Myo15-2IQ (N-terminal EGFP fusion) under no-nucleotide (apo) conditions. (Scale bar, 20 nm.) Note the altered position of the EGFP moiety (arrowhead).

Fig. 3.

Myosin 15 is an actin-activated ATPase with barbed-end–directed motility. (A) Steady-state ATPase activation of EGFP-Myo15-2IQ by actin filaments at 20 °C. At 100 mM KCl, k_cat = 6.0 ± 0.5 s⁻¹ and K_ATPase = 164.6 ± 18.9 µM (mean ± SEM). The apparent affinity for actin (K_ATPase) was sensitive to salt concentration, whereas k_cat was not. Data points are mean ± SD from three preparations, [KCl]: 10 mM (red), 50 mM (green), 100 mM (blue). Basal ATPase activity was subtracted from each data point. (B) Histogram of actin filament velocities in a Myo15-2IQ-EGFP gliding assay. The average filament velocity was 429 ± 74 nm·s⁻¹ at 30 °C (blue bars, n = 2,092 filaments from three preparations, mean ± SD, R² = 0.985) and 278 ± 36 nm·s⁻¹ at 20 °C (red bars, n = 1,712 filaments from one preparation, mean ± SD, R² = 0.952). Gaussian fits are overlaid (dotted lines). (C) Montage of images from a time-lapse series of motile polarity-marked actin filaments using TIRF microscopy at 30 °C. Actin filaments were polymerized from TRITC-gelsolin seeds (green) and stabilized using Alexa 647-phalloidin (blue). A maximum projection (MAX) of the time-lapse stack shows the gelsolin-capped barbed-end trailing at the rear of the actin filament. All observed filaments (n = 73) were barbed-end directed. (Scale bar, 5 µm.)

Fig. 4.

Single molecule measurements of myosin 15 in the optical trap. (A) Sample records of single molecules of Myo15-2IQ-EGFP interacting with an actin filament in the three-bead assay at 23 ± 1 °C. Attached states (horizontal black bars) are detected as a reduction in the variance of bead motion, resulting from increased system stiffness as Myo15-2IQ-EGFP binds to the actin filament. No processive stepping events were observed. (B) Histogram of measured bead displacements in the three-bead assay. The distribution is offset from zero due to the myosin power stroke. A Gaussian fit (dotted line) estimates the power stroke = 7.9 ± 1.3 nm (n = 611 observations, four independent determinations, mean ± SEM, R² = 0.897). (C) Dwell time histogram of interactions with the actin filament. Fitting to a single exponential (dotted line) defines the detachment rate from actin, k_det = 6.2 ± 0.5 s⁻¹ (mean ± SEM, R² = 0.925).