A Toxoplasma gondii class XIV myosin, expressed in Sf9 cells with a parasite co-chaperone, requires two light chains for fast motility

Abstract

Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 μm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 μm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors.

Keywords: ATPase; Class XIV Myosin; Molecular Chaperone; Molecular Motor; Myosin Chaperones; Myosin Motor Complex; Protein Expression; Protein Folding; Toxoplasma gondii; UCS Proteins.

FIGURE 1.

Schematic of T. gondii myosin motor complex. TgMyoA (i.e. TgMyoA heavy chain (HC) with its bound light chains TgMLC1 and TgELC1) and TgGAP45 are anchored to the IMC via transmembrane protein TgGAP50. This multiprotein complex is referred to as the myosin motor complex. Short actin filaments are located between the parasite plasma membrane and the IMC and are thought to be connected to ligands on the host cell surface through linker protein(s) that bind to the cytosolic tails of surface adhesins. TgMyoA (attached to the IMC) displaces the actin filaments (attached to the substrate), causing the parasite to move relative to the substrate. Note that alternative models of parasite motility are emerging (50, 51) in which TgMyoA plays a different but still important role in generating the force required for motility. The figure was adapted from Ref. .

FIGURE 2.

Schematic of the expressed proteins. A, the TgMyoA heavy chain construct contains the motor domain (red) and the light chain-binding region (black) followed by a Bio tag and FLAG tag (gray). B, TgUNC consists of three domains, TPR (yellow), central (cyan), and the UCS region (blue), followed by a Myc tag. The three different TgUNC constructs used during co-expression with TgMyoA heavy chain are shown below the schematic. aa, amino acids.

FIGURE 3.

Co-expression of TgMyoA with the chaperone TgUNC in Sf9 cells produces soluble heavy chain. Sf9 cells were co-infected with recombinant baculovirus coding for the TgMyoA heavy chain (HC) and its light chain TgMLC1. The Western blot shows the total (T) and soluble (S) protein fractions after 72-h infection in the absence (left two lanes) or presence (right two lanes) of co-expressed TgUNC. TgUNC was detected using anti-Myc antibody, whereas TgMyoA heavy chain was detected with anti-FLAG antibody.

FIGURE 4.

Characterization of purified TgMyoA. A, SDS gel analysis of purified motor proteins. Lane 1, purified protein resulting from co-expression of TgMyoA heavy chain (HC), TgMLC1 light chain, and TgUNC. TgUNC only binds to unfolded protein and does not co-purify with TgMyoA. Lane 2, molecular mass standards. Lane 3, purified protein resulting from co-expression of TgMyoA heavy chain, TgMLC1, TgELC1, and TgUNC. B, sedimentation velocity of TgMyoA heavy chain expressed with TgMLC1. A sedimentation coefficient of 7.7 S was determined by curve fitting to one species. The symmetrical nature of the boundary indicates that a homogeneous species is present. OD, optical density.

FIGURE 5.

The number of bound light chains determines the speed of actin movement in an in vitro motility assay. The Gaussian fit of speeds for TgMyoA with TgMLC1 only was 1.5 ± 0.2 μm/s (mean ± S.D., n = 2,341 filaments) (solid blue triangles) compared with 3.4 ± 0.7 μm/s (mean ± S.D., n = 4,774 filaments) for TgMyoA with both light chains (solid red circles). Conditions were as follows: 25 mm imidazole, pH 7.5, 50 mm KCl, 1 mm EGTA, 4 mm MgCl₂, 10 mm DTT, 5 mm MgATP, 0.5–0.7% (w/v) methylcellulose, and oxygen scavengers at 30 °C. Addition of 1.2 mm calcium (i.e. 0.2 mm free calcium) did not affect motility speed (open symbols). In the presence of calcium, TgMyoA with TgMLC1 only moved actin at 1.5 ± 0.2 μm/s (mean ± S.D., n = 619 filaments), and TgMyoA containing both TgMLC1 and TgELC1 moved actin at 3.3 ± 0.6 μm/s (mean ± S.D., n = 1,729 filaments). The values obtained in calcium versus EGTA were indistinguishable (p > 0.1, Kolmogorov-Smirnov test). For each data set, the bin with the highest speed was normalized to 1 for presentation purposes. Data were obtained from three protein preparations of TgMyoA with both light chains and two protein preparations of TgMyoA with TgMLC1 only.

FIGURE 6.

Both TgMLC1 and TgELC1 are bound to the same heavy chain. TgMyoA heavy chain (HC) and TgUNC were co-expressed in Sf9 cells with both 3xMyc-MLC1 and 3xHA-ELC1. The lane labeled S is the total soluble protein after Sf9 cell lysis. TgMyoA was co-immunoprecipitated from cell lysates using either anti-HA antibody (to immunoprecipitate TgELC1; left panels) or anti-TgMLC1 antibody (right panels), confirming that each of the light chains binds to TgMyoA heavy chain. Furthermore, TgMLC1 is present in the TgELC1 pulldowns and vice versa, demonstrating that the two light chains are simultaneously present on the same MyoA heavy chain. TgUNC (∼126 kDa) was detected with anti-Myc antibody, TgMyoA heavy chain (∼104 kDa) was detected with anti-FLAG, 3xMyc-TgMLC1 (∼29 kDa) was detected with anti-Myc in the left panels and anti-TgMLC1 in the right panels, and 3xHA-ELC1 (∼18 kDa) was detected with anti-HA. WB, Western blot; IP, immunoprecipitate.

FIGURE 7.

In vitro motility and steady-state actin-activated ATPase assays. A, in vitro motility speed of TgMyoA with both light chains bound as a function of MgATP concentration. The solid line is a fit to a rectangular hyperbola that defines a maximum speed of 4.6 ± 0.3 μm/s, and K_m = 1.3 ± 0.3 mm MgATP. Error bars represent S.D. B, steady-state actin-activated ATPase assay of TgMyoA with both light chains bound (52 nm) as a function of skeletal actin concentration. Data were fit to the Michaelis-Menten equation: V_max = 84 ± 9.5 s⁻¹, and K_m = 136 ± 22 μm. Conditions were as follows: 10 mm imidazole, pH 7.0, 5 mm NaCl, 1 mm MgCl₂, 1 mm NaN₃, 5 mm MgATP, and 1 mm DTT at 30 °C.

FIGURE 8.

The TPR domain of TgUNC is not required for solubility of TgMyoA. Sf9 cell infections were performed with three TgUNC constructs (see Fig. 2B). Expression and solubility of TgMyoA were determined by Western blotting. Total (T) and soluble (S) fractions were probed with either anti-FLAG (top panel) for TgMyoA heavy chain (HC) or anti-Myc (bottom panel) for TgUNC and its truncations.

FIGURE 9.

In vitro motility speeds of purified TgMyoA expressed with various TgUNC constructs. When TgMyoA was expressed with either full-length TgUNC or ΔTPR, the speed at which it moved actin was similar: full-length TgUNC, 3.4 ± 0.7 μm/s (mean ± S.D., n = 4,774 filaments) and ΔTPR, 3.1 ± 0.5 μm/s (mean ± S.D., n = 2,048 filaments). Speed decreased when TgMyoA was expressed in the presence of the UCS domain only (0.7 ± 0.2 μm/s, mean ± S.D., n = 1,296 filaments). The bin with the highest speed was normalized to 1 for presentation purposes. Data were obtained from three preparations of TgMyoA co-expressed with TgUNC, two protein preparations of TgMyoA co-expressed with ΔTPR, and one protein preparation of TgMyoA co-expressed with the UCS domain.

FIGURE 10.

Schematic representation of the domain structure of TgMyoA. We propose that C-terminal to the motor domain TgMyoA has a typical lever arm stabilized by two bound light chains. To illustrate how lever arm length affects step size, a representative lever arm structure (Mlc1p bound to two adjacent motifs of unconventional myosin V; Protein Data Bank code 1N2D) is shown. Rotation of the lever arm upon phosphate release produces a power stroke that moves actin relative to myosin. The distance that myosin can displace actin per cycle of MgATP hydrolysis increases with lever arm length (compare dashed versus solid arrows). The functional impact of having two bound light chains (the equivalent of TgELC1 depicted here in green and TgMLC1 in blue) is a faster speed of movement of actin, consistent with our data.