The cargo adaptor proteins RILPL2 and melanophilin co-regulate myosin-5a motor activity

Abstract

Vertebrate myosin-5a is an ATP-utilizing processive motor associated with the actin network and responsible for the transport and localization of several vesicle cargoes. To transport cargo efficiently and prevent futile ATP hydrolysis, myosin-5a motor function must be tightly regulated. The globular tail domain (GTD) of myosin-5a not only functions as the inhibitory domain but also serves as the binding site for a number of cargo adaptor proteins, including melanophilin (Mlph) and Rab-interacting lysosomal protein-like 2 (RILPL2). In this study, using various biochemical approaches, including ATPase, single-molecule motility, GST pulldown assays, and analytical ultracentrifugation, we demonstrate that the binding of both Mlph and RILPL2 to the GTD of myosin-5a is required for the activation of myosin-5a motor function under physiological ionic conditions. We also found that this activation is regulated by the small GTPase Rab36, a binding partner of RILPL2. In summary, our results indicate that RILPL2 is required for Mlph-mediated activation of Myo5a motor activity under physiological conditions and that Rab36 promotes this activation. We propose that Rab36 stimulates RILPL2 to interact with the myosin-5a GTD; this interaction then induces exposure of the Mlph-binding site in the GTD, enabling Mlph to interact with the GTD and activate myosin-5a motor activity.

Keywords: Rab; Rab36; actin; allosteric regulation; melanophilin; melanosome; molecular motor; myosin; myosin-5a; organelle; small GTPase; vesicle transport.

Figure 1.

RILPL2 specifically interacts with Myo5a-GTD and enhances the interaction between Myo5a and Mlph-GTBM. A, Myo5a-GTD specifically interacts with RILPL2 but not RILP or RILPL1. Shown is GST pulldown of GST-Myo5a-GTD with RILP, RILPL1, and RILPL2. GST-Myo5a-GTD (2 μm) was incubated with 4 μm His-RILP, His-RILPL1, or His-RILPL2 and then pulled down by GSH-Sepharose. Inputs and pulldown samples were analyzed by SDS-PAGE (4%–20%) and visualized with CBB staining. The bands of ∼43 kDa and ∼26 kDa are the degradation of GST-GTD during the assay. B, RILPL2-RH1 enhances binding of Mlph-GTBM with Myo5a. FLAG-tagged full-length Myo5a (0.5 μm) was incubated with 2 μm GST-Mlph-GTBM and/or 2 μm His-RILPL2-RH1 and then pulled down using GSH-Sepharose. Bottom panel, the inputs and the pulldown samples were analyzed by Western blotting with the indicated antibodies. Top panel, the amount of Myo5a pulled down in the presence of RILPL1-RH1 relative to the absence of RILPL2-RH1 (data are the mean ± S.D. of three independent assays). IB, immunoblot.

Figure 2.

RILPL2-RH1 enhances the activation of Myo5a ATPase activity by Mlph-GTBM. A, activation of Myo5a ATPase activity by Mlph-GTBM in the absence or presence of RILPL2-RH1. The ATPase activity of Myo5a was measured in the presence of 0–40 μm Mlph-GTBM under EGTA conditions (20 mm MOPS-KOH (pH 7.0), 100 mm KCl, 1 mm DTT, 1 mm MgCl₂, 0.25 mg/ml BSA, 2.5 mm PEP, 20 units/ml pyruvate kinase, 12 μm CaM, 0.5 mm ATP, 40 μm actin, and 1 mm EGTA) in the absence (open squares) or presence (closed squares) of 5 μm RILPL2-RH1. V_max and K_d were obtained by a hyperbolic fit and are shown as means ± SDs from three independent assays. B, the effects of ionic strength on the activation of Myo5a ATPase activity by RILPL2-RH1 and Mlph-GTBM. The ATPase activity of Myo5a was determined under EGTA conditions and 25–500 mm KCl in the absence or presence of Mlph-GTBM and/or RILPL2-RH1. Open circles, in the absence of Mlph-GTBM and RILPL2; closed circles, in the presence of 40 μm Mlph-GTBM; open squares, in the presence of 5 μm His-RILPL2-RH1; closed squares, in the presence of 20 μm Mlph-GTBM and 5 μm His-RILPL2-RH1. All data are means ± SDs from three independent assays.

Figure 3.

RILPL2-RH1 and Mlph-GTBM synergistically activate Myo5a motility activity. A, total internal reflection fluorescence microscopy images showing the movements of Myo5a molecules (green, Cy3B-Myo5a) along actin filaments (red, Alexa 488–labeled F-actin) in the absence or presence of 20 μm Mlph-GTBM and/or 5 μm RILPL2-RH1. The moving spots are indicated by arrowheads (see also Movies S1–S4). For clarity, only a part of the image (150 × 150 pixels) is shown. Scale bars = 5 μm. B, normalized frequency of Cy3B fluorescent spots moving along Alexa 488–labeled F-actin within a fixed time frame and area. Values are normalized to a 1-mm length of actin filament in 1 min. Values are means ± SDs of three different movies.

Figure 4.

RILPL2-RH1 and Mlph-GTBM synergistically induce Myo5a to form the extended conformation. The S values of Myo5a (∼2 μm Myo5a in 20 mm MOPS (pH 7.0), 100 mm KCl, 1 mm EGTA, 1 mm MgCl₂, and 1 mm DTT) in the absence or presence of RILPL2-RH1 and Mlph-GTBM were measured by analytical ultracentrifugation. Control, in the absence of RILPL2-RH1 and Mlph-GTBM; +RH1, in the presence of 10 μm RILPL2-RH1; +GTBM, in the presence of 10 μm Mlph-GTBM; +RH1+GTBM, in the presence of 5 μm RILPL2-RH1 and 5 μm Mlph-GTBM.

Figure 5.

Rab36 stimulates Mlph-GTBM/RILPL2–mediated activation of Myo5a ATPase activity by enhancing RILPL2's interaction with Myo5a. A, Rab36 interacts with RILPL2 in a GTP-dependent manner. GST or GST-RILPL2 (20 μm, 20 μl) was bound onto 10 μl of GSH-Sepharose and then incubated with 200 μl of 2 μm His-Rab36 preloaded with GTPγS or GDP. The GSH-Sepharose–bound proteins were eluted by GSH and then analyzed by SDS-PAGE and visualized with CBB staining. B, the effects of Rab36 on Mlph-GTBM/RILPL2–mediated activation of Myo5a ATPase activity. The ATPase activity of Myo5a was measured under EGTA conditions as described in Fig. 1A, except that 5 μm His-RILPL2-RH1, 5 μm Mlph-GTBM, 5 μm His-RILPL2, and/or 20 μm Rab36-GTPγS were added. C, Rab36 enhances RILPL2's interaction with Myo5a-GTD. GST-Myo5a-GTD (4 μm) was incubated with 10 μm His-RILPL2 and/or 10 μm His-RILPL2-RH1 and/or 15 μm His-Rab36-GTPγS and then pulled down using GSH-Sepharose. Left panel, the inputs and the pulldown samples were separated by SDS-PAGE and visualized with CBB staining. Right panel, the molar ratio of RILPL2 versus RILPL2-RH1 pulled down with GST-Myo5a-GTD in the presence of 0–15 μm His-Rab36-GTPγS. The amounts of RILPL2 and RILPL2-RH-1 pulled down were quantified using the National Institutes of Health ImageJ program. The molar ratio of RILPL2 versus RILPL2-RH1 was calculated based on their molecular masses. Data are means ± SDs of at least three independent assays.

Figure 6.

Proposed model for the regulation of Myo5a by RILPL2 and Mlph. A, the structures of the Myo5b-GTD homodimer and Myo5a-GTD/RILPL2-RH1/Mlph-GTBM ternary complex. Top, the structure of the Myo5b-GTD homodimer (PDB code 4J5M) formed by two symmetry-related molecules. Bottom, the structure of the Myo5a-GTD/RILPL2-RH1/Mlph-GTBM ternary complex (PDB code 4KP3). The Mlph-GTBM–binding site and the putative motor domain–binding site are indicated. Sub-1 and Sub-2 indicate subdomain 1 and 2 of Myo5a-GTD, respectively. B, a three-state model for the activation of Myo5a motor function by Rab36, RILPL2, and Mlph-GTBM. In the inhibited state, the two Myo5a-GTDs form a dimer and bind to the two heads of Myo5a, forming a triangular folded conformation. RILPL2 contains two domains, RH1 and RH2, which interact with Myo5a-GTD and the GTP-bound Rab36, respectively. In the absence of Rab36, RH2 prevents RH1 from binding to Myo5a-GTD. Binding of GTP-bound Rab36 to RH2 relieves RH2's inhibition on RH1. RH1 then binds to the GTD dimer of Myo5a in the inhibited state and induces the GTD to expose the Mlph-GTBM–binding site, forming the preactivated state. Finally, Mlph-GTBM binds to the Mlph-GTBM–binding site of Myo5a-GTD in the preactivated state and induces Myo5a to transform to the activated state in the extended conformation.