Figure 6. Spir-2 facilitates myosin V recruitment to vesicle membranes.

(A) A putative interaction mode of the GTDs within the MyoV off state was derived from the MyoVb-GTD structure (PDB ID 4LX0). Two GTDs interact with each other via N-terminal linkers occupying the Spir/MLPH binding pockets on the neighboring GTD, Spir and MLPH GTBMs are shown in ribbon. (B) Schematic representation of myosin V activation by GTBM binding. (C and D) N-terminal Spir fragments target MyoVa to vesicle membranes. (C) N-terminal Spir fragments (Myc-Spir-2-KIND-WH2, Myc-Spir-2-KW; Myc-Spir-2-KIND-WH2-GTBM, Myc-Spir-2-KWM) and full-length MyoVa motor proteins (eGFP-MyoVa-FL, eGFP-MyoVa-FL-Q1753R) have an even cytoplasmic and nuclear localization when transiently expressed in HeLa cells (single expression). At least 5 cells were recorded for each condition and one representative cell is presented here. Scale bars represent 10 µm. (D) Expression of full-length GFP-tagged MyoVa (eGFP-MyoVa-FL; green (middle) and as heat map (left)) shows an even cytoplasmic distribution that is not changed by co-expression of the Spir-2 fragment (Myc-Spir-2-KIND-WH2), which is not able to bind to MyoV and lipid membranes (upper panel). In contrast, co-expression of a myosin V binding Spir-2 fragment (Myc-Spir-2-KIND-WH2-GTBM; red, middle panel) leads to targeting of MyoVa to vesicle membranes and to overlapping Spir-2 and MyoVa localization (higher magnification insets). Heat maps represent grey values for MyoVa fluorescence intensities rising from '0' (black) to '4096' (red) to document equal expression levels of MyoVa proteins in the depicted cells. To address Rab11 dependence on motor protein targeting, the GFP-tagged melanocyte specific F isoform of the Q1753R mutant MyoVa (eGFP-MyoVa-QR) that does not bind Rab11 was expressed. The expressed MyoVa mutant has an even cytoplasmic distribution that was not changed upon co-expression of the myosin V binding Spir-2 fragment (Myc-Spir-2-KIND-WH2-GTBM; red, lower panel). Representative cells are shown. All cells observed under single and co-expression conditions had a vesicular or cytoplasmic localization as shown by the representative cells presented here. 5 cells were recorded for each condition and one representative cell is presented here. Scale bars represent 10 µm.

DOI: http://dx.doi.org/10.7554/eLife.17523.018

Figure 6—figure supplement 1. Rab11 binding may affect the GTD:motor-domain interaction in the folded inhibited state of MyoV.

(A) Motor domain and Rab11 binding sites are located on the opposite sides of MyoV-GTD SD-2. Conserved MyoVa-GTD residues participating in motor domain binding in the inhibited folded conformation of MyoVa (Yao et al., 2015) are shown as spheres and labeled in blue. E1789 and E1791 belongs to a loop connecting MyoV H11 and H12. (B) Comparison of apo-MyoVa and Rab11 bound MyoVa H11-H12 loop. Close-up view of the Rab11 bound H11-H12 MyoV loop region (orange) superimposed on the apo-MyoVa structure (white). The H11-H12 loop also participates in Rab11 binding and adopts a different conformation in the Rab11:MyoVa complex. When it is not engaged in interactions, this MyoV-GTD loop might swap between different conformations. In the MyoVa:Rab11 complex, MyoVa E1791 makes a hydrogen bond (dashed line) with the conserved W1711 side chain stabilizing the residue in the conformation compatible with Rab11 binding. Thus, Rab11 binding to the folded MyoV full-length motor may retract the H11-H12 loop from the motor domain-binding interface and destabilize the full length GTD-motor domain intramolecular interactions, favoring activation of the motor.

DOI: http://dx.doi.org/10.7554/eLife.17523.019