Myosin VI targeting to clathrin-coated structures and dimerization is mediated by binding to Disabled-2 and PtdIns(4,5)P2

Abstract

Vesicle transport is essential for the movement of proteins, lipids and other molecules between membrane compartments within the cell. The role of the class VI myosins in vesicular transport is especially intriguing because they are the only class that has been shown to move "backwards" towards the minus end of actin filaments1. Myosin VI is found in distinct intracellular locations and implicated in processes such as endocytosis2,3, exocytosis, maintenance of Golgi morphology4,5 and cell movement6. We have shown that the C-terminal tail is the key targeting region and have identified three binding sites: a WWY motif for Dab2 binding, a RRL motif for GIPC/Optineurin binding and a site that binds specifically and with high affinity (K d 0.3 μM) to PIP 2 -containing liposomes. This is the first demonstration that myosin VI binds lipid membranes. Lipid binding induces a large structural change in the tail (31% increase in helicity) and when associated with lipid vesicles it can dimerise. In vivo targeting and recruitment of myosin VI to clathrin-coated structures (CCSs) at the plasma membrane is mediated by Dab2 and PIP 2 binding.Dab2 is a myosin VI binding partner present on endocytic CCSs at the plasma membrane7,8. To establish whether binding to Dab2 is involved in targeting myosin VI to CCSs we tested an extensive series of myosin VI tail deletion fragments and point mutants using the mammalian 2-hybrid assay7. A relatively conservative single amino acid change from a tryptophan to a leucine (W1184L, WWY→WLY) was found to abolish myosin VI binding to Dab2 (Fig. 1a). 'Pull down' experiments using GST-tagged wild type myosin VI tail or tail containing the WWY→WLY mutation together with in vitro translated Dab2 confirmed the identity of the Dab2 binding site (Fig. 1c). To check whether the Dab2 binding site was essential for targeting myosin VI to CCSs in vivo we over-expressed GFPtagged mutant tail constructs in HeLa cells. We observed that myosin VI containing a mutated Dab2 binding site (WWY→WLY) was not targeted to CCSs (Fig. 1d,e). It was previously shown2,8 that the presence of a large insert just before the globular C-terminal domain (Fig. S1) in conjunction with Dab2 binding was also required for targeting myosin VI to CCSs at the plasma membrane.GIPC is another myosin VI binding partner9 that is found in both clathrin-coated10 and uncoated endocytic vesicles3. To test the involvement of GIPC in targeting myosin VI to CCSs we mapped the GIPC binding site on the myosin VI tail by deletion and alanine scanning mutagenesis. We observed that the mutation RRL to AAA in the C-terminal tail Correspondence should be addressed to: J.K-J (e-mail: jkj@mrc-lmb.cam.ac.uk). (Fig. 1b) abolished GIPC binding, but had no effect on Dab2 binding (Fig. 1a,b). Optineurin, a myosin VI binding protein associated with the Golgi complex and secretion, also specifically binds to the RRL binding site in the myosin VI tail4. Although phosphorylation of the threonines in the TINT sequence 15 residues upstream of RRL (Fig. S1b...

The MYO6 interactome reveals adaptor complexes coordinating early endosome and cytoskeletal dynamics

“…To verify the novel MYO6 interaction network, we introduced mutations into the known cargo‐binding and lipid‐binding sites, the WWY, RRL and PIP 2 binding motifs 10, 11, 14 and assessed differences in the relative abundance of BirA*‐MYO6 binding partners using a SILAC‐based approach (Fig 1F). Analysis of these cells by confocal microscopy also revealed that the ΔRRL and ΔPIP2, but not the ΔWWY mutation, perturbed MYO6 targeting to APPL1 endosomes (Fig EV1A).…”

A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis

“…Other proteins showed a similar pattern of biphasic recruitment and thus defined a dynamin module. These included actin-binding proteins such as the actin-and clathrin-binding protein Hip1R [44] as well as the motor protein myosin6, which binds actin and the adaptor protein Dab2 [45]. Other proteins involved in actin dynamics and grouped in the dynamin module included the Arp2/ 3 activator N-WASP [46,47], Eps8, an actin capping protein that forms a complex with Abi1 and binds N-WASP [33] and the motor protein myosin1E [48].…”

Proteomics approach to study the functions of Drosophila myosin VI through identification of multiple cargo-binding proteins

“…The myosin VI binding region of human Dab2 is outside of the conserved phosphotyrosine-binding domain, and the residues critical for binding (46) do not appear to be in the Dab sequence. In addition, Drosophila M6 contains LWY in the position of the WWY motif required for Dab2 binding (47); therefore, an interaction between the Drosophila proteins is not necessarily expected.…”