Fig 1: Biochemical analyses of the interaction between Myosin VI and Tom1. a A schematic diagram showing the domain arrangements of Myosin VI, Tom1, NDP52, TAX1BP1, and Optineurin. In this drawing, domains involved in the protein–protein interaction are highlighted with black lines, and the relevant interactions between two proteins are indicated by two-way arrows. b Superposition plots of the 1H-15N HSQC spectra of Tom1(392–463) titrated with the un-labeled C-terminal CBD of Myosin VI proteins at different molar ratios. For clarity, the insert shows the enlarged view of a unique peak corresponding to the side chain of Tom1 W423 residue in the overlaid 1H-15N HSQC spectra. c–e ITC-based measurements of the binding affinities of the C-terminal CBD of Myosin VI with Tom1(392–463) (c), Tom1(392–437) (d), and Tom1(437–463) (e). Kd values are the fitted dissociation constants with standard errors, when using the one-site binding model to fit the ITC data. ‘N.D.’ stands for that the Kd value is not detectable. Source data are provided as a Source Data file. f Overlay plots of the multi-angle light-scattering data of the C-terminal CBD of Myosin VI, Tom1(437–463), and the C-terminal CBD of Myosin VI in complex with Tom1(437–463). The derived molecular masses of the C-terminal CBD of Myosin VI and Tom1(437–463) are shown in red and in blue, respectively, while the derived molecular mass of the C-terminal CBD of Myosin VI and Tom1(437–463) complex is shown in black. The molecular masses errors are the fitted errors obtained from the data analysis software, and are showed in the brackets. The results clearly demonstrate that the C-terminal CBD of Myosin VI and Tom1(437–463) both form a stable monomer and may interact with each other to form a 1:1 stoichiometric complex in solution. Source data are provided as a Source Data file
Fig 2: Myosin VI can link Tom1 with autophagy receptors. a A co-immunoprecipitation assay showing that point mutations of key interface residues observed in the Myosin VI/Tom1 complex structure abolish the specific interaction between Myosin VI(1060–1285) and Tom1 in cells. In this assay, cell extracts were prepared from HEK293T cells co-transfected with different combinations of plasmids as indicated, and 5% of each extracts were used as loading controls (bottom panel). b A co-immunoprecipitation assay revealing that Myosin VI(1060–1285), Tom1 and autophagy receptor TAX1BP1, NDP52, or Optineurin, can form ternary complexes in co-transfected cells. c A co-immunoprecipitation assay showing that mutations of Tom1, which can disrupt the interaction between Myosin VI(1060–1285) and Tom1, can also abolish the formation of the Tom1/Myosin VI/autophagy receptor ternary complex in cells. In this assay, cell extracts were prepared from HEK293T cells co-transfected with different combinations of plasmids as indicated, and 5% of each extracts were used as loading controls (bottom panel). Source data are provided as a Source Data file. d A proposed model depicting the tethering of endosome and autophagosome mediated by Myosin VI in cooperate with Tom1, the autophagy receptors, NDP52, TAX1BP1, and Optineurin as well as relevant ubiquitin chains, for facilitating the maturation of autophagosome in autophagy
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