Abstract
Myosin VI is an unconventional myosin motor that moves towards the minus end of actin filaments. The motor is known to interact with various binding partners and function as an anchor and a vesicle carrier in many diverse functions, including endocytosis and exocytosis. The multiple functions are likely to be regulated through the binding partners. The functional units of the motor (monomers/dimers) and the effects of binding partners remain unclear. Here we present a quantitative FRET based assay to measure the association of binding partners and determine the effect upon the oligomeric state of myosin VI. We have shown that myosin VI tail forms relatively tight interactions with the binding partners NDP52 (Kd 1 μM) and Dab2 (Kd 5 μM). We also found that these binding partners oligomerize myosin VI, increasing the affinity of oligomerization from >80 μM to 4 μM. We purpose this is achieved by the binding partners relieving auto-inhibition of the cargo-binding domain. Furthermore, we have characterized the interactions of binding partners with full length myosin VI using size-exclusion chromatography and sucrose density gradients. Single molecule photobleaching assays have been performed to determine the stoichiometry of the complexes. Functional measurements have been carried out using ATPase and single molecule motility assays in the presence of the binding partners to determine effects upon the motor activity. Supported by DFG, SFB-863, Freidrich Baur-Stiftung and EMBO.
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