Stam, S.

Weak, transient molecular interactions are ubiquitous amongst biological molecules. Regulatability by intracellular concentrations, localization, and other factors is central to their importance. Here, we measure co-localization between fluorescently labeled proteins, Miro2 and {beta}-Pix, to assess their effective binding affinity and its dependence on protein concentration. Co-localization between the two is quantifiable due to the mitochondrial localization of Miro2, which allows the otherwise cytosolic {beta}-Pix to be drawn to mitochondria. Determining the fraction of overexpressed {beta}-Pix co-localizing with mitochondria as a function of overexpressed Miro2 levels allows calculation of an effective dissociation constant from a mass-action binding model. Both the fit to the model and the effective dissociation constant for Miro2 and {beta}-Pix binding depend on the concentration of {beta}-Pix, which suggests that one or more model assumptions do not hold. Additional experiments reveal that competitive binding and/or {beta}-Pix sequestration by Git1 are possible explanations for the {beta}-Pix concentration dependence. Our modeling strategy is readily extendable to ectopic localization of proteins to mitochondria and other compartments for the assessment of co-localization with a binding partner. This reveals fundamental properties of protein-protein interactions and how they may be regulated by protein concentration and other intracellular conditions.