Ran GTPase-Independent and Stereochemical Control of Kinesin-1 and Mitochondrial Motility by Domains of Ran-Binding Protein-2

Abstract

The microtubule-based motor proteins, dynein and kinesin-1, mediate fast mitochondrial trafficking, but the mechanisms underlying the regulation of mitochondrial motility are ill-defined. The Ran-binding protein 2 (RanBP2) is a pleiotropic and multimodular protein, which couples directly with the kinesin-1 isoforms, KIF5B/KIF5C, via its tripartite domains, the kinesin-binding domain (KBD) and the Ran GTPase-binding domains, RBD2 and RBD3. The coupling of RBD2-KBD-RBD3 to kinesin-1 activates its ATPase activity ∼30-fold and with activation kinetics that is biphasic and cooperative. Here, we employ structure-function, biochemical, kinetic and cell-based assays with time-lapse live-cell microscopy of over 260,000 mitochondrial motility-related events to probe the interplay between Ran GTPase and RBD2-KBD-RBD3 on kinesin-1 activation and mitochondrial motility. We uncover mutually exclusive subdomains in RBDs toward Ran GTPase binding, kinesin-1 activation and modulation of mitochondria motility. The RBDs exhibit Ran-GTP-independent, subdomain and stereochemical-dependent discrimination on the biphasic activation kinetics of kinesin-1 or regulation of mitochondrial motility. Remarkably, RBD2-KBD-RBD3 and KBD alone exert opposing effects on the equilibrium between the stationary and motile phases of mitochondria and multiple biophysical parameters of mitochondrial motility. Further, the regulation of the bidirectional transport of mitochondria by either RBD2-KBD-RBD3 or KBD is highly coordinated, since their effects are accompanied always by changes in motile biophysical parameters of opposite-polarity. These studies uncover Ran GTPase-independent antagonizing and multimodal mechanisms of kinesin-1 activation and regulation of mitochondrial motility by distinct domains of RanBP2. Further, they open new venues toward the pharmacological harnessing of mechanisms regulating kinesins, mitochondrial motility or RanBP2 in a variety of disparate disorders.