Identification and characterization of novel factors regulating kinesin‐1 activation and mitochondria dynamics

Abstract

The Ran‐binding protein 2 (RanBP2) is a mosaic and pleiotropic protein, which associates in vitro and in vivo with the conventional microtubule‐based motor and kinesin‐1 isoforms, KIF5B/KIF5C. We showed previously that the kinesin‐binding domain (KBD) together with the flanking Ran‐binding domains 2 and 3 (RBD2, RBD3) of RanBP2 directly jump‐start and boost the ATPase motor activity of kinesin‐1, a motor implicated in mitochondria transport. The goal of this study is to define the roles of RBD2 and RBD3 of RanBP2 and Ran GTPase in kinesin‐1 activation in vitro and multiple biophysical parameters of mitochondria motility in live cells. We show that the activation of kinesin‐1 by RBD2‐KBDRBD3 is Ran GTPase independent; yet mutations in RBD2‐KBDRBD3 and abolishing its interaction with Ran GTPase suppress the Michaelis‐Menton and partially affect the allosteric activation phases of kinesin‐1. These effects lead to a ~20% reduction of kinesin‐1 activity (kcat). In live cells, KBD alone stimulates the motility, displacement, frequency of change in direction, motility persistence and anterograde velocity of mitochondria, whereas a mutant KBD construct has no effect. In contrast, RBD2‐KBDRBD3 of RanBP2, but not mutant constructs, suppress all these biophysical parameters of mitochondria motility. Further, there is stereochemical specificity between RBD2 and RBD3 in certain parameters of regulation of mitochondria motility. These studies uncover novel Ran GTPase‐independent functions of RBD2, RBD3 and KBD of RanBP2 in kinesin‐1 activation and regulation of mitochondria motility.NIH support GM083165 & EY019492 and RPB