Abstract
Purpose: :
The Ran-binding protein 2 (RanBP2) is at the nexus of multiple molecular processes underpinning energy and protein homeostasis, trafficking and function of mitochondria, and nucleocytoplasmic transport. Haploinsufficiency of RanBP2 promotes delocalization of mitochondria components in photosensory neurons and selective inhibition of the interaction between RanBP2 and the microtubule-based motor proteins, the kinesins, KIF5B and KIF5C, prevents the outward dispersion of the mitochondria in multiple cell lines. The kinesin heavy chains are thought to undergo large changes in conformation and motor activity upon cargo binding. Yet, the mechanisms underlying the regulation of loading and unloading of native cargoes, and impact of these processes on the microtubule-binding and motor activities of kinesins, remain largely elusive. The goal of this work is to probe mechanistically the role of RanBP2, domains thereof, and various cargoes of RanBP2, on the motor activity of KIF5B/KIF5C.
Methods: :
We developed and employed reconstitution and end-point assays with purified proteins and interacting domains thereof, to investigate the role of the kinesin-binding domain of RanBP2, flanking domains thereof, and interacting partners, on the motor activity of KIF5B/KIFC.
Results: :
We found that cargoless KIF5B/KIF5C is present in an inactive and stable conformation in the absence and presence of microtubules. Removal of the RanBP2-interacting tail domain from KIF5B/KIF5C constitutively activates kinesin. Constructs containing the kinesin-binding domain (KBD) of RanBP2 are sufficient to activate KIF5B/KIF5C, while mutations in KBD abolish the activation of KIF5B/KIF5C. Moreover, this process is strongly modulated by the cis-trans prolyl isomerase (PPIase) domain of RanBP2. Surprisingly, the activity of this domain is required to modulate likely the production of conformational state(s) in RanBP2, rather than in KIF5B/KIF5C, that serves in turn to regulate the motor activity of KIF5B/KIF5C.
Conclusions: :
The RanBP2 and tail-domain of KIF5B/KIF5C exert strong and antagonistic modulatory functions in the activation of KIF5B/KIF5C and this is also dependent on the PPIase-dependent change in conformation of RanBP2. The reconstitution assays will serve to probe further the effect(s) of various native cargoes of RanBP2 on the motor activity of KIF5B/KIF5C. These assays will provide in depth mechanistic and pharmacological insights onto the reciprocal regulation of the activity of kinesin and its cargoes underlying trafficking processes in ganglion and photoreceptor neurons.
Keywords: protein structure/function • proteins encoded by disease genes • retina