Association of the Kinesin‐Binding Domain of RanBP2 to KIF5B and KIF5C Determines Mitochondria Localization and Function

Abstract

The Ran-binding protein 2 (RanBP2) is a large mosaic protein with a pleiotropic role in cell function. Although the contribution of each partner and domain of RanBP2 to its biological functions are not understood, physiological deficits of RanBP2 downregulate glucose catabolism and energy homeostasis and lead to delocalization of mitochondria components in photosensory neurons. The kinesin-binding domain (KBD) of RanBP2 associates selectively in the central nervous system (CNS), and directly, with the ubiquitous and CNS-specific kinesins, KIF5B and KIF5C, respectively, but not with the highly homologous KIF5A. Here, we determine the molecular and biological bases of the selective interaction between RanBP2 and KIF5B/KIF5C. This interaction is conferred by a approximately 100-residue segment, comprising a portion of the coiled-coil and globular tail cargo-binding domains of KIF5B/KIF5C. A single residue conserved in KIF5B and KIF5C, but not KIF5A, confers KIF5-isotype-specific association with RanBP2. This interaction is also mediated by a conserved leucine-like heptad motif present in KIF5s and KBD of RanBP2. Selective inhibition of the interaction between KBD of RanBP2 and KIF5B/KIF5C in cell lines causes perinuclear clustering of mitochondria, but not of lysosomes, deficits in mitochondrial membrane potential and ultimately, cell shrinkage. Collectively, the data provide a rationale of the KIF5 subtype-specific interaction with RanBP2 and support a novel kinesin-dependent role of RanBP2 in mitochondria transport and function. The data also strengthen a model whereby the selection of a large array of cargoes for transport by a restricted number of motor proteins is mediated by adaptor proteins such as RanBP2.

Kinesin’s light chains inhibit the head- and microtubule-binding activity of its tail

“…Therefore, our data establish the tail HM site as a regulatory focal point. The RanBP2 protein is also likely to be another extrinsic direct regulator of the HM site (47).…”

Conventional Kinesin Holoenzymes Are Composed of Heavy and Light Chain Homodimers

“…We next examined potential cross-reactivities among kinesin-1 antibodies. The production of bacterially expressed full-length recombinant kinesin-1 polypeptides in bacteria mainly resulted in the formation of truncated species of lower molecular mass (37), thus complicating the identification of properly folded, full-length kinesin-1 polypeptides that would enable appropriate antibody characterization. To circumvent these issues, anti-kinesin-1 antibodies were reacted with lysates derived from COS-7 cells transfected with cDNAs coding for 6-His-tagged, full-length mouse kinesin-1A, kinesin-1B, or kinesin-1C ( Figure 1C).…”

Nuclear pore complex tethers to the cytoskeleton

“…It was suggested that the kinesin plus end directed motor could pay a role in delivering cargo to, or taking it away from, the NPC (Mavlyutov et al 2002), a function that may be more crucial in cells with long distance transport requirements, such as neurons. However, it was shown that the kinesin-Nup358 interactions are related to functions away from the NPC in determining the correct localisation and function of mitochondria (Cho et al, 2007). Interestingly, the kinesin binding domain, together with the two flanking ran binding domains, activate the kinesin motor activity of KIF5C (Cho et al, 2009), suggesting that any kinesin bound to Nup358, whether it is in the cytoplasm or at the NPC, would be an active motor.…”