Identification of RanBP2– and kinesin–mediated trafficking pathways: Implications in photoreceptor and retinal function

Abstract

Abstract: : Purpose: RanBP2 is a large mosaic and scaffold protein unique to vertebrates. Selective domains of RanBP2 mediate the specific interaction of these with proteins such as opsin, RanGTPase, CRM1/exportin–1, importin–ß, components of the 19S cap of the proteasome and the microtubule–based motor proteins, KIF5B/KIF5C. Yet, the function of the leucine–rich (LD) of RanBP2, its function in concert with that of other domains and the role of these in retinal/photoreceptor and animal physiology are unclear. Methods: We employed a combination of cell–based assays, biochemical, yeast and mouse genetic, and physiological approaches to identify molecular partners with specific binding affinity towards the LD of RanBP2, to determine the effect of uncoupling KIF5B/KIF5C association with RanBP2 on the distribution of LD–associated partners, and the role of RanBP2 in retinal physiology and disease. Results: We have identified the association of LD of RanBP2 domain with Cox11, a mitochondrial chaperone implicated in the assembly of the mitochondrial respiratory chain. This interaction occurs in presence of other mitochondrial proteins such as hexokinase (HK) and mHsp70. Moreover, RanBP2 modulates the activity of HK. To ascertain the biological implications of KIF5B/KIF5C interaction with RanBP2 in mitochondrial trafficking and biogenesis, selective disruption of the interaction between RanBP2 and the isotype–specific kinesins, KIF5B/KIF5C, was carried out in cultured retinal lines. This led to the clustering of the mitochondria and RanBP2 followed by extremely severe neuronal atrophy. Finally, disruption of the RanBP2 locus had a strong gene–dosage, and possibly sex–dependent, impact on the subcellular localization of mitochondrial components in photoreceptors and animal survival. Conclusions: Thus, another role emerges for RanBP2 whereby RanBP2 acts as a scaffold chaperone in the cytosol to mediate the biogenesis and transport of mitochondrial components and modulate the activity of some of these. These fundamental biological processes are likely bound to be closely coupled to other trafficking pathways and to the pathogenesis of retinopathies.

Keywords: protein structure/function • transgenics/knock–outs • retina