The Ran-binding protein 2 (RanBP2) is a large multimodular and pleiotropic protein. Several molecular partners with distinct functions interacting specifically with selective modules of RanBP2 have been identified. Yet, the significance of these interactions with RanBP2 and the genetic and physiological role(s) of RanBP2 in a whole-animal model remain elusive. Here, we report the identification of two novel partners of RanBP2 and a novel physiological role of RanBP2 in a mouse model. RanBP2 associates in vitro and in vivo and colocalizes with the mitochondrial metallochaperone, Cox11, and the pacemaker of glycolysis, hexokinase type I (HKI) via its leucine-rich domain. The leucine-rich domain of RanBP2 also exhibits strong chaperone activity toward intermediate and mature folding species of Cox11 supporting a chaperone role of RanBP2 in the cytosol during Cox11 biogenesis. Cox11 partially colocalizes with HKI, thus supporting additional and distinct roles in cell function. Cox11 is a strong inhibitor of HKI, and RanBP2 suppresses the inhibitory activity of Cox11 over HKI. To probe the physiological role of RanBP2 and its role in HKI function, a mouse model harboring a genetically disrupted RanBP2 locus was generated. RanBP2−/− are embryonically lethal, and haploinsufficiency of RanBP2 in an inbred strain causes a pronounced decrease of HKI and ATP levels selectively in the central nervous system. Inbred RanBP2+/− mice also exhibit deficits in growth rates and glucose catabolism without impairment of glucose uptake and gluconeogenesis. These phenotypes are accompanied by a decrease in the electrophysiological responses of photosensory and postreceptoral neurons. Hence, RanBP2 and its partners emerge as critical modulators of neuronal HKI, glucose catabolism, energy homeostasis, and targets for metabolic, aging disorders and allied neuropathies.

The Ran-binding protein 2 (RanBP2) is a vertebrate mosaic protein composed of four interspersed RanGTPase binding domains (RBDs), a variable and speciesspecific zinc finger cluster domain, leucine-rich, cyclophilin, and cyclophilin-like (CLD) domains. Functional mapping of RanBP2 showed that the domains, zinc finger and CLD, between RBD1 and RBD2, and RBD3 and RBD4, respectively, associate specifically with the nuclear export receptor, CRM1/exportin-1, and components of the 19 S regulatory particle of the 26 S proteasome. Now, we report the mapping of a novel RanBP2 domain located between RBD2 and RBD3, which is also conserved in the partially duplicated isoform RanBP2L1. Yet, this domain leads to the neuronal association of only RanBP2 with two kinesin microtubulebased motor proteins, KIF5B and KIF5C. These kinesins associate directly in vitro and in vivo with RanBP2. Moreover, the kinesin light chain and RanGTPase are part of this RanBP2 macroassembly complex. These data provide evidence of a specific docking site in RanBP2 for KIF5B and KIF5C. A model emerges whereby RanBP2 acts as a selective signal integrator of nuclear and cytoplasmic trafficking pathways in neurons.The small nuclear GTPase, Ran, is a key regulator of protein (1-3) and RNA (4 -7) nuclear export and protein nuclear import (2, 6, 8 -11). In contrast to earlier proposals (12, 13) and other GTPase-mediated processes (14), recent data support that nucleocytoplasmic trafficking of cargoes across the nuclear envelope is independent of nucleotide hydrolysis (15-21). Instead, a predicted Ran-GTP to Ran-GDP gradient from the nucleus to the cytosol is proposed to propel the nuclear transport across the nuclear pore in a vectorial fashion (2,22,23). This predicted Ran-nucleotide gradient, together with the selective compartmentalization of key RanGTPase modulators in the nucleus and cytosol, limits the pool of transporters available for polarized delivery of cargoes into and from the nucleus, as these carriers act as sensors of the nucleotide-bound state of Ran (for review see Refs. 24 and 25). Among several Ran-dependent nuclear transporters recently identified, CRM1/exportin-1 (26), a member of the importin ␤/karyopherin-␤ class (22, 23), was shown to associate with Ran-GTP and mediate the nuclear export of substrates containing nuclear export signal sequences (27-31). Three key players with restricted subcellular compartmentalization are thought to play a pivotal role in maintaining the predicted Ran-nucleotide bound gradient across the nuclear envelope. The chromatin-associated Ran-nucleotide exchange factor, RCC1 (32), promotes the production of nuclear Ran-GTP via the exchange of RanGDP to RanGTP (33). In the cytosol, RanGTP hydrolysis seems to be mediated by the costimulation of RanGTPase-activating protein (34) and high affinity 35). This mechanism presumably ensures that loading of cargo destined for nuclear import and unloading of nuclear exported substrates, and loading of cargo for nuclear export and unloading of nuclear imported cargo, re...

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.

We report here on the development and validation of a prototype Invader Plus method for the qualitative detection of herpes simplex virus types 1 and 2 in cerebrospinal fluid (CSF). The method combines PCR and Invader techniques in a single, closed-tube, continuous-reaction format that gives an analytical sensitivity of approximately 10 copies per reaction. The clinical sensitivity and specificity were 100.0% and 98.6%, respectively, when the results of the method were validated against the results obtained with a PCR colorimetric microtiter plate system by use of clinical CSF specimens.

The Ran-binding protein 2 (RanBP2) is a giant scaffold and mosaic cyclophilin-related nucleoporin implicated in the Ran-GTPase cycle. There are no orthologs of the RanBP2 gene in yeast and Drosophila genomes. In humans, this bona fide gene is partially duplicated in a RanBP2 gene cluster and lies in a hot spot for recombination on Chromosome (Chr) 2q. This genetic heterogeneity renders further significance of this genomic region in human disease due to its possible involvement in genetically linked disorders such as juvenile nephronophthisis, congenital hepatic fibrosis, and chorioretinal dysplasia. Structure-function studies on bovine RanBP2 indicate that this protein is involved in integrating nucleocytoplasmic transport pathways with protein biogenesis such as production of functional opsin. To gain further insight into the complex functions of RanBP2 in the development and function of the neuroretina and other tissues, and proceed towards the functional analysis of RanBP2 and its molecular partners in vivo, we have determined the complete genomic organization of the murine RanBP2 gene. The gene consists of 29 exons spread over 50 kb and contains a mega-exon of 4663 bp that encompasses the variable Zn-finger-rich domain of RanBP2. This may account, in part, for a predisposition of recombination of this locus and variability of the number of Zn-fingers across mammalian species. The RanBP2 promoter contains tissue-specific elements. A CpG island encompasses this region up to the first intron, making RanBP2 gene expression susceptible of epigenetic regulation. This murine RanBP2 transcript has a tissue-restricted expression profile, and the conceptual protein is 82% identical to human RanBP2. The gene maps to mouse Chr 10, 30 cM proximal of the centromere.