Abstract
The Ran-binding protein 2 (RanBP2) is a vertebrate mosaic protein composed of four interspersed RanGTPase binding domains (RBDs), a variable and species-specific 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 microtubule-based 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.
Highlights
The zinc finger cluster domain (ZnF) of Ran-binding protein 2 (RanBP2) is flanked by the RBD1 and RBD2, suggesting that upon nuclear exiting and docking of exportin-1-RanGTPcargo complexes to ZnF [48], one or more of the neighboring RanBDs may promote the disassembly of nuclear complexes via RanGTP hydrolysis
In light of the extremely high homology between KIF5C and the kinesin heavy chain (KHC)-related members, KIF5A [56, 60] and KIF5B [61, 62] (Fig. 2 of Supplemental Material), and potential heterodimerization between these members, we searched for tryptic peptide masses unique to these kinesin members by tandem mass spectrometry of the larger pool of p120 digested with trypsin
10 and 9 masses were unique to KIF5B and KIF5C, respectively, and none could be uniquely matched to KIF5A (Fig. 1 of Supplemental Material)
Summary
RanBP1, Ran-binding protein 1; RanBP2, Ran-binding protein 2; RBD, RanGTPase binding domain(s); CLD, cyclophilin-like domains; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; ZnF, zinc finger cluster domain; Ab, antibody; mAb, monoclonal antibody; GTP␥S, guanosine 5Ј-3-O-(thio)triphosphate; KHC, kinesin heavy chain; KLC, kinesin light chain.
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