Adapter Proteins for Opposing Motors Interact Simultaneously with Nuclear Pore Protein Nup358

Abstract

Nup358 is a protein subunit of the nuclear pore complex that recruits the opposing microtubule motors kinesin-1 and dynein [via the dynein adaptor Bicaudal D2 (BicD2)] to the nuclear envelope. This pathway is important for positioning of the nucleus during the early steps of mitotic spindle assembly and also essential for an important process in brain development. It is unknown whether dynein and kinesin-1 interact with Nup358 simultaneously or whether they compete. Here, we have reconstituted and characterized a minimal complex of kinesin-1 light chain 2 (KLC2) and Nup358. The proteins interact through a W-acidic motif in Nup358, which is highly conserved among vertebrates but absent in insects. While Nup358 and KLC2 form predominantly monomers, their interaction results in the formation of 2:2 complexes, and the W-acidic motif is required for the oligomerization. In active motor complexes, BicD2 and KLC2 each form dimers. Notably, we show that the dynein adaptor BicD2 and KLC2 interact simultaneously with Nup358, resulting in the formation of 2:2:2 complexes. Mutation of the W-acidic motif results in the formation of 1:1:1 complexes. On the basis of our data, we propose that Nup358 recruits simultaneously one kinesin-1 motor and one dynein motor via BicD2 to the nucleus. We hypothesize that the binding sites are close enough to promote direct interactions between these motor recognition domains, which may be important for the regulation of the motility of these opposing motors. Our data provide important insights into a nuclear positioning pathway that is crucial for brain development and faithful chromosome segregation.

Coil-to-Helix Transition at the Nup358-BicD2 Interface Activates BicD2 for Dynein Recruitment

“…1b) 30 . A minimal complex was reconstituted with BicD2-CTD and a minimal fragment of human Nup358 containing residues 2148-2240, which is called Nup358min 30,47 (Fig. 1c).…”

“…Furthermore, the affinity of the monomeric cargo adaptor Nup358-min towards BicD2 is enhanced when it is dimerized by a leucine zipper, similar to what was previously observed for binding of the cargo adaptor Egalitarian for Drosophila BicD 24,25 . While Nup358-min is a monomer on its own, it oligomerizes to form 2:2 complexes with either the BicD2-CTD or the KLC2, and also forms a ternary complex with 2:2:2 stoichiometry 47,50 . We thus propose that cargo adaptor-induced dimerization may potentially be a universal feature of the activation mechanism, since BicD2 and KLC2 both form dimers in active dynein and kinesin-1 motors 21,22,47,71 .…”

“…The protein was further purified by size exclusion chromatography as described 47 , using the following buffer: 20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM TCEP. BicD2-CTD was expressed and purified as previously described 30,47,50 . In short, BicD2-CTD was purified by Ni-NTA affinity chromatography, followed by proteolytic cleavage of the His6tag by thrombin.…”

“…In addition to binding to BicD2, Nup358 also recruits the opposite polarity motor kinesin-1 via the subunit kinesin-1 light chain 2 (KLC2) which binds to a W-acidic motif with the sequence LEWD in Nup358 [46][47][48] . While dynein is the predominant motor in G2 phase, kinesin-1 is actively involved in nuclear positioning in G2 phase and modulating overall motility 11 .…”

A designed high-affinity peptide that hijacks microtubule-based transport

“…We envisage wider applications of KinTag and similarly derived peptides in the orchestration of transport of organelles, and other cargoes, to investigate the functional roles of positioning within the cell, building on the the strategy of fusing tandem and triplicate motifs to transmembrane proteins previously explored Farias et al (22), but now using a single, structurally and biophysically defined, short sequence. Moreover, it should be possible to augment binding affinity in existing motifs through amino-acid substitutions to explore the role of manipulating relative motor activity in systems where opposite polarity motors bind the same cargo or cargo adaptor (37). It is conceivable that the design of relatively low molecular weight, high-affinity, peptide or peptidomimetic ligands for kinesin-1 could be useful in the spatial (e.g., axon specific) targeting of exogenous therapeutic cargoes for the treatment of neurological disease.…”