Fig. 1.

Interactions between CLC-5 and KIF3B subunits. A: CLC-5 consists of 18 helices (A to R) with the NH₂- and COOH-terminus (C-term) domains being cytoplasmic (membrane boundaries indicated by arrows) (10, 58). B: yeast 2-hybrid using the CLC-5 C-term, which contains 2 CBS domains (11) to screen a renal cDNA library, identified KIF3B as a putative interactor. Interaction between large T and p53 provided a positive control. C: ³⁵S-methionine-labeled CLC-5 C-term was incubated with GST-tagged KIF3B coiled coil (CC)/globular domain (GD). Resin-bound proteins were resolved by SDS-PAGE and detected by autoradiography. Results from 2 independent in vitro translation experiments using 2 different GST-tagged KIF3B clones are shown. The larger band at ∼24.5 kDa represents the 216 amino acid Myc-tagged CLC-5 C-term, whereas the smaller band at ∼18.5 kDa likely represents another translated form of CLC-5 C-term that originated from use of an alternative methionine at codon 583 and hence lacked the NH₂ terminal 32 amino acids. This smaller CLC-5 C-term, which was less efficiently translated, had both CBS domains and hence was pulled down by the GST-tagged KIF3B CC/GD. D: endogenous coimmunoprecipitation using wild-type mouse whole kidney extracts and a KIF3B monoclonal antibody (mAb). KIF3B, KIF3A, and KAP3 mAb were used to confirm endogenous coimmunoprecipitation of these proteins which form a trimeric complex. It is important to note that the interaction between CLC-5 and KIF3B is likely to be transient, as endosomes will be released to continue recycling, and that only a small proportion of the total cellular CLC-5 will be present, at a given time point, on the surface of endosomes that are being trafficked anterogradely; these features are consistent with the relatively low amount of KIF3B that is immunopreciptiated by CLC-5. E: coimmunoprecipitation studies using HEK293 cells demonstrated interactions between CLC-5 and KIF3B domains. Lane 1: input; lanes 2, 3, and 4: pull-down with no antibody, anti-Myc, or anti-HA antibodies, respectively. F: summary of KIF3B domains showing presence (+) or absence (−) of interactions with CLC-5. G: coimmunoprecipitation studies using HEK293 cells demonstrated interactions between CLC-4 and KIF3B, and CLC-7 and KIF3B. Lane 1: input; lanes 2, 3, and 4: pull-down with no antibody, anti-Myc, or anti-HA antibodies, respectively.