Figure 5. Rab6A inhibits KIF1C microtubule co-sedimentation.

(A) Binding of full-length KIF1C to microtubules in the presence of Rab6A. In vitro synthesized ³⁵S-myc-KIF1C was desalted, incubated with GTPγS-preloaded His-tagged Rabs (4.2 μM), and then with 0.8 µg/µl Paclitaxel stabilized pre-polymerized microtubules in 2.5 mM ADP and 0.5 mM GTPγS. Reactions were centrifuged through a 10% sucrose cushion and pellets were analyzed by scintillation counting. The fraction of full-length KIF1C cosedimenting with microtubules in the presence of the indicated Rabs is shown (error bars = SE [n ≥ 2]). (B) Rab6 affects KIF1C motor domain microtubule co-sedimentation in a concentration-dependent manner. KIF1C-355-His (160 nM) was incubated with increasing concentrations of His-Rab6A Q72L, and then with 2.1 µM Paclitaxel stabilized pre-polymerized microtubules in 2.6 mM ADP and 0.35 mM GTPγS. Reactions were centrifuged through a 35% sucrose cushion. Pellets were analyzed by fluorescent antibody immunoblot. (C) Rab6A affects the strong microtubule binding state of KIF1C. Purified KIF1C-355-His (80 nM) was incubated with His-Rab6A Q72L (4.86 µM) or BSA (7.6 µM), and then with increasing concentrations of microtubules in 2.6 mM AMP-PNP and 0.35 mM GTPγS. Samples were processed and analyzed as in B. (D) Rab6A affects the weak microtubule binding state of KIF1C. Purified KIF1C-355-His (160 nM) was incubated with His-Rab6A Q72L, His-Rab33, or BSA (3.42 µM), and then with increasing concentrations of microtubules in 2.6 mM ADP and 0.35 mM GTPγS. Data were fit using GraphPad Prism software. The fraction of motor sedimented is normalized to the amount of microtubules pelleted, determined by Coomassie blue staining.

DOI: http://dx.doi.org/10.7554/eLife.06029.010

Figure 5—figure supplement 1. Rab6A inhibits KIF1C binding and motility.

KIF1C (9 nM), labeled with rabbit anti-KIF1C and Dylight 649 anti-rabbit Fab, was incubated with Rhodamine labelled-microtubules (7.5 µg/ml) adhered to coverslips with anti-β tubulin ± Rab6A (15 µM) and imaged by TIRF microscopy (on average 14 microtubules/75 s/region, 15 regions per condition quantified). (A) Images from a single frame ± Rab6A. Top panels show microtubule-localized KIF1C molecules ± His-Rab6A; bottom shows KIF1C (green) on microtubules (red). Scale, 10 μm. (B) Rab6A decreases the number of motors on microtubules normalized by microtubule length (p < 0.0001). (C) Rab6A decreases the percentage of moving motors (p < 0.0001, bars = SE). Experiments were done in series so that incubation and imaging times were controlled. Rab6A did not affect KIF1C processivity or speed.

DOI: http://dx.doi.org/10.7554/eLife.06029.011