Figure 4. KIF1C loops 6 and 10 are necessary for Rab6A binding.

(A) Binding of in vitro translated myc-KIF1A (1–361), myc-KIF1A/1C, myc-KIF1C/1A, and myc-KIF1C-350 motor domain chimera constructs to GTPγS-loaded GST-Rab6A Q72L (0.2 μM) presented as a percentage of maximal KIF1C bound (24.7% of input). (B) Crystal structure of KIF1A showing the regions swapped between KIF1A and KIF1C. The C-terminal portion is colored red. (C) Binding of in vitro translated myc-KIF1A (1–361), myc-KIF1C-350 Loop 2/3 swap, myc-KIF1C-350 Loop 6/10 swap, myc-KIF1C-350, and myc-KIF1C-350 K103A constructs to GTPγS-loaded GST-Rab6A Q72L (0.2 μM) presented as a percent of myc-KIF1C-350 bound (35.3% of input). (D) Predicted crystal structure of KIF1C with Loop 2/3 (at left), 6/10 (at right) labeled in red. The KIF1C sequence was overlaid onto the KIF1A crystal structure (PDB 2ZFI) using PHYRE2. (E) Binding of endogenous Rab6A to transiently expressed CFP-KIF1C-350, CFP-KIF1C-350 Loop 6/10 swap, and GFP immunoprecipitated with llama GFP-binding protein, as determined by anti-Rab6A immunoblot. Total endogenous Rab6A (2%) at left compared to bound (33%) at right. Below, bound CFP-KIF1C and GFP as measured by Ponceau S staining. (F) Molecular docking of Rab6A onto the predicted structure of KIF1C. The predicted structure (blue) was docked to the crystal structure of Rab6A (PDB 2Y8E, gray) using ClusPro2. The model for the largest cluster containing 144 members is shown. The switch regions of Rab6 are labeled in yellow and loop 6 and 10 of KIF1C are labeled in purple. Error bars represent SD.

DOI: http://dx.doi.org/10.7554/eLife.06029.006

Figure 4—figure supplement 1. The motor domains of KIF1A and KIF1C share 81% identity.

(A) Structure of KIF1A motor domain bound to tubulin dimer (PDB 2HXH). Loops 2, 3, 6, and 10 are highlighted. (B) Alignment of human KIF1C (top) and KIF1A (bottom) with boxes around regions corresponding to the loop mutants generated and indicated in A. The indicated break demarcates the region swapped between KIF1A and KIF1C to generate chimeric proteins.

DOI: http://dx.doi.org/10.7554/eLife.06029.007

Figure 4—figure supplement 2. Mutations in the KIF1C K-loop do not affect KIF1C loop 6/10 mutant motor domain microtubule localization.

HeLa cells transfected with the indicated CFP-KIF1C 350 construct were precipitated in MeOH and imaged by immunofluorescence microscopy. Two cells each are shown. The NRSK mutant changes ²⁹¹KKRK to ²⁹¹NRSK. The GTKT mutant changes ²⁸⁸MQSKKRKSD to ²⁸⁸GTKT, which mimics the absence of the K-loop and makes the mutant more similar to conventional kinesin, KIF5C (Okada and Hirokawa, 2000). Tubulin staining is on the left. Scale bar is 20 µm.

DOI: http://dx.doi.org/10.7554/eLife.06029.008