Figure 2. Rab6A binds KIF1C at two locations.
(A) KIF1C schematic showing CBD truncation (ΔCBD, AA 1-1060), N-terminal motor domain (1–350), and a construct lacking both motor domain and CBD (ΔmotorΔCBD, AA 450-1060). (B) Binding of in vitro translated myc-KIF1CΔCBD to GTPγS-loaded GST-Rab6A Q72L (5 μM) and pulled down using glutathione Sepharose. Left, input (1%) compared to bound fraction (50%), right. (C) Binding of in vitro translated myc-KIF1C constructs to GTPγS-loaded GST-Rab6A Q72L (0.2 μM). Input (7%), left, compared to bound (50%), right. (D) Rab specificity of motor domain binding. GTPγS-preloaded GST-Rab6A Q72L, Rab9A, and Rab5A Q79L (5 μM) incubated with in vitro translated myc-KIF1C motor domain. Input, 1%, on left compared to bound fraction (48%) on right. (E) myc-KIF1C-350 binds to GST-Rab6A Q72L in a concentration-dependent manner. In vitro translated myc-KIF1C motor domain binding to GTPγS-loaded GST-Rab6A, presented as a fraction of maximal binding detected (25% of total). Data were fit using KaleidaGraph software. In vitro translation optimally yields a reaction concentration of 11.25 nM product.