Figure 3.

The SKIP^WD-KLC2^TPR interface and the effect of KLC2^TPR mutations in cargo binding and cellular recruitment. (A) Details of the SKIP^WD:KLC2^TPR interface. KLC^TRP side-chains stabilizing the SKIP^WD cargo peptide (green) are shown as grey sticks emanating from the orange cartoon. Non-carbon elements are: nitrogen, blue; oxygen, red; sulfur, yellow. Hydrogen bonds are represented by dotted cyan lines. (B) Co-immunoprecipitation assay showing the effect of KLC2^TPR mutations at the SKIP^WD:KLC2^TPR interface on the interaction. (C) Fluorescence polarization measurements showing that R251D, N287L and R312E mutations in KLC2 dramatically reduce the affinity of the TPR domain for the SKIP^WD peptide. K_D values reported here were calculated at 150 mM NaCl. (D) Replacement of key KLC2^TPR residues results in loss of GFP-KLC2 association with Arl8/SKIP positive lysosomal membranes. Scale bar shows 10μm. In merge panels, GFP-KLC2, Arl8 and SKIP are shown in green, red and blue respectively.