Fig. 5.

The targeting of rapsyn to the junctional sites between lysosomal vacuoles requires the myristoylation, coiled-coil and RING-H2 domains. (A) Schematic representation of rapsyn constructs used as EGFP fusions in this study. (B) Lysates from COS cells expressing the indicated rapsyn–EGFP construct were blotted with anti-GFP or anti-rapsyn antibody and showed the corresponding proteins at expected sizes confirming the intact protein fusions. (C) C2C12 myoblasts were co-transfected with Lamp1–mCherry (a lysosomal marker), CFP-H2B (a nuclear marker) and either wild-type or the indicated rapsyn–EGFP mutant and then treated with vacuolin-1. Shown are representative images of cells transfected with Lamp1–mCherry and the indicated rapsyn–EGFP construct. Note that non-myristoylated rapsyn G2A–EGFP is concentrated in the nucleus. Rapsyn ΔCC–EGFP, rapsyn ΔRING-H2–EGFP and rapsyn ΔCC/RING-H2–EGFP are more diffuse in the cytoplasm whereas wild-type rapsyn–EGFP is concentrated at junctional sites between enlarged lysosomes. Scale bar: 10 µm.