Contribution of both Golgi binding domains to the localisation of GMAP210. (A) Schematic representation of the full-length GMAP210 protein and of the truncated mutants used in this experiment. The ALPS-like motif is represented in red, coiled-coil regions in light blue, and the predicted GRAB domain in green. Western blot of all of these constructs expressed in RPE1 cells is presented in (F). (B) Cells expressing the mutant lacking the N-terminal domain (GFP-GMAPΔN (251–1979 aas)) were stained for GM130 (B2) and Golgin245 (B3). Insets show enlarged views. Fluorescence intensity profiles at the bottom revealed perfect co-localisation of this mutant with the cis-Golgi marker GM130. (C) Cells transfected with the mutant lacking the C-terminal domain (GFP-GMAPΔC (1–1778 aas)) were processed as above. Although the mutant mostly associated with the cis-side of the GA, it was also present in areas that did not contain GM130. In addition, it also accumulates in some tubular and vesicular structures devoid of GM130. (D, E) The central coiled-coil domains do not contain Golgi targeting information. Cells expressing the GFP-GMAPCC1 (D) or GFP-GMAPCC2 fusion proteins and labelled for GM130 are shown. Bars = 5 μm.