(A) Scheme of the constructs. The first construct corresponds to the N-terminal region of GMAP-210 (aa 1–375; ALPS = 1–38) fused to a C-ter fluorophore (GFP or mCherry). Alternatively, the ALPS motif of GMAP-210 was introduced upstream of an artificial coiled-coil (ACC1). (B) Subcellular localization of GCC- and ACC-based constructs by confocal fluorescence microscopy. Golgi localization was preserved by replacing the coiled-coil region of GMAP-210 (GCC) by an artificial coiled-coil (ACC1), but was abolished by deletion of the ALPS motif. Immunofluorescence of cells expressing ALPS-ACC1-GFP shows low colocalization with TGN46, intermediate overlap with GM130, and high colocalization with endogenous GMAP-210. All images were acquired with a confocal microscope and were analyzed using Volocity software. Scale bars: 10 µm, Z plane. (C) Immunogold EM of RPE1 cells expressing ALPS-ACC1-GFP. Scale bars: 10 µm (B) 100 nm (C). These experiments were repeated at least three times, except EM, which was repeated twice. Data show representative images.