Golvesin-GFP fusions as distinct markers for Golgi and post-Golgi vesiclesin Dictyostelium cells

Golvesin is a new protein associated with membranes of the Golgi apparatus and post-Golgi vesicles in Dictyostelium cells. An internal hydrophobic seq uence of 24 amino-acid residues is responsible for anchoring golvesin to th e membranes of these organelles. In an attempt to visualize organelle dynam ics in vivo, we have used specific antibody and other labels to localize go lvesin-green fluorescent protein (GFP) constructs to different cellular com partments. With a GFP tag at its N-terminus, golvesin shows the same locali zation as the untagged protein. It is transferred to two post-Golgi compart ments, the endosomal and contractile vacuole systems. Endosomes are decorat ed with GFP-golvesin within less than 10 min of their internalisation, and keep the label during the acidic phase of the pathway. Blockage of the C-terminus with GFP causes entrapment of the protein in the Golgi apparatus, indicating that a free C-terminus is required for transfe r of golvesin to any of the post-Golgi compartments. The C-terminally tagge d golvesin proved to be a reliable Golgi marker in Dictyostelium cells reve aling protrusion of Golgi tubules at peak velocities of 3 to 4 mum.s(-1). T he fusion protein is retained in Golgi vesicles during mitosis, visualizing Golgi disassembly and reorganization in line with cytokinesis. (C) 2000 Ed itions scientifiques ct medicales Elsevier SAS.