GAP-43 traffics from Golgi to plasma membrane by a Brefeldin A-insensitive exocytic pathway

TRENCHI ALEJANDRA; GOMEZ, GUILLERMO A. AND DANIOTTI JOSE L.; GOMEZ, GUILLERMO A. DANIOTTI JOSE L.; GOMEZ, GUILLERMO A. AND DANIOTTI JOSE L.

Rosario, Santa Fé, Argentina.

Congreso; XLII Reunión Anual de la Sociedad Argentina de investigación en Bioquímica y Biología Molecular; 2006

Sociedad Argentina de investigación en Bioquímica y Biología Molecular

GAP-43 is a dually palmitoylated (C3,4) protein that mostly localizes in plasma membrane (PM) and is enriched in growth cones in developing neurons. To elucidate mechanisms for intracellular transport of GAP-43, the N-terminal domain (N13GAP-43) and the full length sequence of GAP-43 (GAP-43 Full) were fused to GFP. Biochemical experiments demonstrated the membrane association of these constructs expressed in CHO-K1 cells. Using confocal microscopy analysis we found that at steady state N13GAP-43 and GAP-43 Full are associated with the recycling endosome and TGN in addition to PM. By selective photobleaching we demonstrated the dynamic nature of N13GAP-43 association with PM and endomembranes. Depalmitoylation of GAP-43 by treatment with an inhibitor of protein palmitoylation drastically reduced its TGN and PM localization. In addition, a double mutant of GAP-43 (C3,4S) displayed a cytosolic diffuse pattern. In synchronized experiments of protein expression we found that a fraction of newly synthesized N13GAP-43 localized at the TGN. It was also demonstrated by using live cell confocal microscopy that N13GAP-43, but not a GPI-anchored protein, trafficked from Golgi to PM by a BFA-insensitive pathway. Together, these results suggest that newly synthesized GAP-43 is acylated early in the secretory pathway and then transported to PM by a mechanism independent of clathrin-coated vesicles.