The growth-associated protein-43 (GAP-43) is a dually acylated protein at cysteines 3 and 4 and contains a polybasic domain (aa 32-55) which is able to bind anionic lipids. The aim of this study was to investigate if the polybasic domain modulates the acylation state of GAP-43. Using biochemical assays and live cell fluorescence microscopy, it was demonstrated that after synthesized in the cytosol, GAP-43 is acylated at the trans-Golgi network (TGN), which is necessary for its stable association with membranes. By comparing N-terminal region (aa 1-13, N13GAP-43) and full-length GAP-43 (GAP-43full), it was found that basic residues of GAP-43 could be modulating both its adsorption to TGN and palmitoyl-acyl transferase accessibility. We speculated that the polybasic domain from GAP-43 could be electrostatically interacting with PIP4, an anionic phospholipid highly concentrated at the TGN through an ADP-ribosylation factor 1 (Arf1)-mediated process. In this sense, we observed that Arf1 inhibition did not affect acylation and TGN and plasma membrane association of N13GAP-43 but severely affected subcellular distribution of GAP-43full and its adsorption to TGN, thereby increasing the cytosolic pool of this protein. We conclude that Arf1 activity, through its effector PI4P, is required for TGN adsorption of GAP-43full via its polybasic domain.
