Sec24- and ARFGAP1-dependent trafficking of GABA transporter-1 is a prerequisite for correct axonal targeting.

The GABA transporter-1 (GAT1) is a prototypical protein of the synaptic specialization. Export of GAT1 from the endoplasmic reticulum (ER) is contingent on its interaction with the COPII (coatomer protein-II) coat subunit Sec24D. Here we show that silencing all four Sec24 isoforms strongly inhibits transport of GAT1 to the cell surface. In contrast, transport of GAT1-RL/AS, a mutant that is deficient in Sec24D recruitment, was not inhibited, suggesting a nonconventional, COPII-independent pathway. However, ARFGAP1 bound directly to the C terminus of both GAT1-RL/AS and wild-type GAT1. Surface expression of GAT1-RL/AS involved ARFGAP1. GAT1-RL/AS appeared to bypass the ER-Golgi-intermediate compartment, but its pathway to the plasma membrane still involved passage through the Golgi. Thus, the GAT1-RL/AS mutant allowed to test whether COPII-dependent ER-export is required for correct sorting of GAT1 to the axon terminal in neuronal cells. In contrast to wild-type GAT1, GAT1-RL/AS failed to be specifically enriched at the tip of neurite extensions of CAD.a cells (a neuroblastoma cell line that can be differentiated into a neuron-like phenotype) and in the axon terminals of hippocampal neurons. These findings indicate that correct sorting to the axon is contingent on ER export via the COPII machinery and passage through the ER-Golgi-intermediate compartment.