Trafficking of the amino acid transporter B (SLC6A14) to the plasma membrane involves an exclusive interaction with SEC24C for its exit from the endoplasmic reticulum.

A plasma membrane amino acid transporter B (ATB), encoded by the SLC6A14 gene, is specific for neutral and basic amino acids. It is up-regulated in several types of malignant cancers. Neurotransmitter transporters of the SLC6 family interact with specific SEC24 proteins of the COPII complex along their pathway from the endoplasmic reticulum (ER) to Golgi. This study focused on the possible role of SEC24 proteins in ATB trafficking. Rat ATB was expressed in HEK293 cells, its localization and trafficking were examined by Western blot, deglycosylation, immunofluorescence (co-localization with ER and trans-Golgi markers) and biotinylation. The expression of ATB at the plasma membrane was decreased by dominant negative mutants of SAR1, a GTPase, whose activity triggers the formation of the COPII complex. ATB co-precipitated with SEC24C (but not with the remaining isoforms A, B and D). This interaction was confirmed by immunocytochemistry and the proximity ligation assay. Co-localization of SEC24C with endogenous ATB was also observed in MCF-7 breast cancer cells. Contrary to the endogenous transporter, part of the overexpressed ATB is directed to proteolysis, a process significantly reversed by a proteasome inhibitor bortezomib. Co-transfection with a SEC24C dominant negative mutant attenuated ATB expression at the plasma membrane, due to proteolytic degradation. These results support a hypothesis that lysine at position +2 downstream of the ER export "RI" motif on the cargo protein is crucial for SEC24C binding and for further trafficking to the Golgi. Moreover, there is an equilibrium between ER export and degradation mechanisms in case of overexpressed transporter.