Abstract
GTRAP3-18 interacts with and reduces the activity of the neuronal specific Na(+)/K(+) glutamate transporter, EAAC1 both in vitro and in vivo. GTRAP3-18 and the related isoform, JM4, are distant relatives of the Rab GTPase-interacting factor PRA1, and share a topology of four transmembrane domains and cytosolic termini. GTRAP3-18 and JM4 are resident endoplasmic reticulum (ER) proteins. The physiological role of GTRAP3-18 is poorly understood. We demonstrate for the first time that GTRAP3-18 is a regulator of ER protein trafficking. Expression of GTRAP3-18 delays the ER exit of EAAC1, as well as other members of the excitatory amino acid transporter family. GTRAP3-18 uses hydrophobic domain interactions in the ER membrane to self-associate and cytoplasmic interactions at the C terminus to regulate trafficking. The features of GTRAP3-18 activity are consistent with recent phylogenic sequence analyses suggesting GTRAP3-18 and JM4 be reclassified as mammalian isoforms of the yeast protein family Yip, Yip6b, and Yip6a, respectively.
Highlights
We identified GTRAP3-183, a 22-kDa protein that is dynamically induced by retinoic acid both in vitro and in vivo and inhibits the activity of EAAC1 in a dose-dependent manner (GeneID 66028) [1]
GTRAP3-18 Prevents Complex Oligosaccharide Formation on EAAC1 in a Dose-dependent Manner—EAAC1 and all members of the EAAT/ASCT gene family are DNA precipitate was added to the cells
Our group identified GTRAP3-18, a protein that is dynamically induced by retinoic acid both in vitro and in vivo and inhibits the activity of EAAC1 in a dose dependent manner [1]
Summary
Antibodies—Rabbit and chicken polyclonal anti-GTRAP3-18 antibodies were raised against peptide sequences from the N and C termini of the protein sequence: N1-GTRAP3-18: KFFPGSDRFARPDFRD,C1-GTRAP3-18: KEQQEDSINKFADYIS, JOURNAL OF BIOLOGICAL CHEMISTRY 6175. Transfection of GTRAP3-18 into mammalian cell lines reconstitutes a level of protein expression found in mouse cortical tissue (lane 2). In the co-transfected EAAC1 and GTRAP3-18 cell lysate (lanes 4 – 6), all the EAAC1 protein is sensitive to both Endo H (lane 2) and PNGase F (lane 3), as EAAC1 is predominately high mannose oligosaccharide. Western blot analysis was performed using c-Myc and HA monoclonal and EAAC1 polyclonal antibodies. G, co-transfection of EAAC1 with a 1ϫ molar ratio of GTRAP3-18 (lanes 4 – 6) decreases expression of the complex oligosaccharide form (see arrows) and reduces cell surface expression (lane 6) compared with EAAC1 (lanes 1–3). All experiments were performed with a minimum n ϭ 3 replicates
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