Membrane Topology of Trafficking Regulator of GLUT4 1 (TRARG1)

Abstract

Trafficking regulator of GLUT4 1 (TRARG1) was recently identified to localize to glucose transporter type 4 (GLUT4) storage vesicles (GSVs) and to positively regulate GLUT4 trafficking. Our knowledge of TRARG1 structure and membrane topology is limited to predictive models, hampering efforts to further our mechanistic understanding of how it carries out its functions. Here, we use a combination of bioinformatics prediction tools and biochemical assays to define the membrane topology of the 173-amino acid mouse TRARG1. These analyses revealed that, contrary to the consensus prediction, the N-terminus is cytosolic and that a short segment at the C-terminus resides in the luminal/extracellular space. Based on our biochemical analyses including membrane association and antibody accessibility assays, we conclude that TRARG1 has one transmembrane domain (TMD) (145-172) and a re-entrant loop between residues 101 and 127.

Serine 474 phosphorylation is essential for maximal Akt2 kinase activity in adipocytes

“…Cells were placed on ice, washed with cold PBS, and harvested in cold HES buffer (20 mM HEPES, 1 mM EDTA, and 250 mM sucrose (pH 7.4)) containing phosphatase and protease inhibitors. All subsequent steps were carried out at 4°C as described previously (67). Briefly, cells were homogenized using a Dounce tissue grinder prior to centrifugation at 500 ϫ g for 10 min.…”

Trafficking regulator of GLUT4-1 (TRARG1) is a GSK3 substrate

“…As part of our previous characterization of TRARG1 topology [26], we revealed that the Nterminal cytosolic domain of TRARG1 (1-100; del_101-173) does not exhibit reduced migration by SDS-PAGE, suggesting that this truncation mutant is not phosphorylated (Fig. S3A-B).…”

“…1D for details of Ser/Thr/Tyr, Lys and Cys mutants used in this study). TRARG1 truncation mutants pcDNA3.1-HA-TRARG1-del_129-173-eGFP and pcDNA3.1-HA-TRARG1-del_101-173-eGFP were generated as previously described [26]. Residues 101-127 were deleted from pcDNA3.1-HA-TRARG1-eGFP to yield the plasmid pcDNA3.1-HA-TRARG1-del_101-127-eGFP, using overlap-extension site-directed mutagenesis.…”

“…All subsequent steps were carried out at 4 °C. Cells were fractionated as previously described [26]. Briefly, cells were homogenized by passing through a 22-gauge needle 5 times and a 27-gauge needle 10 times prior to centrifugation at 500 x g for 10 min to pellet cell debris.…”

“…Using overlap-extension site-directed mutagenesis [47], we mutated all the Cys in HA-TRARG1 sequence into Ser, which resulted in the construct pcDNA3.1-HA-TRARG1-C-S. Using Gibson assembly cloning, we mutated the first four Lys at the N-terminus of HA-TRARG1 into Arg, followed by the mutagenesis of the last three Lys into Arg using overlap-extension site-directed mutagenesis, the resulting construct was designated as pcDNA3.1-HA-TRARG1-K-R. TRARG1 truncation mutants pcDNA3.1-HA-TRARG1-del_129-173-eGFP and pcDNA3.1-HA-TRARG1-del_101-173-eGFP were generated as previously described [26]. Residues 101-127 were deleted from pcDNA3.1-HA-TRARG1-eGFP to yield the plasmid pcDNA3.1-HA-TRARG1-del_101-127-eGFP, using overlap-extension site-directed mutagenesis.…”