A GC-MS/Single-Cell Method to Evaluate Membrane Transporter Substrate Specificity and Signaling

Stephen J Fairweather; Shoko Okada; Gregory Gauthier-Coles; Kiran Javed; Angelika Bröer; Stefan Bröer

doi:10.3389/fmolb.2021.646574

A GC-MS/Single-Cell Method to Evaluate Membrane Transporter Substrate Specificity and Signaling

Front Mol Biosci. 2021 Apr 13:8:646574. doi: 10.3389/fmolb.2021.646574. eCollection 2021.

Authors

Stephen J Fairweather^{1

2}, Shoko Okada³, Gregory Gauthier-Coles¹, Kiran Javed¹, Angelika Bröer¹, Stefan Bröer¹

Affiliations

¹ Research School of Biology, Australian National University, Canberra, ACT, Australia.
² Research School of Chemistry, Australian National University, Canberra, ACT, Australia.
³ Commonwealth Scientific and Industrial Research Institute (CSIRO) Land and Water, Canberra, ACT, Australia.

Abstract

Amino acid transporters play a vital role in metabolism and nutrient signaling pathways. Typically, transport activity is investigated using single substrates and competing amounts of other amino acids. We used GC-MS and LC-MS for metabolic screening of Xenopus laevis oocytes expressing various human amino acid transporters incubated in complex media to establish their comprehensive substrate profiles. For most transporters, amino acid selectivity matched reported substrate profiles. However, we could not detect substantial accumulation of cationic amino acids by SNAT4 and ATB^0,+ in contrast to previous reports. In addition, comparative substrate profiles of two related sodium neutral amino acid transporters known as SNAT1 and SNAT2, revealed the latter as a significant leucine accumulator. As a consequence, SNAT2, but not SNAT1, was shown to be an effective activator of the eukaryotic cellular growth regulator mTORC1. We propose, that metabolomic profiling of membrane transporters in Xe nopus laevis oocytes can be used to test their substrate specificity and role in intracellular signaling pathways.

Keywords: GC-MS; SNAT2; Xenopus laevis oocytes; amino acid signaling; amino acid transporters; mTORC1 signaling; metabolomics; slc38a2.