A GC-MS/Single-Cell Method to Evaluate Membrane Transporter Substrate Specificity and Signaling
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 huma...
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Frontiers Media S.A.
2021-04-01
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| Series: | Frontiers in Molecular Biosciences |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmolb.2021.646574/full |
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| author | Stephen J. Fairweather Stephen J. Fairweather Shoko Okada Gregory Gauthier-Coles Kiran Javed Angelika Bröer Stefan Bröer |
| author_facet | Stephen J. Fairweather Stephen J. Fairweather Shoko Okada Gregory Gauthier-Coles Kiran Javed Angelika Bröer Stefan Bröer |
| author_sort | Stephen J. Fairweather |
| collection | DOAJ |
| description | 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 ATB0,+ 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 Xenopus laevis oocytes can be used to test their substrate specificity and role in intracellular signaling pathways. |
| first_indexed | 2024-12-13T12:01:09Z |
| format | Article |
| id | doaj.art-0def0be3c75442ab837a5efc68a8c8be |
| institution | Directory Open Access Journal |
| issn | 2296-889X |
| language | English |
| last_indexed | 2024-12-13T12:01:09Z |
| publishDate | 2021-04-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Molecular Biosciences |
| spelling | doaj.art-0def0be3c75442ab837a5efc68a8c8be2022-12-21T23:47:05ZengFrontiers Media S.A.Frontiers in Molecular Biosciences2296-889X2021-04-01810.3389/fmolb.2021.646574646574A GC-MS/Single-Cell Method to Evaluate Membrane Transporter Substrate Specificity and SignalingStephen J. Fairweather0Stephen J. Fairweather1Shoko Okada2Gregory Gauthier-Coles3Kiran Javed4Angelika Bröer5Stefan Bröer6Research School of Biology, Australian National University, Canberra, ACT, AustraliaResearch School of Chemistry, Australian National University, Canberra, ACT, AustraliaCommonwealth Scientific and Industrial Research Institute (CSIRO) Land and Water, Canberra, ACT, AustraliaResearch School of Biology, Australian National University, Canberra, ACT, AustraliaResearch School of Biology, Australian National University, Canberra, ACT, AustraliaResearch School of Biology, Australian National University, Canberra, ACT, AustraliaResearch School of Biology, Australian National University, Canberra, ACT, AustraliaAmino 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 ATB0,+ 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 Xenopus laevis oocytes can be used to test their substrate specificity and role in intracellular signaling pathways.https://www.frontiersin.org/articles/10.3389/fmolb.2021.646574/fullmTORC1 signalingSNAT2slc38a2GC-MSmetabolomicsXenopus laevis oocytes |
| spellingShingle | Stephen J. Fairweather Stephen J. Fairweather Shoko Okada Gregory Gauthier-Coles Kiran Javed Angelika Bröer Stefan Bröer A GC-MS/Single-Cell Method to Evaluate Membrane Transporter Substrate Specificity and Signaling Frontiers in Molecular Biosciences mTORC1 signaling SNAT2 slc38a2 GC-MS metabolomics Xenopus laevis oocytes |
| title | A GC-MS/Single-Cell Method to Evaluate Membrane Transporter Substrate Specificity and Signaling |
| title_full | A GC-MS/Single-Cell Method to Evaluate Membrane Transporter Substrate Specificity and Signaling |
| title_fullStr | A GC-MS/Single-Cell Method to Evaluate Membrane Transporter Substrate Specificity and Signaling |
| title_full_unstemmed | A GC-MS/Single-Cell Method to Evaluate Membrane Transporter Substrate Specificity and Signaling |
| title_short | A GC-MS/Single-Cell Method to Evaluate Membrane Transporter Substrate Specificity and Signaling |
| title_sort | gc ms single cell method to evaluate membrane transporter substrate specificity and signaling |
| topic | mTORC1 signaling SNAT2 slc38a2 GC-MS metabolomics Xenopus laevis oocytes |
| url | https://www.frontiersin.org/articles/10.3389/fmolb.2021.646574/full |
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