SNAT7 is the primary lysosomal glutamine exporter required for extracellular protein-dependent growth of cancer cells
Quentin Verdon et al. PNAS, 2017
Abstract
Lysosomes degrade cellular components sequestered by autophagy or extracellular material internalized by endocytosis and phagocytosis. The macromolecule building blocks released by lysosomal hydrolysis are then exported to the cytosol by lysosomal transporters, which remain undercharacterized. In this study, we designed an in situ assay of lysosomal amino acid export based on the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis that detects lysosomal storage. This assay was used to screen candidate lysosomal transporters, leading to the identification of sodium-coupled neutral amino acid transporter 7 (SNAT7), encoded by the SLC38A7 gene, as a lysosomal transporter highly selective for glutamine and asparagine. Cell fractionation confirmed the lysosomal localization of SNAT7, and flux measurements confirmed its substrate selectivity and showed a strong activation by the lysosomal pH gradient. Interestingly, gene silencing or editing experiments revealed that SNAT7 is the primary permeation pathway for glutamine across the lysosomal membrane and it is required for growth of cancer cells in a low free-glutamine environment, when macropinocytosis and lysosomal degradation of extracellular proteins are used as an alternative source of amino acids. SNAT7 may, thus, represent a novel target for glutamine-related anticancer therapies.
Cell Culture and DNA Transfection.
Human cell lines were used in accordance with the regulations and guidelines from the French Ministry of Higher Education and Research (CODECOH). HeLa, A2780, and Mia-PaCa2 cells were grown in DMEM supplemented with 10% FBS, 50 μg·mL−1 streptomycin, and 50 U·mL−1 penicillin (“complete medium”; all products from GIBCO). They were kept in a humidified atmosphere at 37 °C with 5% CO2. For CRISPR/Cas9 genome edition and rescue experiments, HeLa and Mia-PaCa2 cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) and Lullaby (OzBiosciences), respectively, according to the manufacturers’ instructions.
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