Supplementary Fig. 8: Results of knockouts of GPR137B and of its isoform GPR137 in human cells and of gpr137ba knockout in zebrafish.

a, Relative fold change of mTOR translocation in response to amino acids is not reduced in HAP1 GPR137B-KO and GPR137-KO cells compared to parental control. Mean ± s.d. is plotted for n = 3 independent experiments. b, Table showing the per cent amino acid sequence identity between zebrafish Gpr137 homologues and human GPR137B; Gpr137ba shows the highest sequence similarity to the human GPR137B homologue. c, Amino acid alignment of zebrafish Gpr137ba and human GPR137B showing a high degree of conservation between the two sequences. d, Sequence validation of gpr137ba zebrafish generated by TALEN. e, Wild-type and gpr137ba-mutant zebrafish are viable; images represent whole-fish mounts of the indicated genotypes at 5 dpf (not selected for gender). Scale bar, 50 μm. Images are representative of at least three independent experiments. f, Immunoblot on whole zebrafish tissue shows that gpr137ba^−/− zebrafish do not have increased LC3B or p62 levels or reduced phosphorylation of S6. Images are representative of three independent experiments. g, The presence and absence of homologues of GPR137, mTOR, Rags and Ragulator in analysed eukaryotic species (n = 179) that have, or do not have, homologues of human GPR137B using a previously described algorithm⁵³. A hand-drawn illustrative human-centric species tree is shown; species that have GPR137B homologues are highlighted. GPR137B homologues are present in all vertebrates examined and in many invertebrates, as well as in eukaryotic species as divergent as D. discoideum and Monosiga brevicollis, but not in more ancestral plants and protists. GPR137B homologues are also not found in fungi, Caenorhabditis elegans and Drosophila model systems.