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. 2022 Sep 14;8(37):eadd2926. doi: 10.1126/sciadv.add2926

Fig. 4. Hydrophobic residues in the FLCN:RagC interface necessary for GAP activity.

Fig. 4.

(A) RagC (blue) and the FLCN (orange):FNIP2 (green) interface in AFC. FLCN:FNIP2 residues mutated at the interface indicated (red). (B) Tryptophan fluorescence xanthosine triphosphatase (XTPase) assay with FLCN:FNIP2 mutants. FLCNWT:FNIP2 (gray), FLCNF10D:FNIP2 (orange), or FLCN:FNIP2V146E (green) was incubated with RagAGDP:RagCXTP. Plotted are means ± SEM. n = 3 replicates. (C) Immunofluorescence images of human embryonic kidney (HEK) 293T cells stably expressing short hairpin RNAs targeting the indicated genes and FLCN rescue constructs. Cells were starved for amino acids (−AA) or restimulated with complete Dulbecco’s modified essential medium (+AA). (D) Quantification of TFE3 nuclear localization for immunofluorescence images and the positive control FLCN164 identified previously (8) shown in fig. S8 (means ± SD, n = 10 fields). ****P < 0.0001. (E) Fluorescence images of HEK293A cells expressing the indicated FLCN:eGFP construct along with HA-FNIP2 and the lysosomal marker Lysosomal associated membrane protein-2 (LAMP2):RFP. Cells were starved for amino acids (−AA) or starved and then restimulated with amino acids for 15 min (+AA). (F) Quantification of the lysosomal localization of overexpressed FLCN for immunofluorescence images (means ± SD, n = 15 cells). **P < 0.01. (G) Immunofluorescence images of HEK293T cells stably overexpressing the FNIP2WT-eGFP-V5 or FNIP2V146E-eGFP-V5 constructs. Cells were starved for amino acids (−AA) or maintained in complete media (+AA). (H) Quantification of TFE3 nuclear localization for immunofluorescence images (means ± SD, n = 10 fields). ****P < 0.0001. (I) Immunofluorescence images of HEK293T cells stably overexpressing the indicated FNIP2-eGFP-V5 constructs. LAMP2 was used as a lysosomal marker. Cells were starved for amino acids (−AA) or maintained in complete media (+AA). (J) Quantification of the lysosomal localization of overexpressed FNIP2 for immunofluorescence images (means ± SD, n = 15 cells). ***P < 0.001. DAPI, 4′,6-diamidino-2-phenylindole.