Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Sep 30;202(7):1107-22.
doi: 10.1083/jcb.201307084.

Recruitment of folliculin to lysosomes supports the amino acid-dependent activation of Rag GTPases

Constance S Petit  1 , Agnes Roczniak-Ferguson, Shawn M Ferguson
Affiliations

Recruitment of folliculin to lysosomes supports the amino acid-dependent activation of Rag GTPases

Constance S Petit et al. J Cell Biol. .

Abstract

Birt-Hogg-Dubé syndrome, a human disease characterized by fibrofolliculomas (hair follicle tumors) as well as a strong predisposition toward the development of pneumothorax, pulmonary cysts, and renal carcinoma, arises from loss-of-function mutations in the folliculin (FLCN) gene. In this study, we show that FLCN regulates lysosome function by promoting the mTORC1-dependent phosphorylation and cytoplasmic sequestration of transcription factor EB (TFEB). Our results indicate that FLCN is specifically required for the amino acid-stimulated recruitment of mTORC1 to lysosomes by Rag GTPases. We further demonstrated that FLCN itself was selectively recruited to the surface of lysosomes after amino acid depletion and directly bound to RagA via its GTPase domain. FLCN-interacting protein 1 (FNIP1) promotes both the lysosome recruitment and Rag interactions of FLCN. These new findings define the lysosome as a site of action for FLCN and indicate a critical role for FLCN in the amino acid-dependent activation of mTOR via its direct interaction with the RagA/B GTPases.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
FLCN controls TFEB-dependent regulation of lysosomes. (A) Spinning disk confocal imaging of TFEB-GFP localization ± FLCN knockdown. (B) Quantification of the effect of FLCN knockdown on the nuclear/cytoplasmic ratio of TFEB-GFP (n = 3 experiments, mean ± SEM; *, P < 0.05 by ANOVA with Bonferroni post-test). (C) Representative Western blots of anti-GFP immunoprecipitations from a stable TFEB-GFP HeLa cell line ± FLCN knockdown (n = 3). (D) Lysotracker labeling of HeLa cells transfected with the indicated siRNAs or after treatment with torin 1 (250 nM, 24 h). (E) Quantification of Lysotracker staining (n = 3 experiments, ANOVA with Bonferroni post-test, mean ± SEM; **, P < 0.01; ***, P < 0.001). Bars: (A and D) 10 µm. All images acquired by spinning disk confocal microscopy.
Figure 2.
Figure 2.
FLCN regulates mTORC1 activity and localization. (A) Western blot analysis of the phosphorylation of reporters of mTORC1 activity ± FLCN siRNA. (B and C) Quantification of the effects of FLCN siRNA on S6K1 (T389) and S6 (S235/236) phosphorylation (mean ± SEM; ***, P < 0.001, ANOVA with Bonferroni post-test, n = 3–4 experiments). (D) Representative immunofluorescence images showing the localization of mTOR ± FLCN siRNA (line-scanning confocal imaging, n = 3; bar, 10 µm). (E) Representative immunofluorescence images showing the localization of LAMP1 and RagC in cells transfected with control vs. FLCN siRNA. Bars: (D and E) 10 µm; (inset) 5 µm. (F) Representative Western blots and quantification (G) showing the effects of FLCN knockdown on starvation (2 h in serum and amino acid–free RPMI) and amino acid–dependent stimulation (20 min after addition of 1× MEM amino acid supplement) of S6 phosphorylation (S235/236, mean ± SEM; ***, P < 0.001, ANOVA with Bonferroni post-test, n = 3 experiments). All experiments were performed in HeLa cells.
Figure 3.
Figure 3.
FLCN and FNIP1 colocalize on lysosomes. (A) Localization of FLCN-tdTomato (red, diffuse) and LAMP1-mGFP (green, lysosomes). (B) FLCN-interacting protein 1 (FNIP1)-GFP (green) localizes to Alexa 647 dextran-positive (magenta) lysosomes. (C) Cells cotransfected with FLCN-tdTomato (red) and FNIP1-GFP (green) whose lysosomes were loaded with Alexa 647 dextran (magenta). (D) FLCN-tdTomato and FNIP1-GFP colocalization clearly occurs on the surface of the enlarged endosomes/lysosomes that form in vacuolin-treated (5 µM, 1 h) cells. All images were obtained by spinning disk confocal microscopy of live HeLa cells. Bars: (A–D) 10 µm; (insets) 5 µm.
Figure 4.
Figure 4.
Regulation of the lysosomal recruitment of FLCN by amino acid availability and FNIP1. (A) Western blot analysis of the 3xHA-tagged endogenous FLCN and phosphorylated S6 protein ± FLCN knockdown. (B) Representative immunofluorescence experiment showing the effect of FLCN knockdown on the levels of endogenous 3xHA-FLCN. (C) Representative images showing the effect of starvation (2 h serum and amino acid–free RPMI) and amino acid re-feeding (1× MEM amino acid solution, 15 min) on 3xHA-tagged endogenous FLCN and LAMP1 in control conditions or (D) after FNIP1 knockdown. All images obtained by line scanning confocal microscopy. Bars: (B–D) 10 µm; (inset) 5 µm. All experiments were performed in HeLa cells.
Figure 5.
Figure 5.
FLCN selectively interacts with inactive Rag GTPases. (A) Representative immunoblots showing that endogenous FLCN co-purifies with all possible combinations of transfected HA-GST–tagged Rag GTPase heterodimers (n = 4). (B) Summary of the key properties of the Rag GTPase mutants used in our study. (C) Immunoblot analysis of GST pull-downs from cells transfected with either HA-GST tagged wild-type RagA+C or the indicated combinations of the constitutively active (RagA Q66L = RagAGTP + RagC S75L = RagCGDP/nucleotide free) and dominant-negative (RagA T21L = RagAGDP/nucleotide free + RagC Q120L = RagCGTP) mutants. (D) Quantification of the interaction between FLCN and the indicated Rags. The FLCN signal in each pull-down was normalized to the corresponding abundance of RagC in the same pull-down sample (mean ± SEM; *, P < 0.05, ANOVA with Bonferroni post-test, n = 4). (E) Immunoblot analysis of GST pull-downs from cells transfected with the indicated combinations of wild-type or dominant-negative recombinant Rag A and C. (F) Effect of starvation (2 h in Earle’s buffered saline solution) on the interaction between endogenous FLCN and wild-type HA-GST-RagA+C (n = 3). (G) Immunoblot analysis of GST pull-downs from cells transfected with wild-type HA-GST-RagAnucleotide-free+C along with control, FLCN, or FNIP1 siRNA. All experiments were performed in HeLa cells.
Figure 6.
Figure 6.
FLCN binds directly to the GTPase domain of RagA. Left: in vitro binding assay where GST-FLCN was immobilized on GSH-beads and the binding of the indicated 6xHis-tagged-proteins (top) was detected by anti-6xHis immunoblotting. Equal loading of GST-FLCN was verified via anti-GST immunoblotting (bottom). Right: Ponceau S staining showing the migration of the various recombinant 6xHis-tagged proteins used in the in vitro binding assay.
Figure 7.
Figure 7.
Constitutively active Rag GTPase mutants rescue the effects of FLCN KD on TFEB and mTOR localization. (A) Immunofluorescence analysis of TFEB-GFP and HA-GST-Rag GTPase localization in control or FLCN KD cells cotransfected with either wild-type RagB+D or constitutively active (CA) Rag B+D (RagB Q99L + RagD S77L) heterodimers. Dashed lines highlight the Rag-transfected cells. (B) Quantification of the fraction of cells exhibiting predominantly nuclear (nuclear ≥ cytoplasmic signal intensity) TFEB-GFP localization after cotransfection of either control siRNA or FLCN knockdown cells with the indicated Rag GTPases (mean ± SEM, n = 3 experiments with >20 cells/condition/experiment). (C) Immunofluorescence analysis of mTOR localization in control or FLCN KD cells cotransfected with either wild-type RagB+D or CA RagB+D heterodimers. All images obtained by line scanning confocal imaging. Bars, 10 µm. All experiments were performed in HeLa cells.
Figure 8.
Figure 8.
Schematic diagram summarizing the proposed lysosome-localized role for FLCN and FNIP1 in the amino acid-dependent regulation of Rag GTPases, mTORC1 and TFEB.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Allaire P.D., Marat A.L., Dall’Armi C., Di Paolo G., McPherson P.S., Ritter B. 2010. The Connecdenn DENN domain: a GEF for Rab35 mediating cargo-specific exit from early endosomes. Mol. Cell. 37:370–382 10.1016/j.molcel.2009.12.037 - DOI - PMC - PubMed
    1. Baba M., Hong S.B., Sharma N., Warren M.B., Nickerson M.L., Iwamatsu A., Esposito D., Gillette W.K., Hopkins R.F., III, Hartley J.L., et al. 2006. Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proc. Natl. Acad. Sci. USA. 103:15552–15557 10.1073/pnas.0603781103 - DOI - PMC - PubMed
    1. Bar-Peled L., Schweitzer L.D., Zoncu R., Sabatini D.M. 2012. Ragulator is a GEF for the rag GTPases that signal amino acid levels to mTORC1. Cell. 150:1196–1208 10.1016/j.cell.2012.07.032 - DOI - PMC - PubMed
    1. Bar-Peled L., Chantranupong L., Cherniack A.D., Chen W.W., Ottina K.A., Grabiner B.C., Spear E.D., Carter S.L., Meyerson M., Sabatini D.M. 2013. A Tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Science. 340:1100–1106 10.1126/science.1232044 - DOI - PMC - PubMed
    1. Betschinger J., Nichols J., Dietmann S., Corrin P.D., Paddison P.J., Smith A. 2013. Exit from pluripotency is gated by intracellular redistribution of the bHLH transcription factor Tfe3. Cell. 153:335–347 10.1016/j.cell.2013.03.012 - DOI - PMC - PubMed

Publication types

MeSH terms