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. 2012 Jun 12;5(228):ra42.
doi: 10.1126/scisignal.2002790.

The transcription factor TFEB links mTORC1 signaling to transcriptional control of lysosome homeostasis

Agnes Roczniak-Ferguson  1 , Constance S Petit, Florian Froehlich, Sharon Qian, Jennifer Ky, Brittany Angarola, Tobias C Walther, Shawn M Ferguson
Affiliations

The transcription factor TFEB links mTORC1 signaling to transcriptional control of lysosome homeostasis

Agnes Roczniak-Ferguson et al. Sci Signal. .

Abstract

Lysosomes are the major cellular site for clearance of defective organelles and digestion of internalized material. Demand on lysosomal capacity can vary greatly, and lysosomal function must be adjusted to maintain cellular homeostasis. Here, we identified an interaction between the lysosome-localized mechanistic target of rapamycin complex 1 (mTORC1) and the transcription factor TFEB (transcription factor EB), which promotes lysosome biogenesis. When lysosomal activity was adequate, mTOR-dependent phosphorylation of TFEB on Ser(211) triggered the binding of 14-3-3 proteins to TFEB, resulting in retention of the transcription factor in the cytoplasm. Inhibition of lysosomal function reduced the mTOR-dependent phosphorylation of TFEB, resulting in diminished interactions between TFEB and 14-3-3 proteins and the translocation of TFEB into the nucleus, where it could stimulate genes involved in lysosomal biogenesis. These results identify TFEB as a target of mTOR and suggest a mechanism for matching the transcriptional regulation of genes encoding proteins of autophagosomes and lysosomes to cellular need. The closely related transcription factors MITF (microphthalmia transcription factor) and TFE3 (transcription factor E3) also localized to lysosomes and accumulated in the nucleus when lysosome function was inhibited, thus broadening the range of physiological contexts under which this regulatory mechanism may prove important.

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Figures

Fig. 1
Fig. 1
TFEB localizes to lysosomes and accumulates in the nucleus in response to perturbation of lysosomal function. (A) Live imaging (spinning disk confocal) of TFEB-GFP (green) and DQ-BSA (red, lysosomal marker) in HeLa M cells shows an enrichment of the TFEB-GFP signal on lysosomes. Insets show higher magnification views. (B) TFEB-GFP localization without (left) or with chloroquine (CQ, 50μM, 15 hours) treatment (right). (C) Percentage of cells exhibiting lysosomal localization (p<0.01, t-test, n=3 experiments, 40 cells/condition/experiment). (D) Percentage of cells showing nuclear enrichment (p<0.01, t-test, n=3 experiments, 40 cells/condition/experiment). (E) Western blotting of total, cytoplasmic and nuclear subcellular fractions obtained from Hela M cells stably expressing TFEB-GFP +/−CQ treatment (50μM, 15 hours). Lamin A/C and tubulin represent control proteins for the nuclear and cytoplasmic fraction respectively. (F) Effect of CQ on TFEB-GFP levels (p<0.01, n=3, t-test). (G) Nuclear enrichment of TFEB-GFP +/− CQ (p<0.01, n=3, t-test). (H) Western blot for TFEB-GFP from cells grown under basal conditions +/− phosphatase treatment of the lysates. Arrows indicate the relative positions of the phosphorylated TFEB (upper arrow) and the dephosphorylated TFEB (lower TFEB). (I) Wildtype TFEB-GFP localization versus the Δ30TFEB-GFP mutant. Scale bars = 10μm
Fig. 2
Fig. 2
Phosphorylation dependent interaction of TFEB with 14-3-3 proteins. (A) Affinity purification and mass spectrometry analysis of heavy labeled HeLa M cells stably expressing TFEB-GFP versus control light labeled HeLa M cells. Averaged peptide intensities are plotted against heavy/light (H/L) SILAC ratios. Significant outliers are colored as indicated in the legend; other identified proteins are shown in dark blue. Representative of results from 2 independent experiments. (B) Western blotting of anti-GFP immunoprecipitations from cells expressing the indicated TFEB-GFP constructs. (C) Effect of the S211A mutation on the subcellular location of TFEB-GFP. Scale bar = 10μm. (D) Western blotting of anti-GFP immunoprecicipations from cells expressing wildtype versus Δ30TFEB-GFP.
Fig. 3
Fig. 3
Regulation of TFEB by mTORC1. (A) Live cell imaging of TFEB-GFP following starvation (Earl's Buffered Saline Solution), rapamycin (200nM) and torin 1 (2μM) treatments (2 hours). (B) Timecourse of the changes in the nuclear to cytoplasmic ratio of TFEB-GFP that arise due to torin 1 (2μM) treatment (n=3 experiments, average of 321 cells/condition/experiment, *p<0.01, ANOVA with Bonferroni post-test). (C) Timecourse showing the change in the electrophoretic mobility of native TFEB of Hela cells treated with 2μM torin 1 for the indicated times. Arrows indicate the relative positions of the phosphorylated TFEB (t=0) and the dephosphorylated TFEB (t=60 and beyond). (D) Western blots of anti-GFP immunoprecipitations from TFEB-GFP expressing cells subjected to the indicated treatments.
Fig. 4
Fig. 4
RagC and mTOR are required for regulation of TFEB localization via phosphorylation-dependent control of 14-3-3 interaction. (A) Live cell imaging of TFEB-GFP localization following RagC and mTOR knockdowns (Scale bar = 10μm). (B) Quantification of the effects of RagC and mTOR knockdowns on the nuclear/cytoplasmic ratio of TFEB-GFP (n=3 experiments, average of 169 cells analyzed per condition per experiment, *p<0.001, ANOVA with Bonferroni post-test). (C) Western blots of anti-GFP immunoprecipitations from TFEB-GFP cells following RagC and mTOR knockdowns.
Fig. 5
Fig. 5
An interaction between TFEB and mTOR on the cytoplasmic surface of lysosomes. (A) Immunofluorescent staining showing the colocalization of TFEB-GFP and LAMP1 +/− torin 1 treatment (2μM, 2 hours). Cells were permeablized for 10 seconds with 0.1% saponin prior to fixation to extract the diffuse cytoplasmic pool of TFEB. This strategy facilitates visualization and quantification of the lysosomal signal for TFEB. (B) Quantification of the intensity ratios for LAMP1 and TFEB-GFP in cells treated +/− torin 1 (2 μM, 2 hours, n=3 experiments, average of 21,874 lysosome analyzed per condition/per experiment, * p<0.05, t-test). (C) Immunofluorescence images showing extensive colocalization of TFEB and mTOR on lysosomes following torin 1 treatment (2 μM, 2 hours). (D) Western blot of anti-GFP immunoprecipitations from control HeLa M cells versus a TFEB-GFP stable line +/− torin 1 pretreatment (2μM, 2 hours). (E) Western blots of anti-GFP immunoprecipitations from torin 1 treated cells that demonstrate the lack of interaction between Δ30TFEB-GFP, mTOR and raptor. (F) Detection of native TFEB following subcellular fractionation of Hela cells +/− torin treatment (2μM, 2 hours). Arrows indicate the relative positions of the phosphorylated TFEB (upper) and the dephosphorylated TFEB (lower). (G) Quantification of the abundance of nuclear TFEB in the preceding fractionation experiments (n=3 experiments, *p<0.05, t-test). All scale bars = 10 μm.
Fig. 6
Fig. 6
Mutation of a predicted NLS in TFEB blocks nuclear accumulation in response to mTOR inhibition. (A) Live imaging of WT TFEB-GFP versus TFEBΔNLS-GFP localization after torin 1 treatment (2μM, 2 hours, scale bar=10μm). (B) Immunoblots of TFEBΔNLS-GFP immunoprecipitates +/− torin 1 treatment (2μM, 2 hours).
Fig. 7
Fig. 7
MITF and TFE3 localize to lysosomes and accumulate in the nucleus in response to inhibition of lysosome function. (A) Live imaging of MITF-GFP [“D” isoform (35) which is most similar to TFEB (Fig. S4A)] and TFE3-GFP reveals an enrichment on lysosomes (labeled by DQ-BSA) and relatively low levels in the nucleus under basal cell growth conditions. (B) Both MITF-GFP and TFE3-GFP are lost from lysosomes and accumulate in the nucleus in response to CQ (50 μM, 15 hours). (C) Subcellular fractionation and immunoblotting show the increase in nuclear levels of the MITF that is natively expressed in HeLa cells in response to CQ. (D) Live cell imaging of the localization of the MITF-M isoform fused to GFP under basal conditions. See also Fig. S4 for further MITF isoform-specific results and quantification.
Fig. 8
Fig. 8
Regulation of TFEB subcellular localization by mTOR interactions. This diagram summarizes how the localization of TFEB to lysosomes via mTOR interactions results in serine 211 phosphorylation and subsequent cytoplasmic sequestration by interactions with 14-3-3 proteins.
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