To investigate the mechanisms linking lysosomal status to the regulation of TFEB subcellular localization, we expressed GFP-tagged TFEB (TFEB-GFP) in HeLa M cells and imaged live cells by spinning disk confocal microscopy. Under basal conditions, the abundance of this protein in the cytoplasm was very high compared to the nucleus ( and Movie 1
). In addition to the diffuse cytoplasmic signal, there was a distinct enrichment of TFEB on lysosomes (, S1A
and Movie 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 (more ...)
The localization of TFEB to lysosomes suggested the existence of regulatory mechanisms that link TFEB activity to lysosomal status and was intriguing given the previous observations that TFEB accumulates in nuclei of cells affected by lysosomal storage disorders (12
). We acutely tested the relationship between TFEB localization and lysosomal status by incubating cells stably expressing TFEB-GFP with chloroquine (CQ), a weak base that impairs lysosome function by accumulating in lysosomes and raising their pH (20
). In response to CQ, TFEB lost lysosomal localization and strongly accumulated in the nucleus (). A similar response was observed following treatment with bafilomycin A, a specific inhibitor of the vacuolar H+
pump (Fig. S1B
Further supporting our imaging results, subcellular fractionation revealed an increase in the nuclear levels of TFEB following blockade of lysosomal function (). This CQ-induced nuclear translocation was accompanied by a decrease in the overall levels of TFEB (), an observation that parallels the link between activation of MITF and its proteosomal degradation (21
). Interestingly, the altered migration of TFEB on SDS-PAGE gels following CQ treatment () suggested a change in phosphorylation status. Indeed, a comparable mobility shift for TFEB was induced by phosphatase treatment of control lysates (). As essentially all of the TFEB runs at the higher molecular weight in untreated samples, we conclude that a very substantial fraction of TFEB is phosphorylated under basal conditions.
We next used mutagenesis to characterize the determinants for lysosomal localization of TFEB. Deletion of the first 30 N-terminal amino acids (Δ30TFEB) or targeted mutation of highly conserved amino acids within this region resulted in loss of the lysosomal localization and an increased nuclear abundance of TFEB ( and S1C
). Thus, essential determinants for lysosomal localization of TFEB reside within the N-terminus of the protein.
To identify additional proteins contributing to the regulated subcellular localization of TFEB, we used a combination of stable isotope labeling with amino acids in cell culture (SILAC) labeling, affinity chromatography and quantitative proteomics. This strategy identified 14-3-3 proteins (all 7 isoforms were present) as major binding partners of TFEB (). Consistent with the ability of TFEB to heterodimerize with the closely related TFE3 and MITF transcription factors (22
), these proteins also co-purified with TFEB (). The presence of 14-3-3 proteins in TFEB-GFP immunoprecipitations was also evident following SDS-PAGE and Coomassie staining (Fig. S2A
) and was further detected with a pan-14-3-3 antibody (Fig. S2B
). Interactions with 14-3-3 had previously been reported to regulate the nuclear abundance of MITF (23
) and TFE3 was identified as a 14-3-3 binding protein in a proteomic screen for 14-3-3 binding proteins (24
). Thus, 14-3-3 interactions are a shared property within this family of transcription factors.
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 (more ...)
14-3-3 proteins typically interact with their targets via short phosphoserine containing motifs (25
). The 14-3-3 binding site on MITF had been mapped to serine 173 which aligns with serine 211 of TFEB [(23
), Fig. S2C
]. This site closely conforms to the RSxpSxP consensus 14-3-3 binding motif (25
). We tested the contribution of serine 211 (S211) to 14-3-3 binding and regulation of subcellular localization by mutating it to alanine and found that this mutation abolished interactions with 14-3-3 proteins (). Conversely, mutation of S142 [a nearby MAPK phosphorylation site (13
)] had no effect on the 14-3-3 interaction (). Likewise, immunoblotting with an antibody specific for phosphorylated 14-3-3 binding motifs revealed a signal on TFEB that was selectively reduced with the S211A mutant (). The residual signal that remained for the anti-14-3-3 binding motif antibody in the S211A mutant represents its modest cross-reactivity with additional phosphorylation sites on TFEB (see below, ). We used this selective recognition of S211 phosphorylation by the anti-14-3-3 binding motif antibody in subsequent experiments to measure the phosphorylation status of S211 in TFEB immunoprecipitates.
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 (more ...)
Having found that S211 phosphorylation is essential for the interaction between 14-3-3 and TFEB, we next focused on the functional significance of TFEB S211 and the resulting 14-3-3 interactions by characterizing the localization of the S211A mutant. Live cell imaging revealed that TFEB-S211A had a much more prominent nuclear localization (). Interestingly, while the overall cytoplasmic levels were reduced, the lysosomal localization remained robust. Therefore, we conclude that phosphorylation of S211 and the resulting interaction with 14-3-3 proteins have a major role in regulating the nuclear abundance of TFEB while the lysosomal recruitment of TFEB is 14-3-3-independent.
To investigate the relationship between lysosomal targeting and S211 phosphorylation, we immunoprecipitated the Δ30TFEB mutant which lacks lysosomal targeting and found that this loss of lysosomal targeting is accompanied by greatly reduced S211 phosphorylation and 14-3-3 binding (). These results demonstrate the importance of lysosomal localization in controlling the phosphorylation state of TFEB-S211 and by extension in promoting the 14-3-3 interactions that retain a large pool of TFEB in the cytoplasm.
The accumulation of TFEB in the nucleus under conditions of starvation-induced autophagy has been linked to the TFEB-mediated regulation of genes encoding proteins important for autophagy (13
). Given our observations of the cytoplasmic retention of TFEB by S211-dependent 14-3-3 interactions, we suspected that the signaling pathway responsible for S211 phosphorylation should be inhibited when autophagy is induced. Based on this consideration, we focused our attention on the mTOR kinase as: (i) mTOR localizes to the cytoplasmic surface of lysosomes as part of the mTORC1 complex (26
) and (ii) the loss of mTOR lysosomal localization and activity under starvation conditions is a major trigger for promoting autophagy (15
). To test for a role for mTOR in regulating TFEB, we investigated the localization of TFEB under conditions of starvation as well as mTOR inhibition. Starvation resulted in the accumulation of TFEB in the nucleus and this was accompanied by the loss of TFEB's lysosomal localization (). Rapamycin, an allosteric mTORC1 inhibitor (29
), had minimal effects on TFEB localization (). However, while rapamycin is a very widely used inhibitor of the lysosome-localized mTORC1 complex, it has been recognized that the ability of this drug to inhibit mTORC1 is highly cell type- and substrate-dependent (29
). Therefore, we also tested the effect of torin 1, a more recently developed ATP-competitive inhibitor that blocks the activity of mTOR towards all substrates (31
) and observed strong nuclear translocation of TFEB, enhanced lysosome association (see also ), and a reduction in the diffuse cytoplasmic pool (). The loss of lysosomal localization of TFEB following starvation but not mTOR inhibition was a surprise as both treatments (as well as CQ treatment) resulted in the inhibition of mTORC1 activity (Fig. S3A
). Analysis of the time course of TFEB nuclear accumulation in response to mTOR inhibition showed that the effect was significant within 30 minutes and was maximal after ~1 hour of treatment (). This change in subcellular localization was paralleled by the dephosphorylation of the native TFEB protein ( and S3B
) and the time course for TFEB nuclear accumulation and dephosphorylation was comparable to that observed for 4E-BP1 (), a well characterized mTORC1 substrate (14
). To further investigate how these different TFEB localization and migration patterns relate to S211 phosphorylation and 14-3-3 interactions, we immunoprecipitated TFEB from starved and torin 1 treated cells and compared them to untreated controls and CQ-treated samples. Similar to CQ treatment, both starvation and torin 1 incubation resulted in loss of 14-3-3 binding and S211 phosphorylation and this effect was most robust in response to torin 1 (). As mTOR inhibition by torin 1 completely eliminated the detection of TFEB by the anti-14-3-3 binding site antibody () while the S211A mutant reduced but did not abolish this signal (), there must be additional mTOR-dependent phosphorylation sites on TFEB. Given that the effects of mTOR inhibition on TFEB localization, phosphorylation and 14-3-3 interaction closely phenocopied those of the S211A mutation () we conclude that TFEB-S211 phosphorylation is a major mechanism for mTOR-dependent regulation of TFEB.
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 (more ...)
To further test mTOR's role in regulating TFEB localization, we performed siRNA-mediated knockdowns of mTOR and RagC [a critical component in the recruitment of the mTORC1 complex to lysosomes (26
)]. Knockdown of either RagC or mTOR (Fig. S3C
) resulted in an increase in the nuclear abundance of TFEB (), reduced lysosomal localization (), reduced 14-3-3 interactions () and diminished TFEB phosphorylation ().
The contrasting results from mTOR inhibition versus siRNA knockdown experiments on the lysosomal localization of TFEB suggested that the recruitment of TFEB to lysosomes is dependent on the physical presence of mTOR but not necessarily its kinase activity, implying that the mTORC1 complex could participate in the recruitment of TFEB to lysosomes. Consistent with this hypothesis, the amount of mTOR on lysosomes parallels that of TFEB because it is reduced in response to starvation (27
) and increased in response to torin 1 (32
). Thus, while we had not detected an interaction between TFEB and mTORC1 components in the SILAC experiment described above (), we reasoned that since the localization of TFEB to lysosomes is greatly enhanced in response to mTOR inhibition () and that mTOR and TFEB colocalize very well on lysosomes under such conditions (), potential interactions between TFEB and mTORC1 components should also be enhanced. Indeed, mTOR and raptor selectively co-immunoprecipitated with TFEB following torin 1 exposure (). To further investigate the relationship between lysosomal targeting of TFEB and mTOR interactions, we tested the mTOR and raptor binding ability of the Δ30TFEB mutant that does not localize to lysosomes () and found that it does not interact with mTOR (). We next investigated the effects of mTOR inhibition on the subcellular localization of the natively expressed TFEB protein and found that its nuclear abundance was strongly increased in both Hela () and ARPE-19 cells (Fig. S3D
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 (more ...)
Collectively, our results support a model wherein mTORC1-dependent S211 phosphorylation of TFEB results in 14-3-3 interactions that promote the cytoplasmic retention of TFEB. In an effort to understand how nuclear import of TFEB is regulated, we searched for nuclear localization signals [NLSs, (33
)] in TFEB and identified a candidate sequence between amino acids 241–252 (Fig. S2C
). To test the hypothesis that 14-3-3 binding to the nearby S211-containing motif occludes this NLS we mutated basic residues (R245–R248) within the predicted NLS to alanine. If the loss of 14-3-3 interactions following mTOR inhibition triggers nuclear accumulation through unmasking of this adjacent NLS, then the torin 1 stimulated increase in nuclear TFEB levels should not occur with this mutant. Indeed, this is what we observed () in spite of the fact that torin 1 still inhibited the 14-3-3 binding and S211 phosphorylation of this mutant ().
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 (more ...)
The close sequence conservation between TFEB, MITF and TFE3 (Fig. S2C
), the ability of these proteins to form functional heterodimers (22
) and evidence of interactions between both MITF and TFE3 with 14-3-3 proteins (23
) led us to consider to possibility that MITF and TFE3 might also be regulated by lysosome status. In support of such a prediction, both MITF (isoforms A and D) and TFE3 also exhibited a predominantly cytoplasmic signal with focal concentration on lysosomes under basal cell culture conditions ( and S4
). As observed for TFEB, both MITF and TFE3 translocate to the nucleus in response to CQ treatment ( and S4B–D
). Furthermore, like the Δ30TFEB mutant (), the MITF-M isoform that is predominantly expressed in melanocytes and which naturally possesses a truncated amino terminus due to alternative promoter usage [(35
), Fig. S4A
], also lacked lysosome localization and was enriched in the nucleus under basal conditions (). Based on these findings, the regulatory mechanisms that we initially uncovered in our investigation of TFEB regulation appear to be broadly conserved within this family of transcription factors.
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 (more ...)