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

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Abstract

Folliculin localizes to lysosomes and directly interacts with Rag GTPases, promoting mTORC1 activation, when amino acids are abundant.

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.

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A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB

Carmine Settembre, Roberto Zoncu, Diego L Medina … (2012)

Introduction The lysosome maintains cellular homeostasis and mediates a variety of physiological processes, including cellular clearance, lipid homeostasis, energy metabolism, plasma membrane repair, bone remodelling, and pathogen defense. All these processes require an adaptive and dynamic response of the lysosome to environmental cues. Indeed, physiologic cues, such as ageing and diet, and pathologic conditions, which include lysosomal storage diseases (LSDs), neurodegenerative diseases, injuries, and infections may generate an adaptive response of the lysosome (Luzio et al, 2007; Ballabio and Gieselmann, 2009; Saftig and Klumperman, 2009). Our understanding of the mechanisms that regulate lysosomal function and underlying lysosomal adaptation is still in an initial phase. A major player in the regulation of lysosomal biogenesis is the basic Helix-Loop-Helix (bHLH) leucine zipper transcription factor, TFEB (Sardiello et al, 2009). Among the identified TFEB transcriptional targets are lysosomal hydrolases that are involved in substrate degradation, lysosomal membrane proteins that mediate the interaction of the lysosome with other cellular structures, and components of the vacuolar H+-ATPase (v-ATPase) complex that participate in lysosomal acidification (Sardiello et al, 2009; Palmieri et al, 2011). TFEB is also a main player in the transcriptional response to starvation and controls autophagy by positively regulating autophagosome formation and autophagosome–lysosome fusion both in vitro and in vivo (Settembre et al, 2011). TFEB activity and its nuclear translocation correlate with its phosphorylation status (Settembre and Ballabio, 2011; Settembre et al, 2011). However, it is still unclear how the cell regulates TFEB activity according to its needs. An intriguing hypothesis is that the lysosome senses the physiological and nutritional status of the cell and conveys this information to the nucleus to drive the activation of feedback gene expression programs. A ‘sensing device', which is responsive to the lysosomal amino acid content and involves both the v-ATPase and the master growth regulator mTOR complex 1 (mTORC1), was recently identified on the lysosomal surface (Zoncu et al, 2011a). The interaction between amino acids and v-ATPase regulates Rag guanosine triphosphatases (GTPases), which in turn activate mTORC1 by translocating it to the lysosomal surface (Sancak et al, 2008, 2010; Zoncu et al, 2011a). According to this mechanism, the lysosome participates in the signalling pathways regulated by mTOR, which controls several cellular biosynthetic and catabolic processes (Zoncu et al, 2011b). We postulated that TFEB uses the v-ATPase/mTORC1 sensing device on the lysosomal surface to modulate lysosomal function according to cellular needs. Consistent with this hypothesis, we found that TFEB interacts with mTOR on the lysosomal membrane and, through this interaction, it senses the lysosomal content. Therefore, TFEB acts both as a sensor of lysosomal state, when on the lysosomal surface, and as an effector of lysosomal function when in the nucleus. This unique lysosome-to-nucleus signalling mechanism allows the lysosome to regulate its own function. Results TFEB responds to the lysosomal status We postulated that TFEB activity was regulated by the physiological status of the lysosome. Therefore, we tested whether disruption of lysosomal function had an impact on TFEB nuclear translocation. TFEB subcellular localization was analysed in HeLa and HEK-293T cells transiently transfected with a TFEB–3 × FLAG plasmid and treated overnight with several inhibitors of lysosomal function. These treatments included the use of chloroquine (CQ), an inhibitor of the lysosomal pH gradient, and Salicylihalamide A (SalA), a selective inhibitor of the v-ATPase (Xie et al, 2004), as well as overexpression of PAT1, an amino acid transporter that causes massive transport of amino acids out of the lysosomal lumen (Sagne et al, 2001). Immunofluorescence analysis showed a striking nuclear accumulation of TFEB–3 × FLAG in treated cells (Figure 1A and B). We repeated this analysis using an antibody detecting the endogenous TFEB (Supplementary Figure S1). Similarly to their effect on exogenously expressed TFEB, both amino acid starvation and lysosomal stress induced nuclear translocation of endogenous TFEB (Figure 1C). These observations were confirmed by immunoblotting performed after nuclear/cytoplasmic fractionation (Figure 1D). Immunoblotting also revealed that TFEB nuclear accumulation was associated with a shift of TFEB–3 × FLAG to a lower molecular weight, suggesting that lysosomal stress may affect TFEB phosphorylation status (Figure 1D). mTORC1 regulates TFEB subcellular localization Based on the observation that mTORC1 resides on the lysosomal membrane and its activity is dependent on both nutrients and lysosomal function (Sancak et al, 2010; Zoncu et al, 2011a), we postulated that the effects of lysosomal stress on TFEB nuclear translocation may be mediated by mTORC1. Consistent with this idea, chloroquine or SalA inhibited mTORC1 activity as measured by level of p-P70S6K, a known mTORC1 substrate (Figure 2A; Zoncu et al, 2011a). The involvement of mTOR appears in contrast with our previous observation that Rapamycin, a known mTOR inhibitor, did not affect TFEB activity. However, recent data indicate that Rapamycin is a partial inhibitor of mTOR, as some substrates are still efficiently phosphorylated in the presence of this drug (Thoreen et al, 2009). Therefore, we used kinase inhibitors Torin 1 and Torin2, which belong to a novel class of molecules that target the mTOR catalytic site, thereby completely inhibiting mTOR activity (Feldman et al, 2009; Garcia-Martinez et al, 2009; Thoreen et al, 2009). We stimulated starved cells, in which TFEB is dephosphorylated and localized to the nucleus, with an amino acid rich medium supplemented with Torin 1 (250 nM), Rapamycin (2.5 μM), or ERK inhibitor U0126 (50 μM). Stimulation of starved cells with nutrients alone induced a significant TFEB molecular weight shift and re-localization to the cytoplasm (Figure 2B). Nutrient stimulation in the presence of the ERK inhibitor U0126 at a concentration of 50 μm induced only a partial TFEB molecular weight shift, suggesting that phosphorylation by ERK partially contributes to TFEB cytoplasmic localization. Treatment with 2.5 μM Rapamycin also resulted in a partial molecular weight shift but did not affect TFEB subcellular localization (Figure 2B), consistent with our previous observations (Settembre et al, 2011). However, Torin 1 (250 nM) treatment entirely prevented the molecular weight shift induced by nutrients and, in turn, resulted in massive TFEB nuclear accumulation. This conclusion is in contrast with a recent study that showed that mTOR-mediated TFEB phosphorylation promoted, rather than inhibited, its nuclear translocation (Pena-Llopis et al, 2011). Instead our data indicate that mTOR is a potent inhibitor of TFEB nuclear translocation and that TFEB is a rapamycin-resistant substrate of mTORC1. In a previous study, we showed that ERK2 phosphorylates TFEB and that starvation and ERK2 inhibition promote TFEB nuclear translocation (Settembre et al, 2011). We tested whether lysosomal stress caused TFEB nuclear translocation also via ERK inhibition. Overnight treatment of HeLa cells with either chloroquine or SalA did not have any effect on ERK activity (Figure 2A), suggesting that mTOR-mediated regulation is predominant. To quantify the effects of ERK and mTOR on TFEB subcellular localization, we developed a cell-based high content assay using stable HeLa cells that overexpress TFEB fused to the green fluorescent protein (TFEB–GFP) (see Materials and methods for details). We tested 10 different concentrations of each inhibitor (U0126, Rapamycin, Torin 1, and Torin 2) ranging from 2.54 nM to 50 μM. Figure 2C and D shows the TFEB nuclear/cytoplasmic distribution for each concentration of each compound in duplicate represented as dose–response curves using a non-linear regression fitting (see Materials and methods for details). Consistent with the above-described data, the most potent compounds that activate TFEB nuclear translocation were Torin 1 (EC50; 147.9 nM) and its analogue Torin 2 (EC50; 1666 nM). ERK inhibitor U0126 showed only a partial effect, while Rapamycin had no effects at any of the concentrations that are routinely used (10 nM–10 μM). Furthermore, Torin 1 treatment potently induced nuclear accumulation of endogenous TFEB in HEK-293T cells (Figure 2E), confirming the observations obtained with the TFEB–GFP construct. As Torin 1 inhibits both mTORC1 and mTORC2 complexes, we next evaluated the contribution of each complex to TFEB regulation. Three main observations suggest that TFEB is predominantly regulated by mTORC1: (1) stimulation of starved cells with amino acids, which activate mTORC1 but not mTORC2, induced an extensive TFEB molecular weight shift, which is highly suggestive of a phosphorylation event (Supplementary Figure S2); (2) knockdown of RagC and RagD, which mediate amino acid signals to mTORC1, caused TFEB nuclear accumulation even in cells kept in full nutrient medium (Figure 2F); (3) in cells with disrupted mTORC2 signalling (Sin1−/− mouse embryonic fibroblasts (MEFs)) (Frias et al, 2006; Jacinto et al, 2006; Yang et al, 2006) TFEB underwent a molecular weight shift and nuclear translocation upon Torin 1 treatment that were similar to control cells (Figure 2G). Together, these data indicate that mTORC1, not mTORC2, regulates TFEB by preventing its nuclear translocation. Finally, co-immunoprecipitation assays in HEK-293T cells expressing TFEB–3 × FLAG showed that TFEB binds both to mTOR and to the mTORC1 subunit raptor but not to the mTORC2 subunits rictor and mSin1, indicating that TFEB and mTORC1 interact both functionally and physically (Figure 2H). mTORC1 controls TFEB subcellular localization via phosphorylation of S142 We previously identified phosphorylation at Serine 142 as a key event for TFEB nuclear translocation during starvation (Settembre et al, 2011). To test whether mTORC1 phosphorylates TFEB at S142, we generated a phosphospecific antibody that recognizes TFEB only when phosphorylated at S142. No signal was detected by this antibody in cells that overexpress the S142A mutant version of TFEB, thus confirming its specificity (Supplementary Figure S3). Using this antibody, we observed that TFEB was no longer phosphorylated at S142 in HeLa cells stably overexpressing TFEB–3 × FLAG and cultured in nutrient-depleted media, consistent with our previous results (Figure 3A). Subsequently, we analysed the levels of S142 phosphorylation in starved cells supplemented with normal media with or without either Torin 1 or Rapamycin. While Torin 1 clearly blunted nutrient-induced S142 phosphorylation, rapamycin did not, suggesting that S142 represents a rapamycin-resistant mTORC1 site (Figure 3A). Indeed, an mTOR kinase assay revealed that mTORC1 phosphorylates highly purified TFEB in vitro with comparable efficiency to other known mTORC1 substrates, and this phosphorylation dropped dramatically when mTORC1 was incubated with the S142A mutant version of TFEB (Figure 3B). These results clearly demonstrate that TFEB is an mTOR substrate and that S142 is a key residue for the phosphorylation of TFEB by mTOR. Recent findings suggest that mTORC1 phosphorylates its target proteins at multiple sites (Hsu et al, 2011; Peterson et al, 2011; Yu et al, 2011). To identify additional serine residues that may be phosphorylated by mTOR, we searched for consensus phosphoacceptor motif for mTORC1 (Hsu et al, 2011) in the coding sequence of TFEB (Figure 3C and D). We mutagenized all TFEB amino acid residues that were putative mTORC1 targets into alanines. We then tested the effects of each of these mutations on TFEB subcellular localization and found that, similarly to S142A, a serine-to-alanine mutation at position 211 (S211A) resulted in a constitutive nuclear localization of TFEB (Figure 3E). Mutants for the other serine residues behaved similarly to wild-type TFEB (Figure 3E; Supplementary Figure S4; Settembre et al, 2011). Together, these data indicate that, in addition to S142, S211 also plays a role in controlling TFEB subcellular localization and suggest that S211 represents an additional target site of mTORC1. mTORC1 and TFEB interact on the lysosomal surface Based on the observations that TFEB is a substrate for mTORC1 (Figure 3A and B) and that the two proteins physically interact (Figure 2H), we tested whether the interaction of TFEB and mTORC1 occurs on the lysosomal membrane. Careful examination of HeLa cells that express TFEB–GFP showed that, while under normal growth conditions the majority of cells displayed a predominantly cytoplasmic TFEB localization, a subset of cells showed clearly discernible intracellular puncta of TFEB–GFP fluorescence, suggesting a lysosomal localization (Supplementary Figure S5). These observations were confirmed in MEFs that transiently express TFEB–GFP along with the late endosomal/lysosomal marker mRFP–Rab7 (Figure 4A). In a subset of cells, TFEB–GFP clearly colocalized with mRFP–Rab7-positive lysosomes and this association persisted over time as lysosomes trafficked inside the cell (Figure 4A and B; Supplementary Movie S1). We reasoned that the partial localization of TFEB to lysosomes may be due to a transient binding to mTORC1, followed by mTORC1-dependent phosphorylation and translocation of TFEB to the cytoplasm. To test this idea, we treated TFEB–GFP HeLa cells with Torin 1, as a way to ‘trap' TFEB in its bound state to inactive mTORC1. Confirming our hypothesis, Torin 1 caused a massive and dramatic accumulation of TFEB–GFP on lysosomes (Supplementary Figure S5). Similarly, Torin 1 treatment of MEFs resulted in a time-dependent accumulation of TFEB–GFP on lysosomes within minutes of drug delivery, followed by a more gradual accumulation into the nucleus (Figure 4C; Supplementary Movies S2 and S3). Interestingly, we also noticed that Torin 1 treatment caused a significant accumulation of endogenous mTOR on lysosomes compared with untreated cells (Figure 4D). Thus, two mechanisms contribute to clustering of TFEB on lysosomes upon Torin 1 treatment: (1) trapping of the mTORC1–TFEB complex in the inactive state and (2) increase of the amount of mTORC1 bound to the lysosomal surface. The accumulation of inactive mTORC1 on the lysosomal surface may reflect a feedback mechanism through which mTORC1 regulates its own targeting to lysosomal membranes via its kinase activity (Zoncu et al, 2011b). To investigate the lysosomal trapping of TFEB in a dynamic and quantitative way, we performed Fluorescence Recovery After Photobleaching (FRAP) experiments on TFEB–GFP-positive lysosomes (Figure 4E and F; Supplementary Movie S4). In control cells, photobleaching of TFEB–GFP-positive lysosomes was followed by a rapid (t 1/2=0.35 min) and substantial (60%) recovery of the initial fluorescence. Conversely, in Torin 1-treated cells, where TFEB–GFP-positive lysosomes were much more prominent and numerous, the fluorescence recovery was slower (t 1/2=0.57 min) and smaller (30% recovery of the initial fluorescence). Thus, a large fraction of TFEB was trapped onto the lysosomal surface through binding to inactive mTORC1 and was no longer able to exchange with the cytoplasm. In conclusion, these data indicate that TFEB and mTORC1 bind to each other on the lysosomal surface, where phosphorylation of TFEB by mTORC1 occurs. mTORC1 regulates TFEB via the Rag GTPases The observation that TFEB is regulated by mTORC1 prompted us to determine whether the activation state of the Rag GTPases, which together with the v-ATPase mediate mTORC1 activation by amino acids, played a role in the control of TFEB subcellular localization. Point mutants of the Rags are available, which fully mimic either the presence of amino acids (‘RagsCA') or their absence (‘RagsDN') (Sancak et al, 2008). We took advantage of these mutants to directly test the requirement for mTORC1 in sequestering TFEB to the lysosome and we asked whether the RagsDN mutants, which cause loss of mTORC1 from the lysosomal surface (Sancak et al, 2010), were able to suppress Torin 1-induced lysosomal accumulation of TFEB as well as TFEB-mTORC1 binding. In co-immunoprecipitation assays, Torin 1 clearly boosted the binding of both raptor and mTOR to TFEB–3 × FLAG (Figure 4G). However, co-expression of the RagsDN mutants reduced the binding of TFEB–3 × FLAG to mTORC1 components down to background levels, both in control and in Torin 1-treated cells (Figure 4G). Consistent with these results, immunofluorescence experiments in HEK-293T co-expressing TFEB–3 × FLAG and the RagsDN mutants showed that TFEB failed to cluster on lysosomes following Torin 1 treatment (Figure 4H). Together, these data strongly suggest that TFEB and mTORC1 only interact when they are both found on the lysosomal surface. Next, we tested whether the activation status of the Rags controlled TFEB nuclear translocation. In HEK-293T cells that co-express TFEB–3 × FLAG and a control small GTPase (Rap2A), amino acid withdrawal caused a massive translocation of TFEB to the nucleus (Figure 5A and D), as previously reported (Settembre et al, 2011). Consistent with mTORC1 re-activation, a brief (20 min) re-stimulation of starved cells with amino acids drove TFEB out of the nucleus in the majority of cells (Figure 5A). In contrast, in cells that co-express both TFEB–3 × FLAG and the RagsCA mutants, TFEB localization was always and completely cytoplasmic, regardless of the nutrient state of the cells (Figure 5B and D). Finally, in cells that co-express both TFEB and the RagsDN mutants, TFEB was almost exclusively found in the nucleus and did not translocate to the cytoplasm upon amino acid stimulation (Figure 5C and D). Thus, the activation state of the Rags completely overrides the nutritional status of the cells and is sufficient to determine TFEB localization. It was previously shown that the RagsCA rescue the inhibitory effect of various lysosomal stressors on mTORC1 activation (Zoncu et al, 2011a). Thus, we asked whether the RagsCA mutants were able to prevent the TFEB nuclear translocation promoted by these stressors (Figures 1A–D and 5E). In cells that co-express both TFEB–3 × FLAG and the RagsCA mutants, TFEB remained entirely cytoplasmic upon treatment with Chloroquine and SalA (Figure 5F and G), while it was nuclear in the vast majority of cells that express a control GTPase and were subject to the same drug treatments (Figure 5E and G). Importantly, treatment of cells co-expressing TFEB–GFP and RagCA with Torin 1 reverted the RagCA-induced cytoplasmic localization of TFEB and massively drove TFEB to the nucleus, further demonstrating that the action of the Rag mutants on TFEB is mediated by mTORC1 (Supplementary Figure S6). In summary, these results demonstrate that TFEB localization is directly regulated by the amino acid-mTORC1 signalling pathway via the activation state of Rag GTPases. The lysosome regulates gene expression via TFEB As the interaction of TFEB with mTORC1 on the lysosomal membrane controls TFEB nuclear translocation, we tested whether the ability of TFEB to regulate gene expression was also influenced by this interaction. The expression of several lysosomal/autophagic genes that were shown to be targets of TFEB (Palmieri et al, 2011) was tested in primary hepatocytes from a conditional knockout mouse line in which TFEB was deleted in the liver (Tcfebflox/flox; alb-CRE), and in a control mouse line (Tcfebflox/flox). Cells were treated with either chloroquine or Torin 1, or left untreated. These treatments inhibited mTOR as measured by the level of p-S6K, whereas the levels of p-ERK were unaffected (Figure 6A). Primary hepatocytes isolated from TFEB conditional knockout mice cultured in regular medium did not show significant differences in the expression levels of several TFEB target genes compared with control hepatocytes (Supplementary Figure S7). However, while the expression of TFEB target genes was upregulated in hepatocytes from control mice after treatment with chloroquine, this upregulation was significantly blunted in hepatocytes from TFEB conditional knockout mice (Figure 6B). Similarly, the transcriptional response upon Torin 1 treatment was significantly reduced in hepatocytes from TFEB conditional knockout mice (Figure 6C). Together, these results indicate that TFEB plays a key role in the transcriptional response induced by the lysosome via mTOR. Discussion Our study demonstrates that TFEB, a master gene for lysosomal biogenesis, is regulated by the lysosome via the mTOR pathway. mTORC1 and TFEB meet on the lysosomal membrane where mTORC1 phosphorylates TFEB. We previously reported that ERK2 phosphorylates TFEB and, in cells treated with an MEK inhibitor, the TFEB nuclear fraction was increased (Settembre et al, 2011). In the same study, we reported that the mTOR inhibitor rapamycin had little or no effects on TFEB subcellular localization. Here, we compared three different types of kinase inhibitors—MEK inhibitor U0126 and mTOR inhibitors rapamycin, Torin 1, and Torin 2—in their ability to cause a shift in TFEB molecular weight and to induce TFEB nuclear translocation. As shown in Figure 2, Torin 1 and Torin 2 induced TFEB nuclear translocation more efficiently compared to U0126. The more pronounced shift of TFEB molecular weight, which was observed in cells treated with Torin 1, suggests that mTORC1 induces TFEB phosphorylation at multiple sites, either directly or indirectly. In a recent high throughput mass spectrometry study, TFEB was predicted to be phosphorylated at 11 different residues, thus suggesting a complex regulation of its activity with several phosphorylation sites potentially involved (Dephoure et al, 2008). Here, we have used an mTORC1 in-vitro kinase assay and a phosphoantibody to demonstrate that serine S142, which we previously found to be phosphorylated by ERK2, is also phosphorylated by mTOR and that this phosphorylation has a crucial role in controlling TFEB subcellular localization and activity. In addition, we have mutated 12 different serines, which were candidate mTOR phosphorylation sites, into alanines, thus abolishing the corresponding TFEB phosphorylation sites. Testing the effects of each of these mutations on TFEB subcellular localization led to the identification of an additional residue, serine S211, which plays a role in TFEB subcellular localization, confirming the predicted complexity of TFEB regulation by phosphorylation. Phosphorylation of TFEB by mTOR had already been reported in a previous study (Pena-Llopis et al, 2011). However, in that study the authors concluded that mTOR promoted, rather than inhibited, TFEB activity. Several lines of evidence indicate that mTOR inhibits TFEB activity. First, TFEB is entirely nuclear when cells are either starved or treated with Torin1, both conditions in which mTOR activity is profoundly inhibited. Second, treatment of starved cells with nutrients, a condition that boosts mTORC1 activity, resulted in TFEB cytoplasmic accumulation, with TFEB being undetectable in the nuclear fraction. Third, treatment with drugs such as chloroquine or SalA, which inhibit mTORC1 function, induced TFEB nuclear accumulation. Fourth, transfection of mutant Rag proteins that inhibit mTORC1 resulted in nuclear accumulation of TFEB and, conversely, mutant Rags that constitutively activate mTORC1 prevented TFEB nuclear accumulation upon starvation, chloroquine and SalA treatment. Fifth, TFEB is in the nucleus in its low-phosphorylated form, an observation that is consistent with a model in which inhibition, rather than activation, of a kinase induces TFEB nuclear translocation. It is difficult to explain the discrepancy between our observations and those reported by Pena-Llopis et al. We considered the possibility that the TSC2-deficient cells that were used in that study may behave differently to other cellular systems in the assays performed. To test this possibility, we analysed TFEB regulation by amino acids, chloroquine and Torin 1 in TSC2−/− cells but obtained the same results that we observed in other cell types both on exogenous TFEB–GFP and on endogenous TFEB (Supplementary Figures S8 and S9, respectively). Our data indicate that mTORC1 negatively regulates TFEB via the amino acid/Rag GTPase pathway. The phosphorylation status of TFEB and its subcellular localization were entirely determined by the activation state of the Rag GTPases, which regulate mTORC1 activity downstream of amino acids (Kim et al, 2008; Sancak et al, 2008). In particular, constitutively active Rags rescued nuclear translocation of TFEB caused by starvation and lysosomal stress, while inactive Rag mutants caused TFEB to accumulate in the nucleus even in fully fed cells. These results imply that, among the many regulatory inputs to mTORC1, the amino acid pathway is particularly important in controlling TFEB activity and plays not only a permissive but also an instructive role. This idea is further supported by our observation that constitutive activation of the growth factor inputs to mTORC1 that occurs in TSC2−/− cells cannot prevent TFEB nuclear accumulation caused by starvation and lysosomal stress. Future work will be required to address how each upstream input to mTOR contributes to TFEB regulation. Nonetheless, compounded with recent evidence showing that amino acid sensing by the v-ATPase/Rag GTPase/mTORC1 may begin in the lysosomal lumen (Zoncu et al, 2011a) our findings substantiate the role of TFEB as the end point of a lysosome-sensing and signalling pathway. Our data shed light into the logic that underlies the control of TFEB localization. In fully fed cells, a fraction of TFEB could always be found on lysosomes, although the majority appeared to freely diffuse in the cytoplasm. The lysosomal localization of TFEB is associated with its ability to physically bind mTORC1, as shown by co-immunoprecipitation assays. Moreover, time-lapse analysis of TFEB–GFP in cells treated with Torin 1 showed that TFEB clustered on lysosomes shortly after the onset of drug treatment, and then progressively appeared in the nucleus (Supplementary Movies S2 and S3). Together, these results suggest the following model of control of TFEB subcellular localization and activity (Figure 7). At any given time, a fraction of TFEB rapidly and transiently binds to the lysosomal surface, where it is phosphorylated by mTORC1 and thus kept in the cytoplasm. Nutrient withdrawal, v-ATPase inhibition, and lysosomal stress inactivate the Rag GTPases, causing loss of mTORC1 from the lysosome and resulting in failure to re-phosphorylate TFEB. Unphosphorylated TFEB progressively accumulates in the nucleus, where it activates lysosomal gene expression programs aimed at correcting the defective nutrient and/or pH status of the lysosome. In this model, the lysosome represents a bottleneck where mTORC1 tightly regulates the amount of TFEB that is allowed to reach the nucleus. mTORC1 may regulate a yet undiscovered TFEB function at the lysosome. This possibility is supported by the observation that blocking mTORC1 activity with Torin 1 resulted in a dramatic accumulation of TFEB not only in the nucleus but also on lysosomes, which was visible as increased binding to mTORC1 in co-IP assays, as well as reduced mobility in FRAP experiments. Future work will address what function, if any, TFEB performs on the lysosomal surface. Interestingly, recent evidence indicating that TFEB regulates multiple aspects of lysosomal dynamics, including the propensity of lysosomes to fuse with the plasma membrane (Medina et al, 2011), suggests that the range of biological functions of TFEB still needs to be fully elucidated. Our data further emphasize the emerging role of the lysosome as a key signalling centre. In particular, a molecular machinery that connects the presence of amino acids in the lysosomal lumen to the activation of mTORC1 indicates a new role for the lysosome in nutrient sensing and cellular growth control (Rabinowitz and White, 2010; Singh and Cuervo, 2011; Zoncu et al, 2011a). It also suggests that mTORC1 participates in a lysosomal adaptation mechanism that enables cells to cope with starvation and lysosomal stress conditions (Yu et al, 2010). This mechanism responds to a wide range of signals that relay the metabolic state of the cell, as well as the presence of various stress stimuli. For instance, loss of lysosomal proton gradient, caused by either energy depletion or pathological conditions, may suppress mTORC1 activity, either by blocking the proton-coupled transport of nutrients to and from the lysosome, or by directly affecting the v-ATPase (Marshansky, 2007). Similarly, lysosomal membrane permeabilization observed in certain LSDs and neurodegenerative diseases may result in nutrient leakage and suppression of mTORC1 (Dehay et al, 2010; Kirkegaard et al, 2010). We found that the transcriptional response of lysosomal and autophagy genes to starvation and mTOR inhibition by Torin 1 was hampered in hepatocytes from mice carrying a liver-specific conditional knockout of TFEB, demonstrating that TFEB is a main mediator of this response. Therefore, TFEB translates a lysosomal signal into a transcriptional program. This lysosome-to-nucleus signalling mechanism, which operates a feedback regulation of lysosomal function, presents intriguing parallels with the sterol sensing pathway in the endoplasmic reticulum, where cholesterol depletion and ER stress cause the nuclear translocation of the Sterol Responsive Element Binding Protein (SREBP) transcription factor, which then activates gene expression programs that enhance cholesterol synthesis and ER function (Wang et al, 1994; Peterson et al, 2011). Another example is represented by the mitochondria retrograde signalling pathway, in which mitochondrial dysfunction activates factors such as NFκB, NFAT, and ATF, through altered Ca2+ dynamics (Butow and Avadhani, 2004). Finally, as TFEB overexpression was able to promote substrate clearance and to rescue cellular vacuolization in LSDs (Medina et al, 2011), the identification of a lysosome-based, mTOR-mediated, mechanism that regulates TFEB activity offers a new tool to promote cellular clearance in health and disease. Materials and methods Cell culture HeLa and HEK-293T cells were purchased from ATCC and cultured in DMEM supplemented with 10% fetal calf serum, 200 mM L-glutamine, 100 mM sodium pyruvate, penicillin 100 units/ml, streptomycin 100 mg/ml, 5% CO2 at 37°C. Primary hepatocytes were generated as follow: 2-month-old mice were deeply anaesthetized with Avertin (240 mg/kg) and perfused first with 25 ml of HBSS (Sigma H6648) supplemented with 10 mM HEPES and 0.5 mM EGTA and after with a similar solution containing 100 U/ml of Collagenase (Wako) and 0.05 mg/ml of Trypsin inhibitor (Sigma). Liver was dissociated in a petri dish, cell pellet was washed in HBSS and plated at density of 5 × 105 cells/35 mm dish and cultured in William's medium E supplemented with 10% FBS, 2 mM glutamine, 0.1 μM Insulin, 1 μM Dexamethasone and pen/strep. The next day, cells were treated as described in the text. Sin1−/− and control MEFs were generated as previously described (Jacinto et al, 2006) and maintained in DMEM supplemented with 10% FBS, glutamine and pen/strep. TSC2+/+ p53−/− and TSC2−/− p53−/− MEFs, kindly provided by David Kwiatkowski (Harvard Medical School), were maintained in DMEM supplemented with 10% heat-inactivated FBS, glutamine and pen/strep. Generation of a Tcfebflox mouse line We used publicly available embryonic stem (ES) cell clones (http://www.eucomm.org/) in which Tcfeb was targeted by homologous recombination at exons 4 and 5. The recombinant ES cell clones were injected into blastocysts, which were used to generate a mouse line carrying the engineered allele. Liver-specific KO was generated crossing the Flox/Flox mice with a transgenic line expressing the CRE under the Albumin promoter (ALB-CRE) obtained from the Jackson laboratory. All procedures involving mice were approved by the Institutional Animal Care and Use Committee of the Baylor College of Medicine. Plasmids and cell transfection Cells were transiently transfected with DNA plasmids pRK5-mycPAT1, pRK5-HAGST-Rap2A, pRK5-HAGST-RagB and its Q99L (CA) and T54N (DN) mutants, pRK5-HAGST-RagD and its Q121L (DN) and S77L (CA) mutants; pTFEB-GFP, and pCMV-TFEB-3 × FLAG using lipofectamine2000 or LTX (Invitrogen) according to the protocol from manufacturer. Site-direct mutagenesis was performed according to the manufacturer instructions (Stratagene) verifying the correct mutagenesis by sequencing. Drugs and cellular treatments The following drugs were used: Rapamycin (2.5 μM, otherwise indicated) from Sigma; Torin 1 and Torin 2 (250 nM, otherwise indicated) from Biomarine; U0126 (50 μM) from Cell Signaling Technology; Chloroquine (100 μM) from Sigma; Salicylihalamide A (2 μM) was a kind gift from Jeff De Brabander (UT Southwestern). Immunoblotting and antibodies The mouse anti-TFEB monoclonal antibody was purchased from My Biosource catalogue No. MBS120432. To generate anti-pS142 specific antibodies, rabbits were immunized with the following peptide coupled to KLH: AGNSAPN{pSer}PMAMLHIC. Following the fourth immunization, rabbits were sacrificed and the serum was collected. Non-phosphospecific antibodies were depleted from the serum by circulation through a column containing the non-phosphorylated antigene. The phosphospecific antibodies were next affinity purified using a column containing the phosphorylated peptide. Cells were lysed with M-PER buffer (Thermo) containing protease and phosphatase inhibitors (Sigma); nuclear/cytosolic fractions were isolated as previously described (Settembre et al, 2011). Proteins were separated by SDS–PAGE (Invitrogen; reduced NuPAGE 4–12% Bis-tris Gel, MES SDS buffer). If needed, the gel was stained using 20 ml Imperial Protein Stain (Thermo Fisher) at room temperature for 1 h and de-stained with water. Immunoblotting analysis was performed by transferring the protein onto a nitrocellulose membrane with an I-Blot (Invitrogen). The membrane was blocked with 5% non-fat milk in TBS-T buffer (TBS containing 0.05% Tween-20) and incubated with primary antibodies anti-FLAG and anti-TUBULIN (Sigma; 1:2000), anti-H3 (Cell Signaling; 1:10 000) at room temperature for 2 h whereas the following antibodies were incubated ON in 5% BSA: anti-TFEB (My Biosource; 1:1000), anti-P TFEB (1:1000) ERK1/2, p-ERK1/2, p-P70S6K, P70S6K (Cell Signaling; 1:1000). The membrane was washed three times with TBS-T buffer and incubated with alkaline phosphatase-conjugated IgG (Promega; 0.2 mg/ml) at room temperature for 1 h. The membrane was washed three times with TBS buffer and the expressed proteins were visualized by adding 10 ml Western Blue® Stabilized Substrate (Promega). In-vitro kinase assays FLAG–S6K1, TFEB–3 × FLAG, and TFEBS142A–3 × FLAG were purified from transiently transfected HEK-293T cells treated with 250 nM Torin 1 for 1 h and lysed in RIPA lysis buffer. The cleared lysates were incubated with FLAG affinity beads (Sigma) for 2 h, washed four times in RIPA containing 500 mM NaCl, and eluted for 1 h at 4°C using a competing FLAG peptide. mTORC1 was purified from HEK-293T cells stably expressing FLAG raptor in 0.3% CHAPS using FLAG affinity beads. Kinase assays were preincubated for 10 min at 4°C before addition of ATP, and then for 30 min at 30°C in a final volume of 25 μl consisting of kinase buffer (25 mM HEPES, pH 7.4, 50 mM KCl, 10 mM MgCl2) active mTORC1, 250–500 nM substrate, 50 μM ATP, 1 μCi [γ-32P]ATP, and when indicated 250 nM Torin 1. Reactions were stopped by the addition of 6 μl of sample buffer, boiled for 5 min, and analysed by SDS–PAGE followed by autoradiography. Immunoprecipitation assays HEK-293T cells that express FLAG-tagged proteins were rinsed once with ice-cold PBS and lysed in ice-cold lysis buffer (150 mM NaCl, 40 mM HEPES (pH 7.4), 2 mM EGTA, 2.5 mM MgCl2, 0.3% CHAPS, and one tablet of EDTA-free protease inhibitors (Roche) per 25 ml). The soluble fractions from cell lysates were isolated by centrifugation at 13 000 r.p.m. for 10 min in a microfuge. For immunoprecipitations, 35 μl of a 50% slurry of anti-FLAG affinity gel (Sigma) was added to each lysate and incubated with rotation for 2–3 h at 4°C. Immunoprecipitates were washed three times with lysis buffer. Immunoprecipitated proteins were denatured by the addition of 35 μl of sample buffer and boiling for 5 min, resolved by 8–16% SDS–PAGE, and analysed by immunoblotting. Immunofluorescence assays on HEK-293T cells HEK-293T cells were plated on fibronectin-coated glass coverslips in 35 mm tissue culture dishes, at 300 000 cells/dish. In all, 12–16 h later, cells were transfected with 100 ng of TFEB–3 × FLAG, along with 200 ng Rap2A or Rag GTPase mutants. The next day, cells were subjected to drug treatments or starvation, rinsed with PBS once and fixed for 15 min with 4% paraformaldehyde in PBS at RT. The slides were rinsed twice with PBS and cells were permeabilized with 0.05% Triton X-100 in PBS for 5 min. After rinsing twice with PBS, the slides were incubated with primary antibody in 5% normal donkey serum for 1 h at room temperature, rinsed four times with PBS, and incubated with secondary antibodies produced in donkey (diluted 1:1000 in 5% normal donkey serum) for 45 min at room temperature in the dark, washed four times with PBS. Slides were mounted on glass coverslips using Vectashield (Vector Laboratories) and imaged on a spinning disk confocal system (Perkin-Elmer). High content nuclear translocation assay TFEB–GFP cells were seeded in 384-well plates, incubated for 12 h, and treated with 10 different concentrations of ERK inhibitor U0126 (Sigma-Aldrich) and mTOR inhibitors Rapamycin (Sigma-Aldrich), Torin 1 (Biomarin), and Torin 2 (Biomarin), ranging from 2.54 nM to 50 μM. After 3 h at 37°C in RPMI medium, cells were washed, fixed, and stained with DAPI. For the acquisition of the images, 10 pictures per each well of the 384-well plate were taken by using confocal automated microscopy (Opera high content system; Perkin-Elmer). A dedicated script was developed to perform the analysis of TFEB localization on the different images (Acapella software; Perkin-Elmer). The script calculates the ratio value resulting from the average intensity of nuclear TFEB–GFP fluorescence divided by the average of the cytosolic intensity of TFEB–GFP fluorescence. The results were normalized using negative (RPMI medium) and positive (HBSS starvation) control samples in the same plate. The data are represented by the percentage of nuclear translocation at the different concentrations of each compound using Prism software (GraphPad software). The EC50 for each compound was calculated using non-linear regression fitting (Prism software). Live cell imaging and photobleaching protocol MEFs were transiently transfected with TFEB–GFP and mRFP–Rab7 by nucleofection (Lonza). Cells were plated on glass bottom 35 mm dishes (MatTek Corp.) at a density of 300 000 cells/dish. The next day, cells were transferred to a physiological imaging buffer (130 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 2.5 mM MgCl2, 25 mM HEPES) supplemented with 5 mM glucose and imaged on a spinning disk confocal microscope (Andor Technology) with a 488-nm and a 561-nm laser through a × 63 objective. To achieve photobleaching of individual TFEB–GFP-positive lysosomes, areas of interest were drawn around selected spots, and movie acquisition was started. Sixty seconds later, the spots were photobleached with a high power (50 mM) 488 nm pulse (100 μs/pixel illumination) using the Andor FRAPPA unit. FRAP analysis The fluorescence recovery of photobleached TFEB-GFP-positive lysosomes was analysed using custom-written plugins in ImageJ (National Institutes of Health). Circular areas of interest were drawn around the spots to be analysed, and the integrated fluorescence within these areas was measured throughout the movie. Fluorescence intensity traces from 5 to 10 spots per condition were normalized to the initial value and time aligned, and their mean and s.d. were calculated using Microsoft Excel. Final plots and curve fitting were made with Prism (GraphPad). RNA extraction, quantitative PCR, and statistical analysis Total RNA was extracted from cells using TRIzol (Invitrogen). Reverse transcription was performed using TaqMan reverse transcription reagents (Applied Biosystems). Lysosomal and autophagic gene-specific primers were previously reported (Settembre et al, 2011). Fold change values were calculated using the DDCt method. Briefly, GAPDH and Cyclophillin were used as ‘normalizer' genes to calculate the DCt value. Next, the DDCt value was calculated between the ‘control' group and the ‘experimental' group. Lastly, the fold change was calculated using 2(-DDCt). Biological replicates were grouped in the calculation of the fold change values. Unpaired T-Test was used to calculate statistical significance. Asterisks in the graph indicate that the P-value was <0.05. mTORC1 phosphosite prediction In order to identify possible phosphosites that may be targeted by mTORC1, we developed a simple method that quantifies the agreement between regions around serine or threonine sites in TFEB and the mTORC1 phosphorylation motif (Hsu et al, 2011). The method calculates the score according to a position-specific score matrix for an amino acid at given distance from the phosphosite of interest. The position starts from −5 and runs to +4. The phosphosite is set at position 0. If there is another serine or threonine in this interval, that residue's score is skipped in the sum. We used MyDomains tool in prosite/expasy.org to sketch the functional domains of TFEB. Domain information was retrieved from UniProt/SwissProt database. Human TFEB and its orthologue sequences were aligned by ClustalW (version 2.0.12), using the default parameters. Supplementary Material Supplementary Movie 1 Supplementary Movie 2 Supplementary Movie 3 Supplementary Movie 4 Supplementary Information Review Process File

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Mutations in a novel gene lead to kidney tumors, lung wall defects, and benign tumors of the hair follicle in patients with the Birt-Hogg-Dubé syndrome.

Michael Nickerson, Michelle B Warren, Jorge R. Toro … (2002)

Birt-Hogg-Dubé (BHD) syndrome is a rare inherited genodermatosis characterized by hair follicle hamartomas, kidney tumors, and spontaneous pneumothorax. Recombination mapping in BHD families delineated the susceptibility locus to 700 kb on chromosome 17p11.2. Protein-truncating mutations were identified in a novel candidate gene in a panel of BHD families, with a 44% frequency of insertion/deletion mutations within a hypermutable C(8) tract. Tissue expression of the 3.8 kb transcript was widespread, including kidney, lung, and skin. The full-length BHD sequence predicted a novel protein, folliculin, that was highly conserved across species. Discovery of disease-causing mutations in BHD, a novel kidney cancer gene associated with renal oncocytoma or chromophobe renal cancer, will contribute to understanding the role of folliculin in pathways common to skin, lung, and kidney development.

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Regulation of TFEB and V-ATPases by mTORC1

Samuel Peña-Llopis, Silvia Vega-Rubín-de-Celis, Jacob Schwartz … (2011)

Introduction Target of rapamycin (TOR) complex 1 (TORC1) is a pivotal regulator of cell growth conserved from yeast to humans and implicated in cancer (Wullschleger et al, 2006; Abraham and Eng, 2008). Mammalian TORC1 (mTORC1) includes the atypical serine/threonine kinase mTOR and an adaptor protein, regulatory-associated protein of mTOR (Raptor) (Hara et al, 2002; Kim et al, 2002; Loewith et al, 2002). mTORC1 regulates cell growth, at least in part, by promoting cap-dependent translation and its best-characterized substrates are the eukaryotic initiation factor 4E binding protein-1 (4E-BP1) (Sonenberg and Hinnebusch, 2009) and S6 kinase 1 (S6K1) (Fingar and Blenis, 2004). mTORC1 is specifically inhibited by rapamycin (also called sirolimus) (Jacinto et al, 2004; Sarbassov et al, 2004) and rapamycin analogues are used clinically for many applications including cancer treatment (Abraham and Eng, 2008). Indeed, two rapamycin analogues, temsirolimus (Hudes et al, 2007) and everolimus (Motzer et al, 2008), are approved by the FDA for the treatment of advanced RCC (renal cell carcinoma). mTORC1 is regulated by a complex formed by the proteins tuberous sclerosis complex 1 (TSC1) and 2 (TSC2), which is essential for the relay of signals from oxygen (Brugarolas et al, 2004; Connolly et al, 2006; Kaper et al, 2006; Liu et al, 2006; DeYoung et al, 2008), energy stores (Inoki et al, 2003b; Corradetti et al, 2004; Shaw et al, 2004) and growth factors (Jaeschke et al, 2002; Kwiatkowski et al, 2002; Zhang et al, 2003a). mTORC1 regulation by hypoxia involves the protein regulated in development and DNA damage response 1 (REDD1), a conserved protein with a novel fold (Vega-Rubin-de-Celis et al, 2010), which, when overexpressed is sufficient to inhibit mTORC1 in a TSC1/TSC2-dependent manner (Brugarolas et al, 2004). Interestingly, in some settings, hypoxia signals are transduced via AMP-activated protein kinase (Liu et al, 2006; Wolff et al, 2011), which phosphorylates TSC2 and is normally involved in the relay of energy signals (Inoki et al, 2003b; Gwinn et al, 2008). mTORC1 regulation by growth factors involves TSC2 phosphorylation by Akt (Dan et al, 2002; Inoki et al, 2002; Manning et al, 2002; Potter et al, 2002), extracellular signal-regulated kinase (Erk) (Ma et al, 2005) and ribosomal S6 kinase (Rsk) (Roux et al, 2004). TSC2 functions as a GTPase-activating protein towards Ras homologue enriched in brain (Rheb) (Castro et al, 2003; Garami et al, 2003; Tee et al, 2003; Inoki et al, 2003a; Zhang et al, 2003b), a small GTPase that directly interacts with and activates mTORC1 (Sancak et al, 2007; Avruch et al, 2009). mTORC1 regulation by nutrients is independent of TSC1/TSC2 (Zhang et al, 2003a; Smith et al, 2005; Roccio et al, 2006) and involves its localization to a cellular compartment where it becomes receptive to activation by Rheb (Sancak et al, 2008, 2010). Amino-acid stimulation drives mTORC1 to the surface of the late endosome/lysosome in a manner that depends on a Rag GTPase heterodimer (RagA [or B] bound to RagC [or D]) and a multimeric complex termed the Ragulator (Kim et al, 2008; Sancak et al, 2010). Importantly, constitutive mTORC1 targeting to this compartment renders mTORC1 insensitive to amino-acid withdrawal (Sancak et al, 2010). The significance of the late endosome/lysosome in mTORC1 activation has been recently established in experiments manipulating endosome maturation (Flinn et al, 2010; Li et al, 2010). Disrupting endosome maturation with a constitutively active Rab5 or through depletion of hVps39 inhibited the activation of mTORC1 by growth factors and amino acids (Flinn et al, 2010; Li et al, 2010). A critical component of the endosome required for its acidification and maturation is the vacuolar H+-ATPase (V-ATPase) (Marshansky and Futai, 2008). V-ATPases are multisubunit complexes formed by a membrane-embedded V0 domain (a, d, e, c, c′ and c″ subunits) responsible for proton translocation, and a cytosolic V1 domain (A–H subunits), which provides energy through ATP hydrolysis (Forgac, 2007). V-ATPases are present in virtually every eukaryotic cell and V-ATPase function is essential for survival under a variety of conditions (Beyenbach and Wieczorek, 2006). However, little is known about the regulation of V-ATPases and in particular about how their expression is controlled. Here, we have uncovered a regulatory network linking V-ATPases and endocytosis to mTORC1 that involves the transcription factor EB (TFEB). TFEB is a basic helix-loop-helix (bHLH) leucine zipper transcription factor of the Myc family, microphthalmia transcription factor subfamily (Steingrimsson et al, 2004), that is translocated in a subset of renal tumours (Davis et al, 2003; Kuiper et al, 2003; Srigley and Delahunt, 2009). Results V-ATPase enrichment among mTORC1-regulated genes We identified V-ATPase genes in an unbiased screen for genes whose expression was tightly regulated by mTORC1. We used a robust experimental paradigm involving the combination of two interventions, one genetic and one pharmacologic. In Tsc2 +/+ immortalized mouse embryo fibroblasts (MEFs), but not Tsc2 −/−, mTORC1 is inhibited by serum deprivation (0.1% fetal bovine serum, FBS) (Figure 1A). In Tsc2 −/− cells, abnormally increased mTORC1 activity can be corrected by treatment with rapamycin (Figure 1B). By contrast, rapamycin has little effect on mTORC1 in Tsc2 +/+ cells in which mTORC1 is already inhibited by low serum (Figure 1B). Thus, under serum-deprived conditions, mTORC1 activity is low in Tsc2 +/+ cells (untreated or rapamycin treated), high in Tsc2 −/− cells, but lowered by rapamycin; a pattern referred to as a ‘low/low/high/low' or ‘LLHL' (Figure 1B). This experimental setup creates an ‘asymmetric' response to rapamycin (in Tsc2 −/− versus Tsc2 +/+ cells), which, as illustrated hereafter, can be harnessed to great advantage. Using an Affymetrix MOE430A array platform and duplicate samples, genes were identified with an LLHL expression pattern (upregulated in untreated Tsc2 −/− cells compared with a baseline formed by Tsc2 +/+ cells, untreated or rapamycin treated, and Tsc2 −/− rapamycin-treated cells). With a false discovery rate-corrected P-value (FDR Q) cutoff of 0.05 and a threshold of 1.5-fold, we identified 78 probe sets (henceforth referred to as probes). They corresponded to 75 genes (Figure 1C and D). We hypothesized that if the association of probes with the LLHL pattern was random, there would be similar numbers of probes with the three alternate patterns (HLLL, LHLL and LLLH). Strikingly, however, no probes fitted an HLLL or LHLL pattern, and only 22 probes conformed to an LLLH pattern (Figure 1C). Given these results and assuming a binomial distribution with equal probabilities for all patterns, the probability of finding 78 probes with an LLHL pattern by chance alone was below 1.6 × 10−28. To validate the microarray findings, qRT–PCR was performed on a subset of genes with the weakest LLHL association (highest P-values). In every instance, the LLHL pattern was confirmed and the fold change was even greater than expected (Figure 1E). Among the LLHL probes, we found six for V-ATPase genes (Figure 1D; Supplementary Table S1). The probability that there would be six V-ATPase probes among the 78 probes identified by chance alone was 20% reduction in mean expression levels in Tsc2 −/− MEFs by Tfeb knockdown), including several V-ATPases (Supplementary Table S3). LLHL ‘flattening' was confirmed for several V-ATPase genes by qRT–PCR (Figure 4E). Interestingly, the decrease in the number of genes with an LLHL pattern after Tfeb knockdown was specific for the LLHL pattern. In fact, Tfeb knockdown resulted in an increase in the number of genes with a LLLH pattern (35 versus 323; Figure 4C; Supplementary Table S4). These results suggest that Tfeb is an important effector downstream of mTORC1 involved in the regulation of a substantial number of genes. Regulation of endocytosis by V-ATPases, TFEB and mTORC1 Having determined that mTORC1 regulated V-ATPase expression in cells in culture and in mice and having established that TFEB was required for mTORC1-induced V-ATPase expression, we sought to examine the functional significance of V-ATPase regulation by mTORC1. Because V-ATPases are involved in vesicle trafficking (Marshansky and Futai, 2008), we evaluated the effects of mTORC1 on endocytosis. For these experiments, we used albumin, the most abundant protein in plasma and a protein that is taken up by macrophages and hydrolyzed to amino acids, which are then released in the circulation and are utilized by different cells (Guyton and Hall, 1996). While albumin uptake occurs through different pathways in different cell types (Mayor and Pagano, 2007), albumin is taken up by MEFs through a caveolin-dependent mechanism (Razani et al, 2001). FITC-conjugated bovine serum albumin (FITC-BSA) was efficiently taken up by MEFs, where it was found in punctate structures reminiscent of vesicles, which also contained V-ATPases (Figure 5A). In keeping with the idea that mTORC1 regulates endocytosis, FACS analyses showed higher levels of albumin uptake in Tsc2 −/− than Tsc2 +/+ cells (Figure 5B and C). In addition, treatment of Tsc2 −/− cells with rapamycin downregulated albumin uptake (Figure 5C). Albumin uptake required V-ATPases as determined by experiments using the V-ATPase inhibitor bafilomycin A1 (Bowman and Bowman, 2002) (Figure 5B and C). In addition, albumin uptake was downregulated by siRNA-mediated depletion of essential components of either the V0 or the V1 domain (alone or in combination) in highly transfectable HeLa cervical carcinoma cells (Figure 5D and E). Next, we tested whether Tfeb may be similarly involved in endocytosis. Indeed, Tfeb knockdown resulted in a statistically significant decrease in albumin uptake (Figure 5F). The effects of Tfeb knockdown on albumin uptake in Tsc2 −/− cells were similar to those achieved by treatment with rapamycin (Figure 5F). Taken together, these data show that mTORC1 promotes endocytosis in a Tfeb- and V-ATPase-dependent manner. Regulation of Tfeb phosphorylation and subcellular localization by the Tsc1/Tsc2 complex and rapamycin Having established the importance of Tfeb downstream of mTORC1, we explored the mechanism whereby mTORC1 regulated Tfeb. We observed that not only the expression levels of Tfeb appeared to be upregulated by mTORC1, but also that mTORC1 affected Tfeb electrophoretic mobility on SDS–PAGE (Figures 4B and 6A). In Tsc2 −/− cells, Tfeb was prominently found in a fast-migrating form(s) (Figures 4B and 6A). Treatment of Tsc2 −/− cells with rapamycin shifted Tfeb up giving rise to a pattern similar to that observed in wild-type cells (Figure 6A). Similar results were observed in Tsc1 +/+ and Tsc1 −/− cells (Figure 6B). To determine how quickly Tfeb mobility was affected by rapamycin, a time course was performed. As shown in Figure 6C, rapamycin treatment for 1 h was sufficient to change the distribution of Tfeb. Changes in Tfeb migration may reflect changes in phosphorylation, and phosphoproteomic studies have shown TFEB to be phosphorylated in >10 sites (Dephoure et al, 2008; Mayya et al, 2009). To determine whether Tfeb migration was affected by phosphorylation, we evaluated the effects of λ phosphatase treatment. As shown in Figure 6D, the migration of Tfeb was accelerated by treatment of cell lysates with λ phosphatase. Parenthetically, by comparison to similarly processed samples incubated without λ phosphatase for the same amount of time, phosphatase exposure not only accelerated Tfeb migration, but also led to a downregulation of total Tfeb protein levels (Figure 6D). Notably, even fast-migrating Tfeb in Tsc2 −/− cells was subject to a downshift following phosphatase treatment (Figure 6D). These results indicate that even in Tsc2 −/− cells, Tfeb retained some level of phosphorylation. We considered that the changes in Tfeb phosphorylation may be linked to changes in its subcellular localization and performed subcellular fractionation experiments. Whereas in Tsc2 +/+ cells, nuclear Tfeb was undetectable, Tfeb was found in nuclear fractions of Tsc2 −/− cells (Figure 6E). In the nucleus, Tfeb appeared to be present almost exclusively in a fast-migrating form (Figure 6E). To determine whether nuclear Tfeb retained some level of phosphorylation, nuclear extracts were treated with λ phosphatase. As shown in Figure 6F, this resulted in a further shift, indicating that nuclear Tfeb was also phosphorylated. Since phosphatase treatment collapsed both nuclear and cytosolic Tfeb onto an undistinguishable band, the differences in migration likely reflect differences in phosphorylation. Next, we sought to determine how rapamycin affected nuclear Tfeb in Tsc2 −/− cells. Rapamycin treatment caused a shift up in nuclear Tfeb mobility and a progressive decline in nuclear Tfeb (Figure 6G). Taken together, these data show that the migration and nuclear localization of Tfeb are coordinately regulated by Tsc1/Tsc2 and rapamycin and suggest that mTORC1 controls the phosphorylation state and nuclear localization of Tfeb. Regulation of TFEB phosphorylation and nuclear localization by mTORC1 To extend these studies, we examined the regulation of TFEB by mTORC1 in HeLa cells. HeLa cells were transduced with lentiviruses encoding a TSC2 shRNA or a scrambled control and, as for MEFs, cultured for 24 h in 0.1% FBS with or without rapamycin. The results were remarkably similar to those observed in MEFs. TSC2 inactivation led to a downshift in TFEB and this was reverted by rapamycin (Figure 7A and B). As in MEFs, phosphatase treatment accelerated TFEB migration even in cells depleted of TSC2 (Figure 7C). Subcellular fractionation studies showed the presence of fast-migrating TFEB in nuclear fractions of cells depleted of TSC2, but not in controls (Figure 7D) and nuclear TFEB retained some level of phosphorylation (Figure 7E). Thus, the regulation of TFEB phosphorylation and nuclear localization by TSC1/TSC2 and rapamycin was conserved across cell types. To unequivocally establish that TFEB regulation by TSC1/TSC2 and rapamycin was indeed mTORC1 dependent, we inactivated mTORC1 by knocking-down Raptor. HeLa cells were transduced with a Raptor shRNA (or a scrambled control) and, following selection, were subjected to TSC2 depletion using siRNA. As shown in Figure 7F, in cells depleted of Raptor, TSC2 RNAi failed to accelerate TFEB mobility. In a complementary set of experiments, Raptor siRNA (like rapamycin) retarded TFEB mobility in HeLa cells depleted of TSC2 (Figure 7G). Regardless of the means to deplete Raptor, Raptor inactivation prevented the accumulation of faster-migrating TFEB (Figure 7F and G). Taken together, our data indicate that mTORC1 coordinately regulates TFEB phosphorylation and nuclear localization in multiple cell types. mTORC1-dependent nuclear localization of TFEB requires C-terminal serine-rich motif To dissect TFEB regulation by mTORC1, we first identified conditions in which ectopically expressed TFEB behaved like endogenous TFEB. Epitope-tagged TFEB, when expressed at low levels in HeLa cells, preserved its regulation by mTORC1; it was enriched in the nuclei of TSC2-depleted cells and the levels were decreased by rapamycin (Figure 8A). To determine what regions were important for TFEB regulation by mTORC1, we examined a series of deletions (Figure 8B). TFEB233–476 and TFEB290–476 behaved like full-length TFEB, indicating that the N-terminal half of TFEB was dispensable for mTORC1-dependent nuclear localization (Figure 8C). Within this region, mutation of a serine residue (S142) proposed to be phosphorylated by ERK2 and to regulate TFEB nuclear localization (Settembre et al, 2011) had no appreciable effect (Figure 8D and E). Deletion of residues 1–320 resulted in a protein that was enriched in nuclei (Figure 8C). Interestingly, deletion of just 15 amino acids from the C-terminus abrogated mTORC1-dependent nuclear localization (Figure 8C). By contrast, deletion of seven amino acids had no effect (Figure 8C). These data indicated that there were critical residues between 462–469. This corresponded to a serine-rich region (462SSRRSSFS469). To further evaluate, this serine-rich motif, we mutated all S to A (5 × SA). Interestingly, TFEB5 × SA was largely cytoplasmic and failed to be driven into the nucleus in cells with active mTORC1 (Figure 8D and E). Thus, a serine(s) within this motif is necessary for mTORC1-dependent nuclear TFEB localization. Conversely, mutation of the same residues to aspartic acid resulted in an enrichment of TFEB in the nucleus (Figure 8D and E). TFEB5 × SD localized to the nucleus even in cells with inactive mTORC1. Interestingly, the percentage of cells with TFEB5 × SD in the nucleus was the same as for wild-type TFEB in mTORC1-active cells (Figure 8E; see also Figure 8A). These data show that the presence of phosphomimetic residues in the serine-rich motif obviates the mTORC1 requirement for TFEB nuclear localization. The simplest explanation for these data is that a serine(s) in the C-terminal serine-rich motif is phosphorylated in an mTORC1-dependent manner and that phosphorylation is both necessary and sufficient to drive TFEB into the nucleus (see model in Figure 8F). Complex TFEB regulation beyond mTORC1 by multiple signals Next, we examined TFEB in wild-type MEFs and HeLa cells grown under standard conditions (10% FBS). TFEB was found in both a slow and fast-migrating form(s) in MEFs, but predominantly in a slow migrating form(s) in HeLa cells (Figure 9A). We evaluated the effects of multiple interventions on TFEB mobility. Glucose withdrawal resulted in a downshift in TFEB in both MEFs and HeLa cells (Figure 9A). Amino acid withdrawal induced acute changes in TFEB migration in HeLa cells and subsequently led to the accumulation of slow migrating TFEB, which was also observed in MEFs (Figure 9A). The mobility of TFEB was also affected by serum deprivation in both MEFs and HeLa cells (Figure 9A). Finally, changes in TFEB mobility were also induced by treatment with a reducing agent, DTT. DTT led to a progressive shift in TFEB to a fast-migrating form(s) in both HeLa and MEFs (Figure 9A). Overall, these data show that TFEB is extensively and dynamically regulated by multiple stimuli. While post-translational modifications other than phosphorylation may be involved in the changes observed in TFEB migration, TFEB could be down-shifted by phosphatase treatment in every instance (data not shown). The dynamic changes in TFEB mobility induced by the various interventions could not be explained by their expected effects on mTORC1 suggesting that TFEB regulation involves multiple pathways and is likely to be rather complex. This is consistent with the finding that TFEB is phosphorylated in >10 sites (Dephoure et al, 2008; Mayya et al, 2009; Yu et al, 2011). Finally, since TFEB is an oncogene (Davis et al, 2003; Kuiper et al, 2003; Srigley and Delahunt, 2009), we tested whether TFEB was implicated in the regulation of cell proliferation. As shown in Figure 9B, stable depletion of TFEB in both MEFs and HeLa cells lowered the rates of cell expansion. Discussion Using a robust experimental paradigm, we uncovered a novel regulatory network linking endocytosis to TFEB and mTORC1. An unbiased gene expression analysis coupled with a design amenable to refined statistics led us to identify a connection between mTORC1 and V-ATPase genes. Publicly available data sets confirmed the link and experiments in mice showed that mTORC1 regulated V-ATPase expression also in vivo. A recent study had established that TFEB regulated lysosomal biogenesis (Sardiello et al, 2009) and among the genes regulated by TFEB we found several V-ATPases. These results led us to hypothesize that V-ATPase regulation by mTORC1 may be mediated by TFEB. Indeed, TFEB was necessary for V-ATPase upregulation by mTORC1. mTORC1 regulated TFEB phosphorylation and nuclear localization and a C-terminal serine-rich motif was identified that is essential for mTORC1-dependent TFEB nuclear localization. Our data suggest that mTORC1 induces the phosphorylation of a serine(s) within this motif driving thereby TFEB to the nucleus. TFEB regulates the expression of V-ATPases and other lysosomal genes and TFEB was required for mTORC1-induced endocytosis. To our knowledge this is the first study to show that endogenous TFEB is regulated at the subcellular localization level and that mTORC1 promotes TFEB nuclear localization. These data link an oncogenic transcription factor that is a master regulator of lysosomal biogenesis, TFEB, to mTORC1 and endocytosis. An increasing amount of evidence implicates TORC1 in endocytosis. mTOR was identified in an RNAi screen for kinases involved in clathrin-mediated endocytosis (Pelkmans et al, 2005) and Tsc2-deficient cells were previously shown to have increased fluid-phase endocytosis (Xiao et al, 1997). In Drosophila melanogaster, a sensitized screen for genes that modified a tissue-specific dTOR overexpression phenotype identified an important regulator of endocytosis, Hsc70-4, a clathrin uncoating factor, and the authors showed that dTOR was involved in fat body endocytosis (Hennig et al, 2006). Interestingly, besides V-ATPases, mTORC1 regulated the expression of multiple lysosomal genes (Figure 1D; see also Supplementary Table S5). These effects may be mediated, at least in part, by TFEB, which regulates lysosome biogenesis (Sardiello et al, 2009). Thus, one mechanism whereby mTORC1 and TFEB may affect endocytosis is by regulating V-ATPase levels and lysosomes. The lysosome is the final destination not only for endosomal cargo but also for autophagosomes. In response to starvation, mTORC1 is inhibited leading to the formation of autophagosomes that engulf intracellular components for recycling and sustenance (He and Klionsky, 2009; Kroemer et al, 2010). Autophagosomes and their content are delivered to lysosomes, which accumulate in the perinuclear area (Korolchuk et al, 2011). Autophagosomes fuse with lysosomes to generate autolysosomes, a process that rapidly depletes the lysosomal pool (Yu et al, 2010). The degradation of macromolecules and release of their constituents into the cytosol reactivates mTORC1 (Yu et al, 2010). mTORC1 reactivation terminates autophagy and through a poorly understood mechanism repletes the lysosome pool (Yu et al, 2010). However, how the pool of lysosomes is repleted is unknown. Our results suggest that lysosomal reformation in response to autophagy, which is mTORC1 dependent (Yu et al, 2010), may be mediated, at least in part, by TFEB. mTORC1 coordinately regulates TFEB phosphorylation and nuclear localization. mTORC1 promoted TFEB nuclear localization and this process required a C-terminal serine-rich motif. Serine substitution to non-phosphorylatable residues abrogated TFEB regulation by mTORC1. More importantly, mutation to phosphomimetic amino acids was sufficient to reproduce the effect of mTORC1 on TFEB nuclear localization obviating the need for mTORC1. These data support a model in which mTORC1 (or an effector kinase) directly phosphorylates a serine(s) within the C-terminal serine-rich motif driving thereby TFEB to the nucleus. Consistent with this notion, nuclear TFEB was phosphorylated. This model may seem at odds with the observation that nuclear TFEB is fast-migrating and possibly hypo-phosphorylated by comparison to cytoplasmic (presumably cytosolic) TFEB. However, as our data suggest, nuclear TFEB may be phosphorylated on sites that are not phosphorylated on cytosolic TFEB, and thus no inferences can be drawn about the degree of phosphorylation based on SDS–PAGE migration. Furthermore, while we have identified a critical motif involved in mTORC1 regulation, other sites in TFEB appear to be regulated by mTORC1 (Yu et al, 2011). Our results further emphasize the link between mTORC1 and the late endosome/lysosome and provide evidence for the existence of bidirectional regulatory loops. mTORC1 localizes to the surface of the late endosome/lysosome in a highly choreographed manner (Korolchuk et al, 2011; Narita et al, 2011). The late endosome/lysosome is necessary for mTORC1 activation (Flinn et al, 2010; Li et al, 2010; Sancak et al, 2010) and mTORC1 is reactivated under conditions of persistent starvation following macromolecule degradation in autolysosomes (Yu et al, 2010). However, not only is mTORC1 downstream of the lysosome, but, inasmuch as mTORC1 regulates TFEB and V-ATPases, mTORC1 is also upstream. mTORC1 reactivation by autolysosomes requires Spinster, a lysosomal efflux sugar transporter. Spinster has been proposed to act by regulating lysosomal pH (Rong et al, 2011), which is controlled primarily by the V-ATPase. Interestingly, not only has luminal pH been implicated in mTORC1 regulation, but changes in cytosolic pH occur in response to alterations in nutrient conditions (Korolchuk et al, 2011) and have been implicated in the regulation of lysosome topology and mTORC1 (Korolchuk et al, 2011). Recently, TFEB was found to regulate autophagy gene expression (Settembre et al, 2011). Starvation led to ERK2 inactivation, which in turn, decreased TFEB phosphorylation and increased its nuclear localization. This model is in principle compatible with our results. TFEB is phosphorylated in over 10 sites (Dephoure et al, 2008; Mayya et al, 2009; Yu et al, 2011), and mechanisms likely exist that drive TFEB into the nucleus independently of the activation state of mTORC1. However, the studies do differ in one aspect. Whereas mutation of the putative ERK2 phosphorylation site, S142, to A is reported to be sufficient to drive TFEB into the nucleus (Settembre et al, 2011), we find that, at least under low serum conditions, the subcellular localization of TFEB is not appreciably altered by a S142A mutation. The experimental conditions studied by Settembre et al (2011), which involve evaluating ectopically expressed TFEB in cells in the absence of glucose, amino acids and serum, are quite different from ours. Individual deprivation of glucose, amino acids or serum has profound, dynamic and diverse effects on TFEB (see Figure 9A). Given these observations and the complexity of TFEB phosphorylation (Dephoure et al, 2008; Mayya et al, 2009; Yu et al, 2011), how starvation of all three sources will affect TFEB is unclear. Consistent with this complexity, the changes in TFEB migration observed following deprivation of individual factors could not be explained by the predicted changes on mTORC1 activity. In fact, we speculate that the discovery that mTORC1 regulates TFEB may have been possible only because of the tight experimental system we used. Besides V-ATPases and lysosomal genes, many other genes downstream of mTORC1 were regulated in a Tfeb-dependent manner. Approximately 25% of genes induced by mTORC1 required Tfeb. Experiments are ongoing to determine how many of these genes are directly regulated by Tfeb. A similar experimental setup previously identified Hif-1α as an mTORC1 effector (Brugarolas et al, 2003), and Hif-1α is an important mediator of a metabolic gene expression programme (Duvel et al, 2010). Along with sterol regulatory element-binding protein-1 (Porstmann et al, 2008), a transcription factor whose nuclear localization is regulated by mTORC1, and Hif-1α, TFEB is an important mediator of mTORC1 effects on gene expression. The discovery that mTORC1 regulates TFEB has clinical implications. It may explain the expression of melanocyte lineage markers in tumours of patients with TSC (Martignoni et al, 2008), a syndrome arising from mutations in either the TSC1 or TSC2 genes, resulting in constitutive mTORC1 activation (El-Hashemite et al, 2003; Kenerson et al, 2007). Patients with TSC are predisposed to develop renal angiomyolipomas, which express melanocytic markers such as HMB45 and Melan-A (Martignoni et al, 2008), and since expression of these proteins in melanomas is thought to be driven by TFEB (or related family members with whom TFEB can heterodimerize) (Steingrimsson et al, 2004), these markers may be similarly driven by TFEB in renal angiomyolipomas. Interestingly, TFEB functions as an oncogene in the kidney. The TFEB gene is translocated in a subset of renal tumours where it is subjected to a robust heterologous promoter (Davis et al, 2003; Kuiper et al, 2003). Translocation carcinomas tend also to aberrantly express melanocytic markers (Srigley and Delahunt, 2009), which may be similarly driven by TFEB. TFEB activation may also account for the expression of cathepsin-K, a lysosomal protease that has been recently proposed as a marker for this tumour type (Martignoni et al, 2009). Interestingly, translocation carcinomas are often recognized by immunohistochemical studies showing high levels of nuclear TFEB (Srigley and Delahunt, 2009). Because TFEB nuclear localization is regulated by mTORC1 and as we show, sirolimus excludes TFEB from the nucleus, sirolimus, or its derivatives, temsirolimus and everolimus, may be effective against this tumour type. While this study focused on a small subset of genes, many genes were identified whose expression was regulated by mTORC1 not only positively but also negatively (Supplementary Figures S8–S10; Supplementary Table S1). Moreover, genes were also found whose expression was regulated by Tsc2 independently of mTORC1 (LLHH and HHLL patterns). As previously reported (Brugarolas et al, 2003), this would point towards mTORC1-independent functions of the Tsc1/Tsc2 complex (Supplementary Figure S8; Supplementary Table S1). In addition, the expression of some genes appeared to be regulated by rapamycin to a significantly greater extent than by Tsc2 (HLHL and LHLH patterns) (Supplementary Figure S8; Supplementary Table S1). Importantly, in all these settings, analyses of alternative patterns showed the associations to be statistically significant (Supplementary Figure S8). Incidentally, among the genes with an HLHL pattern, there was enrichment for genes implicated in glycolysis, pentose phosphate pathway and fatty acid biosynthesis (Supplementary Table S5). These pathways were recently reported to be regulated by mTORC1 (Duvel et al, 2010) and our results suggest that they are affected by rapamycin to a greater extent than by Tsc1/Tsc2. In summary, our work uncovered a novel regulatory network connecting mTORC1 to a master regulator of lysosome biogenesis, TFEB, which is essential for mTORC1-induced endocytosis. Materials and methods Cell culture, drug and hypoxia treatments MEFs Tsc2 +/+; p53 −/− (Tsc2 +/+) and Tsc2 −/−; p53 −/− (Tsc2 −/−) were a gift of DJ Kwiatkowski (Brigham and Women's Hospital, Harvard Medical School, Boston, MA). Cells were grown in high glucose Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% FBS (HyClone) and 1% (v/v) penicillin/streptomycin (P/S) (Gibco) in a humidified incubator at 37°C and 5% CO2. For experiments, cells were plated to achieve ∼70% confluency at the time of treatment. For low-serum experiments, cells were washed twice in PBS and medium was changed to the same base medium but supplemented with 0.1% FBS and cells were harvested after 24 h. Where indicated, rapamycin (LC Laboratories) was used at 25 nM in methanol and methanol was used as vehicle. Where indicated, cells were treated with actinomycin D (Sigma) at 10 μg/ml, bafilomycin A1 (LC Laboratories) at 100 nM, or DTT (10 mM). For growth curves, cells were plated on day 0, maintained in 10% FBS and triplicates were counted every 24 h. For hypoxia experiments, cells were exposed to 1% O2 and 5% CO2 at 37°C in a hypoxia chamber (Coy Laboratory Products). For starvation time courses, cells were washed with PBS twice and starved of amino acids (amino acid-free media), glucose (glucose-free media) or serum (0.1% FBS) for different amounts of time. Hif-1α, Tfeb, Raptor and TSC2 shRNAs Recombinant lentiviruses were generated in HEK293T cells by transient transfection using calcium phosphate with the envelope plasmid pMD2.G (Database ID p486), packaging plasmid psPAX2 (p485) and the specific plasmid of interest. pMD2.G, psPAX2 and pLKO.1 or pGIPZ vectors were transfected at a ratio of 3:8:10. Supernatants were collected for 2 days, passed through a 0.45-μm filter, and used to infect MEFs or HeLa cells for 24 h in medium containing 8 μg/ml polybrene (Sigma). MEFs and HeLa cells were selected with 2 μg/ml puromycin for at least 1 week and HeLa cells were further enriched by FACS. The following vectors were used: pLKO.1 vectors with a Hif-1α shRNA (5′-AGAGGTGGATATGTCTGGG-3′) (p481) generously provided by LB Gardner (Nemetski and Gardner, 2007), a mouse Tfeb shRNA from Open Biosystems (sequence A, TRCN0000085548, 5′-CGGCAGTACTATGACTATGAT-3′) (p633) or a designed Tfeb shRNA (sequence B, 5′-GGCAGTACTATGACTATGATG-3′) (p652), a human TFEB shRNAs from Open Biosystems (sequence 110, TRCN0000013110, 5′-GAGACGAAGGTTCAACATCAA-3′) (p700) or (sequence 111, TRCN0000013111, 5′-GAACAAGTTTGCTGCCCACAT-3′) (p701), a raptor shRNA from Addgene (Addgene plasmid 1858) (p741) or a scrambled sequence (5′-GGGTCTGTATAGGTGGAGA-3′) (p480). pGIPZ vectors containing a TSC2 (p496) or a scrambled (p483) shRNA were from Open Biosystems. Western blotting and antibodies Cell lysates and western blotting was performed as previously described (Vega-Rubin-de-Celis et al, 2010), using antibodies from the following sources: S6K1, phospho-S6K1 (T389), S6, phospho-S6 (S235/236), total 4E-BP1, phospho-4E-BP1 (T37/46), phospho-4E-BP1 (S65) from Cell Signaling; HIF-1α, TSC1 and Menin from Bethyl laboratories; TSC2, ATF4 and c-Myc from Santa Cruz; Raptor from Millipore; Erk1/2, phospho-Erk1/2 (T183/Y185) and Tubulin from Sigma; Cyclophilin B from Abcam; GLUT-1 from Novus Biologicals; and Grp78 from BD Biosciences. Atp6v1a and Atp6v1b2 antibodies were a generous gift from D Brown (Massachusetts General Hospital, Boston, MA); and Atp6v0a1 and Atp6v0a3 antibodies were kindly provided by V Marshansky (Massachusetts General Hospital, Boston, MA). Tfeb antibodies were developed in collaboration with Bethyl Laboratories. For the remaining Materials and methods, please see the Supplementary data section. Accession numbers Microarrays were deposited in GEO (http://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE27982 and GSE28021. Supplementary Material Supplementary Data Supplementary Table S1 Supplementary Table S3 Supplementary Table S4 Review Process File

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Journal ID (nlm-ta): J Cell Biol

Journal ID (iso-abbrev): J. Cell Biol

Journal ID (hwp): jcb

Title: The Journal of Cell Biology

Publisher: The Rockefeller University Press

ISSN (Print): 0021-9525

ISSN (Electronic): 1540-8140

Publication date (Print): 30 September 2013

Volume: 202

Issue: 7

Pages: 1107-1122

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[1 ]Department of Cell Biology and [2 ]Program in Cellular Neuroscience, Neurodegeneration, and Repair, Yale University School of Medicine, New Haven, CT 06510

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Correspondence to Shawn M. Ferguson: shawn.ferguson@ 123456yale.edu

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Publisher ID: 201307084

DOI: 10.1083/jcb.201307084

PMC ID: 3787382

PubMed ID: 24081491

SO-VID: ea95e7e4-7a47-463f-ae30-3526a71b8f7d

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