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Regulation of TFEB and V-ATPases by mTORC1

Samuel Pena-Llopis1,2,3,6,Silvia Vega-Rubin-de-Celis1,2,3,6,Jacob C Schwartz4, Nicholas C Wolff1,2,3,Tram Anh T Tran1,2,3, Lihua Zou5,Xian-Jin Xie3, David R Corey4 andJames Brugarolas1,2,3,*1Department of Developmental Biology, University of TexasSouthwestern Medical Center, Dallas, TX, USA, 2Department of InternalMedicine, Oncology Division, University of Texas Southwestern MedicalCenter, Dallas, TX, USA, 3Simmons Comprehensive Cancer Center,University of Texas Southwestern Medical Center, Dallas, TX, USA,4Department of Pharmacology, University of Texas SouthwesternMedical Center, Dallas, TX, USA and 5Cancer Program, Broad Instituteof Harvard and MIT, Cambridge, MA, USA

Mammalian target of rapamycin (mTOR) complex 1

(mTORC1) is an important, highly conserved, regulator

of cell growth. Ancient among the signals that regulate

mTORC1 are nutrients. Amino acids direct mTORC1 to the

surface of the late endosome/lysosome, where mTORC1

becomes receptive to other inputs. However, the interplay

between endosomes and mTORC1 is poorly understood.

Here, we report the discovery of a network that links

mTORC1 to a critical component of the late endosome/

lysosome, the V-ATPase. In an unbiased screen, we found

that mTORC1 regulated the expression of, among other

lysosomal genes, the V-ATPases. mTORC1 regulates

V-ATPase expression both in cells and in mice. V-ATPase

regulation by mTORC1 involves a transcription factor

translocated in renal cancer, TFEB. TFEB is required for

the expression of a large subset of mTORC1 responsive

genes. mTORC1 coordinately regulates TFEB phosphoryla-

tion and nuclear localization and in a manner dependent

on both TFEB and V-ATPases, mTORC1 promotes endocy-

tosis. These data uncover a regulatory network linking an

oncogenic transcription factor that is a master regulator of

lysosomal biogenesis, TFEB, to mTORC1 and endocytosis.

The EMBO Journal (2011) 30, 3242–3258. doi:10.1038/

emboj.2011.257; Published online 29 July 2011

Subject Categories: membranes & transport; signal transduction

Keywords: autophagy; lysosome; microarray; RCC; Tcfeb

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 sub-

strates 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 phos-

phorylation 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 compart-

ment 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 compart-

ment renders mTORC1 insensitive to amino-acid withdrawal

(Sancak et al, 2010). The significance of the late endosome/

lysosome in mTORC1 activation has been recently establishedReceived: 21 December 2010; accepted: 24 June 2011; publishedonline: 29 July 2011

*Corresponding author. Department of Internal Medicine, OncologyDivision, University of Texas Southwestern Medical Center, 6000 HarryHines Boulevard, NB5.102A, Dallas, TX 75390-9133, USA.Tel.: þ 1 214 648 4059; Fax: þ 1 214 648 1960;E-mail: [email protected] authors contributed equally to this work

The EMBO Journal (2011) 30, 3242–3258 | & 2011 European Molecular Biology Organization | Some Rights Reserved 0261-4189/11

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The EMBO Journal VOL 30 | NO 16 | 2011 &2011 European Molecular Biology Organization

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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 Hþ -ATPase

(V-ATPase) (Marshansky and Futai, 2008). V-ATPases are

multisubunit complexes formed by a membrane-embedded

V0 domain (a, d, e, c, c0 and c00 subunits) responsible for

proton translocation, and a cytosolic V1 domain (A–H sub-

units), 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 Tsc2þ /þ 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, rapamy-

cin has little effect on mTORC1 in Tsc2þ /þ cells in which

mTORC1 is already inhibited by low serum (Figure 1B). Thus,

under serum-deprived conditions, mTORC1 activity is low in

Tsc2þ /þ 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 Tsc2þ /þ 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 Tsc2þ /þ 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 per-

formed on a subset of genes with the weakest LLHL associa-

tion (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 o9�10�8. Furthermore, an

analysis of all the V-ATPase probes in the array showed

a striking LLHL pattern for most genes (Figure 2A) and the

results were confirmed in a subset of genes by qRT–PCR

(Figure 2B) and western blot (Figure 2C).

Because Tsc2þ /þ and Tsc2�/� cells are also p53�/�, they

may have diverged from each other over time, and thus, we

examined the role of mTORC1 in V-ATPase gene expression in

primary MEFs. MEFs were generated from Tsc1floxP/floxP

(Tsc1F/F) (Kwiatkowski et al, 2002) as well as littermate

Tsc1þ /þ embryos, and exposed to recombinant adenoviruses

driving the expression of Cre recombinase (Ad-Cre). Ad-Cre

transduction of Tsc1F/F MEFs led to effective recombination

of loxP sites (Supplementary Figure S1A), with loss of the

Tsc1 protein, activation of mTORC1 (Figure 2D) and V-ATPase

upregulation (Figure 2D).

To broaden these observations, we examined publicly

available gene expression data sets. Recently, a study was

reported in which the effects of rapamycin were evaluated

over time in serum-deprived Tsc2�/� and Tsc1�/� immorta-

lized MEFs (Duvel et al, 2010). While in this study rapamycin

was administered only to the mutant cells, we observed a

previously unrecognized downregulation of V-ATPase expres-

sion by rapamycin (Supplementary Figure S2). V-ATPase

expression was reduced by 6 h (Supplementary Figure S2)

and maximal V-ATPase downregulation was observed

at 24 h, the time at which our experiments were conducted

(Figure 1D). Similar results were observed in a second

publicly available data set of immortalized Tsc2�/� MEFs

treated with rapamycin (Cunningham et al, 2007) (Supple-

mentary Figure S3). The analysis of these data sets confirmed

our results that mTORC1 regulated V-ATPase expression.

mTORC1 regulation of V-ATPase expression in

the mouse

To extend our observations to an in vivo setting, we injected

Tsc1F/F mice with Ad-Cre intravenously under conditions

known to induce recombination of target genes in hepato-

cytes (Kucejova et al, 2011). Ad-Cre (but not control Ad-eGFP

or saline) led to effective recombination of loxP sites

(Supplementary Figure S1B), loss of the Tsc1 protein and

robust activation of mTORC1 (Figure 2E). This was accom-

panied by increased expression of V-ATPase genes at both the

mRNA and protein levels (Figure 2E and F; Supplementary

Figure S4A). As Ad-Cre injection affected the expression of

many housekeeping genes including actin, we used as a

reference cyclophilin B, whose expression was seemingly

unaffected by Ad-Cre (Figure 2F; Supplementary Figure S4).

While control mice were infected also with an adenovirus

(Ad-GFP), we could not rule out effects of Cre recombinase

Regulation of TFEB and V-ATPases by mTORC1S Pena-Llopis et al

&2011 European Molecular Biology Organization The EMBO Journal VOL 30 | NO 16 | 2011 3243

beyond loxP site recombination, and additional experiments

were performed using Tsc1þ /þ mice injected with Ad-Cre

as a control (Figure 2F; Supplementary Figure S4B).

Independently of the control, Tsc1 loss was accompanied by

mTORC1 activation and increased V-ATPase expression.

Next, we explored the regulation of V-ATPases by mTORC1

in a previously reported tumour model. Transgenic mice

expressing a constitutively active myristoylated form of

Akt1 (myr-Akt) in the prostate develop prostate intraepithe-

lial neoplasia (PIN) and PIN lesions exhibit robust mTORC1

Symbol P FDR Q FC Gene titleFam50a 7E–7 0.005 1.5 Family with sequence similarity 50, member ATuba4a 3E–6 0.010 1.9 Tubulin, α 4AScpep1 9E–6 0.010 1.5 Serine carboxypeptidase 1

Sord 1E–5 0.010 2.9 Sorbitol dehydrogenaseAnpep 2E–5 0.012 1.9 Alanyl (membrane) aminopeptidasePfkfb3 2E–5 0.013 1.6 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3

Atp6v0b 2E–5 0.013 1.7 ATPase, H+ transporting, lysosomal V0 subunit BNpy 3E–5 0.013 2.1 Neuropeptide Y

Tuba4a 3E–5 0.013 2.3 Tubulin, α 4A4E–5 0.015 1 9 Ribonuclease P 25 subunit (human)

Tsc2 –/–

Veh Rapa Veh RapaTsc2+/+

Tsc2 –/–Tsc2+/+

Tsc2 –/–Tsc2+/+

DA

% FBS 10 0.1 10 0.1

S6K1

Tsc2

0.1% FBS

Rpp25 4E–5 1.9Mid1ip1 5E–5 0.015 1.6 Mid1 interacting protein 1 (gastrulation specific G12-like (zebrafish))

Mrps6 7E–5 0.017 1.9 Mitochondrial ribosomal protein S6Vps26a 7E–5 0.017 1.6 Vacuolar protein sorting 26 homologue A (yeast)

Flcn 8E–5 0.017 1.6 FolliculinMt1 8E–5 0.017 1.6 Metallothionein 1

Cyc1 1E–4 0.018 1.5 Cytochrome c-12410022L05Rik 1E–4 0.018 1.6 RIKEN cDNA 2410022L05 gene

Dclk1 1E–4 0.018 1.8 Doublecortin-like kinase 1Snta1 1E–4 0.018 1.5 Syntrophin, acidic 1Itm2a 1E–4 0.018 1.5 Integral membrane protein 2A

Sesn3 1E–4 0.018 2.2 Sestrin 3Galc 1E–4 0.018 1.6 GalactosylceramidaseNeu1 2E–4 0.018 2.0 Neuraminidase 1Gga2 2E–4 0.018 1.5

B

S6

Tubulin

Rapamycin – + – +

S6K1

S6

Golgi-associated, γ adaptin ear containing, ARF-binding protein 2Esd 2E–4 0.018 1.6 Esterase D/formylglutathione hydrolase

Chchd10 2E–4 0.018 1.9 Coiled-coil-helix-coiled-coil-helix domain containing 10Hmox1 2E–4 0.019 2.3 Heme oxygenase (decycling) 1Acot13 2E–4 0.020 1.6 Acyl-CoA thioesterase 13

A030009H04Rik 3E–4 0.020 1.6 RIKEN cDNA A030009H04 geneSteap3 3E–4 0.020 1.6 STEAP family member 3

Alad 3E–4 0.020 1.8 Aminolevulinate, Δ-, dehydrataseSlc25a33 3E–4 0.020 1.6 Solute carrier family 25, member 33

Cndp2 3E–4 0.020 1.6 CNDP dipeptidase 2 (metallopeptidase M20 family)Ube2l3 3E–4 0.022 1.5 Ubiquitin-conjugating enzyme E2L 3Snapc3 4E–4 0.023 1.6 Small nuclear RNA activating complex, polypeptide 3

Kif1a 4E–4 0.023 1.5 Kinesin family member 1AGats 4E–4 0.023 1.7 GATS protein-like 2

5E–4 0.024 1.6

Tubulin

ATPase, H+ transporting, lysosomal V0 subunit BRenbp 5E–4 0.025 1.9 Renin-binding protein

Il1rn 6E–4 0.026 1.5 Interleukin 1 receptor antagonistUchl3/Uchl4 6E–4 0.026 1.7 Ubiquitin carboxyl-terminal esterase L3 (ubiquitin thiolesterase) and L4

Steap1 6E–4 0.026 2.3 Six transmembrane epithelial antigen of the prostate 1Spon2 6E–4 0.027 1.8 Spondin 2, extracellular matrix proteinCreg1 6E–4 0.027 2.9 Cellular repressor of E1A-stimulated genes 1

Nrk 7E–4 0.027 2.3 Nik-related kinaseLgals3 7E–4 0.027 4.3 Lectin, galactose binding, soluble 3

Hif1a 7E–4 0.027 1.6 Hypoxia-inducible factor 1, α subunitMaoa 7E–4 0.027 1.5 monoamine oxidase A

Gla 7E–4 0.028 2.1 Galactosidase, �Atp6v1g1 8E–4 0.028 1.6 ATPase, H+ transporting, lysosomal V1 subunit G1

Uap1l1 8E–4 0.028 2.2 UDP-N-acteylglucosamine pyrophosphorylase 1-like 1Cxadr 8E–4 0.029 1.8 Coxsackievirus and adenovirus receptor

mTORC1 activity

C No. of significant probesets (genes)

L L H L

Abcb6 9E–4 0.029 1.7 ATP-binding cassette, subfamily B (MDR/TAP), member 6Oxct1 9E–4 0.029 1.5 3-Oxoacid CoA transferase 1Ero1l 9E–4 0.029 2.1 ERO1-like (S. cerevisiae)

Atp6ap2 9E–4 0.030 1.6 ATPase, H+ transporting, lysosomal accessory protein 20610010O12Rik 0.001 0.031 1.6 RIKEN cDNA 0610010O12 gene

Pgp 0.001 0.034 1.6 Phosphoglycolate phosphataseLrpap1 0.001 0.034 1.5 Low-density lipoprotein receptor-related protein associated protein 1

Fmr1 0.002 0.035 1.6 Fragile X mental retardation syndrome 1 homologueCyb5b 0.002 0.036 1.7 Cytochrome b5 type B

Cdsn 0.002 0.039 1.6 CorneodesmosinEmb 0.002 0.039 1.8 Embigin

Ddit3 0.002 0.040 1.6 DNA-damage-inducible transcript 3Gpr137b 0.002 0.041 1.6 G protein-coupled receptor 137BSqstm1 0.003 0.043 1.5 Sequestosome 1

7Tsc2+/+***

E

H L L L L H L L L L H L L L L H

FDR

Atp6v

1a

Ddit4l

Steap

1

Atp6v

0c

Fabp3

Vamp3

Dpp7

0 0 537 (495) 41 (38)

FDR + FC>1.5

0 0 78 (75) 22 (20)

P < 1.6×10–28

Bdh1 0.003 0.043 2.0 3-Hydroxybutyrate dehydrogenase, type 11110008P14Rik 0.003 0.044 1.6 RIKEN cDNA 1110008P14 gene

Slc16a1 0.003 0.045 2.4 Solute carrier family 16 (monocarboxylic acid transporters), member 1Foxa2 0.003 0.046 1.6 Forkhead box A2Cd68 0.003 0.047 2.7 CD68 antigen

Atp6v1a 0.003 0.047 1.5 ATPase, H+ transporting, lysosomal V1 subunit ADdit4l 0.003 0.047 1.7 DNA-damage-inducible transcript 4-like

Steap1 0.003 0.047 1.6 Six transmembrane epithelial antigen of the prostate 1Atp6v0c 0.004 0.048 1.5 ATPase, H+ transporting, lysosomal V0 subunit C

Fabp3 0.004 0.049 1.6 Fatty acid binding protein 3, muscle and heartVamp3 0.004 0.049 1.7 Vesicle-associated membrane protein 3

Dpp7 0.004 0.049 1.7 Dipeptidylpeptidase 7

–3 0 3

Rel

ativ

e m

RN

A le

vels

no

rmal

ized

to

�-a

ctin

0

1

2

3

4

5

6 Tsc2+/+ RapaTsc2–/–

Tsc2 –/– Rapa

***

***

*** ******

***

Normalized expression

P -S6K1 (T389)

P -S6K1 (T389)

P -S6 (S235/236)

P -S6 (S235/236)

Atp6v0b

Figure 1 Identification of mTORC1-regulated genes. Western blot of immortalized MEFs of the indicated genotypes grown for 24 h in the statedamounts of FBS (A), and in the presence of rapamycin (or vehicle) (B). (C) Number of probes (genes) in Affymetrix microarrays correspondingto the indicated patterns (upregulated under one condition compared with the other three by t-test using a Qo0.05 after FDR correction). Dataare shown also incorporating a fold change criterion (41.5-fold; FC41.5). (D) Heatmap representation of normalized expression values forLLHL probes with a fold change 41.5 (ranked by P-value). Genes encoding V-ATPase subunits and V-ATPase-associated proteins arehighlighted in yellow and in bold lysosomal genes are noted. (E) qRT–PCR analysis of genes with the weakest LLHL pattern. Error barsrepresent s.e.m. (n¼ 4); ***Po0.001.


The EMBO Journal VOL 30 | NO 16 | 2011 &2011 European Molecular Biology Organization3244

activation (Majumder et al, 2004). Myr-Akt would be ex-

pected to inactivate Tsc1/Tsc2 and the PIN phenotype is

reversed by treatment with the rapamycin analogue

everolimus (also called RAD001) (Majumder et al, 2004).

While this represented a very different experimental para-

digm and gene expression studies were performed following

12 and 48 h of RAD001 oral administration, a collective

analysis showed a similar mTORC1-dependent induction of

8

10

12

******

*** ***

Tsc2 +/+

Tsc2 +/+ Rapa Tsc2 –/– Tsc2 –/– Rapa

BASymbol P FDR Q FC Gene title

Atp6ap1 0.06 0.20 1.16 V-ATPase, accessory protein 1Atp6ap2 9E–4 0.030 1.62 V-ATPase, accessory protein 2Atp6ap2 0.001 0.032 1.50 V-ATPase, accessory protein 2Atp6ap2 0.009 0.07 1.49 V-ATPase, accessory protein 2

Rapa Rapa

Tsc2 +/+ Tsc2 –/–

– –

Atp6v

1a

Atp6v

1g1

Rel

ativ

e m

RN

A le

vels

no

rmal

ized

to

�-a

ctin

0

2

4

6***

*** ***

Atp6v0a1 0.016 0.10 1.55 V0-ATPase, subunit a1Atp6v0a1 0.030 0.14 1.36 V0-ATPase, subunit a1Atp6v0a1 0.041 0.16 1.25 V0-ATPase, subunit a1Atp6v0a1 0.96 0.98 1.01 V0-ATPase, subunit a1Atp6v0a2 3E–4 0.021 1.25 V0-ATPase, subunit a2Atp6v0a2 0.019 0.11 1.24 V0-ATPase, subunit a2Atp6v0a2 0.78 0.88 –1.04 V0-ATPase, subunit a2

Tcirg1 0.004 0.05 1.29 V0-ATPase, subunit a3Atp6v0b V0-ATPase, subunit bAtp6v0b

2E–5 0.013 1.665E–4 0.024 1.59 V0-ATPase, subunit b

Atp6v0b 0.008 0.07 1.27 V0-ATPase, subunit b0.004 0.048 1.50 V0-ATPase, subunit c

Atp6v0c 0.07 0.23 1.15 V0-ATPase, subunit cAtp6v0c 0.10 0.27 1.11 V0-ATPase, subunit c

Atp6v0d1 0.008 0.07 1.24 V0-ATPase, subunit d1Atp6v0e 0.44 0.64 1.07 V0-ATPase, subunit e

Atp6v0e2 0.57 0.74 –1.04 V0-ATPase, subunit e2Atp6v1a 0.003 0.047 1.50 V1-ATPase, subunit AAtp6v1a 0.007 0.07 1.75 V1-ATPase, subunit A

Atp6v1b2 1.41 V1-ATPase, subunit B2Atp6v1b2 1.37 V1-ATPase, subunit B2Atp6v1c1 1.46 V1-ATPase, subunit C1Atp6v1c1 0.001 0.032 1.44 V1-ATPase, subunit C1Atp6v1c1 0.008 0.07 1.51 V1-ATPase, subunit C1

Rapamycin

Tubulin

Atp6v1b2

Atp6v1a

C

DAtp6v1d 1.45 V1-ATPase, subunit DAtp6v1d 0.002 0.036 1.33 V1-ATPase, subunit DAtp6v1d 0.002 0.039 1.35 V1-ATPase, subunit D

Atp6v1e1 0.001 0.034 1.42 V1-ATPase, subunit E1Atp6v1e1 0.08 0.24 1.18 V1-ATPase, subunit E1Atp6v1e2 0.78 0.88 –1.01 V1-ATPase, subunit E2

Atp6v1f 0.08 0.24 1.25 V1-ATPase, subunit FAtp6v1g1

4E–4 0.0237E–4 0.0279E–4 0.029

7E–5 0.017

8E–4 0.028 1.62 V1-ATPase, subunit G1Atp6v1g1 0.07 0.22 1.38 V1-ATPase, subunit G1Atp6v1g2 0.036 0.15 1.09 V1-ATPase, subunit G2

Atp6v1h 0.004 0.05 1.50 V1-ATPase, subunit H

Rapamycin – +

– + – +

– + – + – +

Vehicle

Tsc1+/+

Tsc2 +/+ Tsc2 –/–

Tsc1F/F Tsc1+/+ Tsc1F/F

Tsc1

Atp6v1a30

Normalized expression–3

P -S6K1(T389)

P -S6(S235/236)

Atp6v1b2E

Days 10 3 3 2 2 3 3 5 5 7 7 10 10

Tsc1

Veh eGFP Cre

Tcirg1(Atp6v0a3)

Adenovirus

S6

Tubulin

Atp6v1a

Atp6v1b2

(T389)

S6K1 2.0 ** *

2.5

3.0Tsc1+/+

Tsc1F/F

F

(T37/46)

(S235/236)

S6

Cyclophilin

(S65)

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V-ATPase expression (Supplementary Figure S5). In fact, a

statistically significant LLHL pattern was found for 13 probes

corresponding to 9 V-ATPase genes. Thus, V-ATPases are

regulated by mTORC1 not only in cells in culture, but also

in mice in both physiological and pathological states.

Transcriptional regulation of V-ATPases by mTORC1

To obtain insight into the mechanism of V-ATPase mRNA

upregulation by mTORC1, we examined the relative contribu-

tion of mRNA synthesis and stability. While as expected,

baseline V-ATPase mRNA levels were higher in Tsc2�/� than

Tsc2þ /þ cells, mRNA decay rates were very similar

(Figure 3A). These data suggested that V-ATPase mRNA

levels were upregulated by mTORC1 through a transcriptional

mechanism. Consistent with this notion, RNA polymerase II

(Pol II) chromatin immunoprecipitation (ChIP) experiments

showed Pol II enrichment at V-ATPase coding sequences in

Tsc2�/� cells (Supplementary Figure S6). This effect persisted

after normalization to b-actin ChIP data suggesting that the

effect was not due to a generalized increase in Pol II tran-

scription by mTORC1 (Figure 3B).

We noted that among the 75 genes regulated by mTORC1

was hypoxia-inducible factor 1a (Hif-1a) (Figure 1D), which

we previously showed is regulated by Tsc1/Tsc2 and mTORC1

(Brugarolas et al, 2003). Hif-1a is a critical determinant of the

activity of the heterodimeric Hif-1 transcription factor

(Kaelin, 2008) and a recent ChIP-chip study reported finding

Hif-1a on V-ATPase d1 sequences (Xia et al, 2009).

Furthermore, several putative hypoxia response elements

(HRE) were identified in the promoters of several V-ATPases

(Supplementary Table S2). To determine whether Hif-1acontributed to V-ATPase regulation, we examined the effects

of both hypoxia and Hif-1a knockdown on V-ATPase expres-

sion. As a positive control, the expression of the canonical

Hif-1 target Glut-1 was evaluated. Hypoxia stabilized Hif-1aprotein and this was accompanied by increased expression

of Glut-1 (Figure 3C and D). As expected, the upregulation of

Glut-1 by hypoxia was markedly blunted by Hif-1a knock-

down (Figure 3C and D). We next examined V-ATPase levels.

While in Tsc2þ /þ cells, we did not observe an increase in V-

ATPase expression following hypoxia (or a consistent down-

regulation following Hif-1a knockdown), in Tsc2�/� cells

there was a trend towards induction by hypoxia and this

was somewhat blunted by Hif-1a knockdown, which also, in

some cases, downregulated baseline V-ATPase expression

levels (Figure 3D). Thus, while Hif-1a may partially contri-

bute to V-ATPase gene regulation in Tsc2�/� cells, Hif-1a does

not seem to be the principal regulator of V-ATPase gene

expression under these conditions.

mTORC1-dependent V-ATPase regulation requires Tfeb

A recent study showed that lysosomal biogenesis is regulated

by TFEB (Sardiello et al, 2009), and we noted that among the

genes reported to be regulated by TFEB, there were several

V-ATPases. Interestingly, the consensus sequence for TFEB

(also called Tcfeb in the mouse), which includes the

E-box (Sardiello et al, 2009), overlaps with HRE (Wenger

et al, 2005) (Supplementary Table S2). We set out to examine

whether TFEB may be involved in mTORC1-dependent reg-

ulation of V-ATPases. An antibody recognizing TFEB was

generated in collaboration with Bethyl, and we evaluated

the effects of Tfeb knockdown on V-ATPase expression.

Among eight Tfeb shRNAs tested, two were found that

substantially lowered Tfeb levels (Figure 4A and B). Tfeb

depletion downregulated V-ATPase expression, particularly in

Tsc2-deficient cells, in which baseline levels were upregu-

lated (Figure 4B; see also Figure 4E). These data show that

Tfeb is required for mTORC1-induced V-ATPase expression.

Tfeb is an important mediator of mTORC1 changes

on gene expression

To determine how broadly TFEB regulated gene expression

downstream of mTORC1, we performed gene expression

studies in MEFs stably depleted of Tfeb. Having previously

used an Affymetrix platform and seeking to determine

the robustness of our findings, Illumina microarrays were

used. Otherwise, the experimental design was identical as

that reported in Figure 1C and D. First, we compared the

results in the Tsc2þ /þ and Tsc2�/� cells transduced with a

scrambled shRNA to those previously obtained with plain

Tsc2þ /þ and Tsc2�/� cells. While the number of LLHL

probes identified with the Illumina chips was much larger

than with the Affymetrix arrays, the distribution across

patterns was strikingly similar; the majority of probes had

an LLHL pattern and no probes were found with a HLLL or

LHLL pattern (compare Figures 4C and 1C). Furthermore,

among the 75 genes previously identified with a LLHL

pattern, a statistically significant LLHL pattern was retained

in 48% (Supplementary Figure S7). Considering that the

microarray platforms were different, that microarrays were

processed at different facilities several years apart, and that

stringent criteria were used (FDR Qo0.05), a 48% retention

rate was considered satisfactory and suggested that many

other genes besides V-ATPases were bona fide targets of

mTORC1.

To specifically determine the contribution of Tfeb to

mTORC1-induced gene expression and the LLHL pattern,

we compared scrambled versus Tfeb shRNA-expressing

cells. Quite surprisingly, Tfeb knockdown markedly

decreased the number of genes with an LLHL pattern (1146

versus 424; Figure 4C). Furthermore, as visually illustrated

in the heatmap (Figure 4D), in 26% of LLHL probes, Tfeb

knockdown resulted in a substantial ‘flattening’ of the LLHL

pattern (420% 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 expres-

sion 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).

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Figure 3 V-ATPases are regulated transcriptionally by mTORC1 independently of Hif-1a. (A) qRT–PCR analysis of V-ATPase mRNA decay ratesin the indicated cell types in low serum (n¼ 3). (B) RNA polymerase II (Pol II) occupancy on V-ATPase gene coding sequences compared withIgG control (n¼ 3). Western blotting (C) and qRT–PCR (D) of Tsc2þ /þ and Tsc2�/� MEFs stably transduced with a lentiviral construct with aHif-1a (or a scrambled) shRNA sequence and exposed to hypoxia for the indicated number of hours (n¼ 3). Error bars represent s.e.m.;*Po0.05; ***Po0.001.



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Tsc2 –/– Tsc2 +/+ Tsc2 –/–Tsc2 +/+

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Figure 4 V-ATPase regulation by mTORC1 requires Tfeb and Tfeb is an important mediator of the changes in gene expression inducedby mTORC1. (A) qRT–PCR of MEFs of the indicated genotypes stably transduced with the indicated shRNA vectors; each Tfeb shRNA (A, B)was compared with the scrambled (Sc) control by an unpaired t-test (n¼ 3). Error bars represent s.e.m.; *Po0.05; ***Po0.001. (B) Westernblot of serum-starved MEFs of the indicated genotypes. (C) Number of probes (genes) corresponding to the different patterns upregulatedunder one condition compared with the other three by t-tests (Qo0.05 after FDR correction) in Illumina microarrays. Data are shown alsoincorporating a fold change criterion (41.5-fold; FC41.5). (D) Heatmap representation of normalized expression values for all 1418 significantLLHL probes with FC41.5 in scrambled shRNA transduced MEFs compared with Tfeb shRNA transduced MEFs. Box shows probes for whichmean expression levels are reduced by 420% following Tfeb knockdown in Tsc2�/� MEFs. (E) qRT–PCR of Tsc2þ /þ and Tsc2�/� MEFs stablytransduced with the indicated shRNA vectors and treated (or not) with rapamycin (n¼ 3). Error bars represent s.e.m.; NS, non-significant;**Po0.01; ***Po0.001.



In keeping with the idea that mTORC1 regulates endocytosis,

FACS analyses showed higher levels of albumin uptake in

Tsc2�/� than Tsc2þ /þ 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

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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 statisti-

cally 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

Tsc1þ /þ 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 phos-

phorylation, and phosphoproteomic studies have shown

TFEB to be phosphorylated in 410 sites (Dephoure et al,

2008; Mayya et al, 2009). To determine whether Tfeb migra-

tion was affected by phosphorylation, we evaluated the

effects of l phosphatase treatment. As shown in Figure 6D,

the migration of Tfeb was accelerated by treatment of

cell lysates with l phosphatase. Parenthetically, by compar-

ison to similarly processed samples incubated without

l 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 Tsc2þ /þ 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

A B

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and Tsc2�/� cells treated (or not) with rapamycin and subsequently fractionated into nuclear [N] or cytoplasmic [C] fractions. (F) Western blots ofnuclear versus cytoplasmic fractions from serum-starved Tsc2�/� cells incubated as indicated with l phosphatase. (G) Western blot of whole-celllysates (WCL) or nuclei from serum-starved Tsc2�/� cells treated with rapamycin for the indicated periods of time.



fast-migrating form (Figure 6E). To determine whether nucle-

ar Tfeb retained some level of phosphorylation, nuclear

extracts were treated with l 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 regula-

tion 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 accumu-

lation 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 deter-

mine 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

A B C

Rapa – + – +Sc TSC2

TFEB

HeLa

shRNA

TSC2 TFEB

Rapa (h) 0 1 2 3 4 6

HeLa TSC2 shRNA

– + – + – + – +

– + – + – + – +

– +

λ PPase

– + – +Rapa

shRNA Sc TSC2

HeLa

TFEB (short)

D E F G

Nuclei Cytosol

HeLaHeLa

TSC2 shRNA TSC2 TSC2shRNA Sc Rptor Sc Rptor

siRNA Sc TSC2

HeLa

Tubulin

�-S6 (S235/236)

�-S6 (S235/236)

Tubulin

S6

siRNA Sc Rptor Sc Rptor

shRNA Sc TSC2

HeLa

Tubulin

TFEB (long)

Rapa

shRNA Sc TSC2 Sc TSC2

Nuclei Cytosol

N C N C

λ PPase

TFEB(short)

TFEB(long)

Menin

TFEB(short)

TFEB(long)

Menin

T b li

TFEB(short)

TFEB(long)

�-S6K1(T389)

S6K1

Raptor

TFEB(short)

TFEB(long)

�-S6K1(T389)S6K1

Raptor

TubulinTubulin Tubulin Tubulin

Figure 7 mTORC1-dependent regulation of TFEB phosphorylation and nuclear localization. (A) Western blot of HeLa cells stably transducedwith lentiviruses containing a TSC2 shRNA (or a scrambled control, Sc) and serum-starved (0.1% FBS) ± rapamycin for 24 h. (B) Western blotof serum-starved TSC2-depleted HeLa cells treated with rapamycin for the indicated hours. (C) Western blots from HeLa cells as in (A)incubated, where stated, with l phosphatase. (D) Western blots of cellular fractions from HeLa cells treated as indicated. (E) Western blot ofnuclear [N] and cytoplasmic [C] fractions from TSC2-depleted serum-starved HeLa cells incubated (or not) with l phosphatase. (F) Westernblot of HeLa cells stably transduced with a lentivirus encoding a Raptor shRNA (or a scrambled control, Sc) and subsequently transfected withsiRNAs and serum starved. (G) Western blot of HeLa cells stably transduced with lentiviral vectors as indicated and subsequently transfectedwith siRNA and serum starved. Rptor, Raptor.



100

A

476233

476

B

Gln-rich Basic HLH LZ Pro-rich

TFEB

– + – +

Per

cent

frac

tion

0

20

40

60

80

Sc TSC2

476290

476321

469

461

320CC/N N

Rapa

shRNA

***

GrowthFactors

TSC1Rapa –– + +

shRNA Sc TSC2 FD

TSC2

Raptor

mTOR

Rheb

(Inactive)

Rapamycin

Aminoacids

Late endosome

GTP

GβL GβLRaptor

mTOR

(Active)

TFEB (1–476)

TFEB (S142A)

V-ATPase

Nucleus

V-ATPaselysosomal genes

TfebP

Tfeb

Tfeb

TFEB (5×SA)

TFEB (5×SD)

Per

cent

nuc

lear

frac

tion

0

20

40

60

80

100

Sc TSC2

Rapa – + – + – + – +

Sc TSC2

NS

E

shRNA

************

Per

cent

nuc

lear

frac

tion

0

20

40

60

80

100

***

Sc TSC2

Rapa – + – + – + – +

Sc TSC2

NS

NS

TFEB (1–476) TFEB (S142A) TFEB (5×SA) TFEB (5×SD)

shRNA

Per

cent

frac

tion

20

40

60

80

100

C

C/NN

***

*****

***

+0

Sc TSC2

TFEB (1–476)

Sc TSC2

TFEB (233–476)

Sc TSC2

TFEB (290–476)

Sc TSC2

TFEB (321–476)

Sc TSC2

TFEB (1–469)

Sc TSC2

TFEB (1–461)

Sc TSC2

TFEB (1–320)

Rapa

shRNA

– +– +– +– +– +– +– +– +– +– +– +– +– +–

P

P P

Figure 8 mTORC1-dependent nuclear TFEB localization requires C-terminal serine-rich motif. (A) Confocal microscopy (representative images andbar graph quantification) of TFEB subcellular localization in HeLa cells stably expressing a scrambled shRNA (Sc) or a shRNA targeting TSC2transfected with myc-tagged full-length TFEB and incubated for 24h in 0.1% FBS with or without rapamycin. Percent of cells with cytoplasmic TFEB[C], nuclear [N] or both [C/N]. ‘*’ Illustrates statistically significant differences in nuclear TFEB (NþC/N) between TSC2-depleted HeLa cells bycomparison to the other three conditions (n¼ 4). (B) Schematic of full-length TFEB and deletion mutants; HLH, helix-loop-helix; LZ, leucine zipper.(C) Quantification of the subcellular distribution of mutant TFEB, compared with wild-type, in HeLa cells. ‘*’ Illustrates statistically significantdifferences in nuclear TFEB (NþC/N) between TSC2-depleted HeLa cells by comparison to the other three conditions for each panel (n¼ 3). Confocalmicroscopy images (D) and bar graph quantification (E) of wild-type or mutant TFEB; 5� SA, S462/463/466/467/469A; 5� SD, S462/463/466/467/469D. Error bars represent s.e.m. (n¼ 3). (F) Model of TFEB regulation by mTORC1. NS, non-significant; *Po0.05; **Po0.01; ***Po0.001.



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 corre-

sponded 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 cytoplas-

mic 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 phosphory-

lated in an mTORC1-dependent manner and that phosphor-

ylation 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 interven-

tions 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 accumu-

lation 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 progres-

sive 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-trans-

lational 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 410 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 pro-

liferation. 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.

A MEFs HeLa

Tfeb

Grp78

Tfeb

TubulinLC3

Tubulin

Tfeb

Tubulin

�-Erk1/2 (T183/Y185)

Erk1/2

Tfeb

TubulinAtf4

2×105

3×105

4×105

8×105

6×105

4×105

2×105

106ScshTfeb AshTfeb B

MEFs

ScshTFEB 110shTFEB 111

HeLaB

Days0 1 2 3 4

Cel

l nu

mb

er

0

105

Days0 1 2 3 4

0

Glucose deprivation (h) 0 0.5 1 3 6 9 0 0.5 1 3 6 9

0 0.5 1 3 6 9 0 0.5 1 3 6 9

0 0.51 3 6 9 0 0.5 1 3 6 9

0 0.5 1 3 6 9 0 0.51 3 6 9

Amino-acid deprivation (h)

0.1% FBS (h)

DTT (h)

Figure 9 Complex TFEB regulation by multiple signals beyondmTORC1. (A) HeLa and MEFs were starved for amino acids, glucoseand serum (0.1% FBS), or treated with 10 mM DTT for the indicatedtimes. (B) Graphs depicting cell counts of MEFs and HeLa cells overtime (Sc, scrambled). Po10�4 and Po10�7 for Tfeb knockdownMEFs and HeLa cells, respectively, using two-factor ANOVA. Errorbars represent s.e.m. (n¼ 3).



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 pre-

viously 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 endo-

cytosis (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 endoso-

mal 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 lyso-

somes 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 reacti-

vation terminates autophagy and through a poorly under-

stood 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 loca-

lization 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 phosphor-

ylates 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 condi-

tions 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 phosphory-

lated 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 follow-

ing 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 experi-

mental 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-1a as

an mTORC1 effector (Brugarolas et al, 2003), and Hif-1a 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 trans-

cription factor whose nuclear localization is regulated by

mTORC1, and Hif-1a, 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 activa-

tion (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 hetero-

dimerize) (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 carcino-

mas 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 carcino-

mas 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 func-

tions 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 signifi-

cantly 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 treatmentsMEFs Tsc2þ /þ ; p53�/� (Tsc2þ /þ ) and Tsc2�/�; p53�/� (Tsc2�/�)were a gift of DJ Kwiatkowski (Brigham and Women’s Hospital,Harvard Medical School, Boston, MA). Cells were grown in highglucose Dulbecco’s modified Eagle’s medium (Sigma) supplemen-ted with 10% FBS (HyClone) and 1% (v/v) penicillin/streptomycin(P/S) (Gibco) in a humidified incubator at 371C and 5% CO2. Forexperiments, cells were plated to achieve B70% confluency at thetime of treatment. For low-serum experiments, cells were washedtwice in PBS and medium was changed to the same base mediumbut supplemented with 0.1% FBS and cells were harvested after24 h. Where indicated, rapamycin (LC Laboratories) was used at25 nM in methanol and methanol was used as vehicle. Whereindicated, cells were treated with actinomycin D (Sigma) at 10 mg/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 experi-ments, cells were exposed to 1% O2 and 5% CO2 at 371C in ahypoxia chamber (Coy Laboratory Products). For starvation timecourses, cells were washed with PBS twice and starved of aminoacids (amino acid-free media), glucose (glucose-free media) orserum (0.1% FBS) for different amounts of time.

Hif-1a, Tfeb, Raptor and TSC2 shRNAsRecombinant lentiviruses were generated in HEK293T cells bytransient transfection using calcium phosphate with the envelopeplasmid pMD2.G (Database ID p486), packaging plasmid psPAX2(p485) and the specific plasmid of interest. pMD2.G, psPAX2 andpLKO.1 or pGIPZ vectors were transfected at a ratio of 3:8:10.Supernatants were collected for 2 days, passed through a 0.45-mmfilter, and used to infect MEFs or HeLa cells for 24 h in mediumcontaining 8 mg/ml polybrene (Sigma). MEFs and HeLa cells wereselected with 2mg/ml puromycin for at least 1 week and HeLa cellswere further enriched by FACS. The following vectors were used:pLKO.1 vectors with a Hif-1a shRNA (50-AGAGGTGGATATGTCTGGG-30) (p481) generously provided by LB Gardner (Nemetski andGardner, 2007), a mouse Tfeb shRNA from Open Biosystems(sequence A, TRCN0000085548, 50-CGGCAGTACTATGACTATGAT-30) (p633) or a designed Tfeb shRNA (sequence B, 50-GGCAGTACTATGACTATGATG-30) (p652), a human TFEB shRNAs from OpenBiosystems (sequence 110, TRCN0000013110, 50-GAGACGAAGGTTCAACATCAA-30) (p700) or (sequence 111, TRCN0000013111, 50-GAACAAGTTTGCTGCCCACAT-30) (p701), a raptor shRNA from Addgene(Addgene plasmid 1858) (p741) or a scrambled sequence (50-GGGTCTGTATAGGTGGAGA-30) (p480). pGIPZ vectors containing a TSC2(p496) or a scrambled (p483) shRNA were from Open Biosystems.

Western blotting and antibodiesCell lysates and western blotting was performed as previouslydescribed (Vega-Rubin-de-Celis et al, 2010), using antibodies fromthe 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-1a, TSC1 and Menin from Bethyllaboratories; TSC2, ATF4 and c-Myc from Santa Cruz; Raptor fromMillipore; Erk1/2, phospho-Erk1/2 (T183/Y185) and Tubulin fromSigma; Cyclophilin B from Abcam; GLUT-1 from Novus Biologicals;and Grp78 from BD Biosciences. Atp6v1a and Atp6v1b2 antibodieswere a generous gift from D Brown (Massachusetts GeneralHospital, Boston, MA); and Atp6v0a1 and Atp6v0a3 antibodieswere kindly provided by V Marshansky (Massachusetts GeneralHospital, Boston, MA). Tfeb antibodies were developed incollaboration with Bethyl Laboratories.

For the remaining Materials and methods, please see theSupplementary data section.



Accession numbersMicroarrays were deposited in GEO (http://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE27982 and GSE28021.

Supplementary dataSupplementary data are available at The EMBO Journal Online(http://www.embojournal.org).

Acknowledgements

We thank Dr WG Kaelin Jr (Dana-Farber Cancer Institute) and themembers of the Brugarolas laboratory for helpful discussions; MGRoth, JM Abrams and MAWhite (UT Southwestern Medical Center)for reading the manuscript; and C Li (Dana-Farber Cancer Institute)for statistical advice. We are indebted to Drs DJ Kwiatkowski(Brigham and Women’s Hospital, Harvard Medical School,Boston, MA) for Tsc2-deficient MEFs and Tsc1F/F mice; LBGardner (New York University) for Hif1-a and scrambled shRNAplasmids; N Esumi (Johns Hopkins University) for TFEB plasmid;and D Brown and V Marshansky (Massachusetts General Hospital,Boston, MA) for V-ATPase antibodies. This work was supported bya fellowship of Excellence (BPOSTDOC06/004) from GeneralitatValenciana (Spain) to SP-L; a fellowship from Fundacion CajaMadrid to SV-R; T32CA124334 and F32CA136087 to TATT; NIBIB

EB 05556 to JCS; NIGMS 77253 to DRC; and the following Grants toJB: K08NS051843, RO1CA129387, RSG115739 from the ACS and aBOC 5FY06582 from the March of Dimes Foundation. Microarraysamples were processed at the Genomics Shared Resource of theHarold C Simmons Cancer Center supported in part by1P30CA142543. JB is a Virginia Murchison Linthicum Scholar inMedical Research at UT Southwestern. The content is solely theresponsibility of the authors and does not represent official viewsfrom any of the granting agencies.

Author contributions: SP-L, SV-RC and JB designed the experi-ments. SP-L performed experiments, all statistical and microarrayanalyses and wrote an initial draft of the manuscript. SV-RC con-ducted confocal microscopy and most TFEB experiments, JCScarried out ChIP assays under the supervision of DRC. NCW wasresponsible for the mouse experiments. TATT helped with shRNAcloning. LZ was involved in preliminary microarray data character-ization. X-JX provided statistical input and revised statistics. JBconceived the study, was responsible for the direction of the projectand wrote the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

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