Regulation of TFEB and V-ATPases by mTORC1EMBO open Regulation of TFEB and V-ATPases by mTORC1...
Transcript of Regulation of TFEB and V-ATPases by mTORC1EMBO open Regulation of TFEB and V-ATPases by mTORC1...
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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.
Regulation of TFEB and V-ATPases by mTORC1S Pena-Llopis et al
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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Figure 2 mTORC1 regulates V-ATPases. (A) Heatmap representation of normalized expression values for all informative V-ATPase probes inthe arrays. qRT–PCR (B) and western blot (C) of V-ATPases in Tsc2þ /þ and Tsc2�/�MEFs in 0.1% FBS and treated (or not) with rapamycin for24 h (n¼ 4). (D) Western blot of Tsc1þ /þ and Tsc1F/F primary MEFs exposed (or not) to Ad-Cre and treated (or not) with rapamycin in 0.1%FBS for 24 h. (E) Western blot from liver extracts (assembled from two gels) of mice injected with the indicated adenovirus (or vehicle) andharvested after the stated number of days. (F) qRT–PCR analysis of V-ATPase gene expression in liver extracts from Tsc1þ /þ or Tsc1F/F miceinjected with Ad-Cre and harvested following the indicated number of days (n¼ 2 per time point). Error bars represent s.e.m.; *Po0.05;**Po0.01; ***Po0.001.
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 3245
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),
Regulation of TFEB and V-ATPases by mTORC1S Pena-Llopis et al
The EMBO Journal VOL 30 | NO 16 | 2011 &2011 European Molecular Biology Organization3246
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).
4
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0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12
Tsc2 +/+
Tsc2 +/+ RapaTsc2 –/–
Tsc2 –/– Rapa
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.
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 3247
56789
10ScAB
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FDR 0 0 0 01984 (1616) 43 (42)
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** ******
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– R – R+/+ −/−– R – R
Scrambled Tfeb+/+ −/−
shRNA
FDR 644 (560) 410 (377)FDR + FC>1.5 0 0 487 (424) 351 (323)
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– + – + – + – + Rapa
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– + – + – + – + Rapa
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Tsc2 +/+ Tsc2 –/– Tsc2 +/+ Tsc2 –/–Tsc2 +/+
Tsc2 –/– Tsc2 +/+ Tsc2 –/–Tsc2 +/+
Tsc2 –/– Tsc2 +/+ Tsc2 –/–Tsc2 +/+
Tsc2 –/– Tsc2 +/+ Tsc2 –/–Tsc2 +/+
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.
Regulation of TFEB and V-ATPases by mTORC1S Pena-Llopis et al
The EMBO Journal VOL 30 | NO 16 | 2011 &2011 European Molecular Biology Organization3248
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
Tsc2 –/–
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–
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Figure 5 mTORC1 regulates endocytosis in a V-ATPase- and Tfeb-dependent manner. (A) Confocal images of serum-starved (0.1% FBS)Tsc2þ /þ and Tsc2�/� MEFs incubated with FITC-BSA and evaluated for V-ATPase colocalization following (or not) treatment with the V-ATPase inhibitor bafilomycin A1. (B) FACS scatterplots highlighting live (propidium iodide (PI) negative) Tsc2þ /þ and Tsc2�/� MEFsincubated with FITC-BSA for the indicated times; bafilomycin-treated samples shown as a control. (C) Bar graph representation of FITC-BSAincorporation in live cells pretreated (or not) with rapamycin (rapa) by reference to FITC-BSA incorporation in untreated cells (n¼ 4).Representative FACS histogram (D) as well as bar graph representation (n¼ 3) and western blot (E) of HeLa cells transfected with siRNAoligonucleotides targeting the V0C, V1A or both V-ATPase subunits, or a scrambled control (Sc), and incubated for 60 min with FITC-BSA; abafilomycin A1-treated sample [B] is included as a control. (F) Bar graph representation of FITC-BSA uptake by FACS analysis in the indicatedcell types in 0.1% serum and treated (or not) with rapamycin (n¼ 3). Error bars represent s.e.m.; NS, non-significant; *Po0.05; **Po0.01.
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 3249
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
Rapa – –+ +– –+ +
Tfeb
Tsc1 +/+ Tsc1 –/–
C D E
�-S6
S6
(S235/236)
Tubulin
Rapa
Tfeb
�-S6(S235/236)
Tubulin
Tfeb
�-S6(S235/236)
Tubulin
Tsc2 –/–
Tsc2 +/+ Tsc2 –/–Tsc2 +/+ Tsc2 –/–
RapaRapa (h)
λ PPase – + – + – + – +
Rapa – +– + – +
– +
Tubulin
Tfeb(short)Tfeb(long)
N C N C N C N C
Tubulin
Menin
Tfeb(short)
Tfeb(long)
GFWCL Nuclei
Rapa (h)
Tfeb (short)N C N C
Tfeb
– + 0 1 2 3 4 6 0 1 2 3 4 6λ PPase
Tsc2 –/–Tsc2 –/–
Tubulin
Tfeb (long)
Menin
�-S6 (S235/236)Menin
Tubulin
0 1 2 3 4 6
Tsc2 +/+ Tsc2 –/–
Figure 6 Coordinated regulation of Tfeb phosphorylation and nuclear localization by the Tsc1/Tsc2 complex and rapamycin. (A, B) Western blotof MEFs of the indicated genotypes in 0.1% serum treated or not with rapamycin for 24 h (as in Figure 1B). (C) Western blot of serum-starved(0.1% serum) Tsc2�/� cells treated with rapamycin for the indicated times. (D) Western blot of Tsc2þ /þ and Tsc2�/� cells under serum starvationconditions treated (or not) with rapamycin and subsequently incubated, as stated, with l phosphatase. (E) Western blot of serum-starved Tsc2þ /þ
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.
Regulation of TFEB and V-ATPases by mTORC1S Pena-Llopis et al
The EMBO Journal VOL 30 | NO 16 | 2011 &2011 European Molecular Biology Organization3250
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.
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 3251
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.
Regulation of TFEB and V-ATPases by mTORC1S Pena-Llopis et al
The EMBO Journal VOL 30 | NO 16 | 2011 &2011 European Molecular Biology Organization3252
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).
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 3253
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.
Regulation of TFEB and V-ATPases by mTORC1S Pena-Llopis et al
The EMBO Journal VOL 30 | NO 16 | 2011 &2011 European Molecular Biology Organization3254
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.
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 3255
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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