Using a robust experimental paradigm, we uncovered a novel regulatory network linking endocytosis to TFEB and mTORC1. An unbiased gene expression analysis coupled with a design amenable to refined statistics led us to identify a connection between mTORC1 and V-ATPase genes. Publicly available data sets confirmed the link and experiments in mice showed that mTORC1 regulated V-ATPase expression also in vivo
. A recent study had established that TFEB regulated lysosomal biogenesis (Sardiello et al, 2009
) and among the genes regulated by TFEB we found several V-ATPases. These results led us to hypothesize that V-ATPase regulation by mTORC1 may be mediated by TFEB. Indeed, TFEB was necessary for V-ATPase upregulation by mTORC1. mTORC1 regulated TFEB phosphorylation and nuclear localization and a C-terminal serine-rich motif was identified that is essential for mTORC1-dependent TFEB nuclear localization. Our data suggest that mTORC1 induces the phosphorylation of a serine(s) within this motif driving thereby TFEB to the nucleus. TFEB regulates the expression of V-ATPases and other lysosomal genes and TFEB was required for mTORC1-induced endocytosis. To our knowledge this is the first study to show that endogenous TFEB is regulated at the subcellular localization level and that mTORC1 promotes TFEB nuclear localization. These data link an oncogenic transcription factor that is a master regulator of lysosomal biogenesis, TFEB, to mTORC1 and endocytosis.
An increasing amount of evidence implicates TORC1 in endocytosis. mTOR was identified in an RNAi screen for kinases involved in clathrin-mediated endocytosis (Pelkmans et al, 2005
) and Tsc2-deficient cells were previously shown to have increased fluid-phase endocytosis (Xiao et al, 1997
). In Drosophila melanogaster
, a sensitized screen for genes that modified a tissue-specific dTOR overexpression phenotype identified an important regulator of endocytosis, Hsc70-4, a clathrin uncoating factor, and the authors showed that dTOR was involved in fat body endocytosis (Hennig et al, 2006
). Interestingly, besides V-ATPases, mTORC1 regulated the expression of multiple lysosomal genes (; see also Supplementary Table S5
). These effects may be mediated, at least in part, by TFEB, which regulates lysosome biogenesis (Sardiello et al, 2009
). Thus, one mechanism whereby mTORC1 and TFEB may affect endocytosis is by regulating V-ATPase levels and lysosomes.
The lysosome is the final destination not only for endosomal cargo but also for autophagosomes. In response to starvation, mTORC1 is inhibited leading to the formation of autophagosomes that engulf intracellular components for recycling and sustenance (He and Klionsky, 2009
; Kroemer et al, 2010
). Autophagosomes and their content are delivered to lysosomes, which accumulate in the perinuclear area (Korolchuk et al, 2011
). Autophagosomes fuse with lysosomes to generate autolysosomes, a process that rapidly depletes the lysosomal pool (Yu et al, 2010
). The degradation of macromolecules and release of their constituents into the cytosol reactivates mTORC1 (Yu et al, 2010
). mTORC1 reactivation terminates autophagy and through a poorly understood mechanism repletes the lysosome pool (Yu et al, 2010
). However, how the pool of lysosomes is repleted is unknown. Our results suggest that lysosomal reformation in response to autophagy, which is mTORC1 dependent (Yu et al, 2010
), may be mediated, at least in part, by TFEB.
mTORC1 coordinately regulates TFEB phosphorylation and nuclear localization. mTORC1 promoted TFEB nuclear localization and this process required a C-terminal serine-rich motif. Serine substitution to non-phosphorylatable residues abrogated TFEB regulation by mTORC1. More importantly, mutation to phosphomimetic amino acids was sufficient to reproduce the effect of mTORC1 on TFEB nuclear localization obviating the need for mTORC1. These data support a model in which mTORC1 (or an effector kinase) directly phosphorylates a serine(s) within the C-terminal serine-rich motif driving thereby TFEB to the nucleus. Consistent with this notion, nuclear TFEB was phosphorylated. This model may seem at odds with the observation that nuclear TFEB is fast-migrating and possibly hypo-phosphorylated by comparison to cytoplasmic (presumably cytosolic) TFEB. However, as our data suggest, nuclear TFEB may be phosphorylated on sites that are not phosphorylated on cytosolic TFEB, and thus no inferences can be drawn about the degree of phosphorylation based on SDS–PAGE migration. Furthermore, while we have identified a critical motif involved in mTORC1 regulation, other sites in TFEB appear to be regulated by mTORC1 (Yu et al, 2011
Our results further emphasize the link between mTORC1 and the late endosome/lysosome and provide evidence for the existence of bidirectional regulatory loops. mTORC1 localizes to the surface of the late endosome/lysosome in a highly choreographed manner (Korolchuk et al, 2011
; Narita et al, 2011
). The late endosome/lysosome is necessary for mTORC1 activation (Flinn et al, 2010
; Li et al, 2010
; Sancak et al, 2010
) and mTORC1 is reactivated under conditions of persistent starvation following macromolecule degradation in autolysosomes (Yu et al, 2010
). However, not only is mTORC1 downstream of the lysosome, but, inasmuch as mTORC1 regulates TFEB and V-ATPases, mTORC1 is also upstream. mTORC1 reactivation by autolysosomes requires Spinster, a lysosomal efflux sugar transporter. Spinster has been proposed to act by regulating lysosomal pH (Rong et al, 2011
), which is controlled primarily by the V-ATPase. Interestingly, not only has luminal pH been implicated in mTORC1 regulation, but changes in cytosolic pH occur in response to alterations in nutrient conditions (Korolchuk et al, 2011
) and have been implicated in the regulation of lysosome topology and mTORC1 (Korolchuk et al, 2011
Recently, TFEB was found to regulate autophagy gene expression (Settembre et al, 2011
). Starvation led to ERK2 inactivation, which in turn, decreased TFEB phosphorylation and increased its nuclear localization. This model is in principle compatible with our results. TFEB is phosphorylated in over 10 sites (Dephoure et al, 2008
; Mayya et al, 2009
; Yu et al, 2011
), and mechanisms likely exist that drive TFEB into the nucleus independently of the activation state of mTORC1. However, the studies do differ in one aspect. Whereas mutation of the putative ERK2 phosphorylation site, S142, to A is reported to be sufficient to drive TFEB into the nucleus (Settembre et al, 2011
), we find that, at least under low serum conditions, the subcellular localization of TFEB is not appreciably altered by a S142A mutation.
The experimental conditions studied by Settembre et al (2011)
, which involve evaluating ectopically expressed TFEB in cells in the absence of glucose, amino acids and serum, are quite different from ours. Individual deprivation of glucose, amino acids or serum has profound, dynamic and diverse effects on TFEB (see ). Given these observations and the complexity of TFEB phosphorylation (Dephoure et al, 2008
; Mayya et al, 2009
; Yu et al, 2011
), how starvation of all three sources will affect TFEB is unclear. Consistent with this complexity, the changes in TFEB migration observed following deprivation of individual factors could not be explained by the predicted changes on mTORC1 activity. In fact, we speculate that the discovery that mTORC1 regulates TFEB may have been possible only because of the tight experimental system we used.
Besides V-ATPases and lysosomal genes, many other genes downstream of mTORC1 were regulated in a Tfeb-dependent manner. Approximately 25% of genes induced by mTORC1 required Tfeb. Experiments are ongoing to determine how many of these genes are directly regulated by Tfeb. A similar experimental setup previously identified Hif-1α as an mTORC1 effector (Brugarolas et al, 2003
), and Hif-1α is an important mediator of a metabolic gene expression programme (Duvel et al, 2010
). Along with sterol regulatory element-binding protein-1 (Porstmann et al, 2008
), a transcription factor whose nuclear localization is regulated by mTORC1, and Hif-1α, TFEB is an important mediator of mTORC1 effects on gene expression.
The discovery that mTORC1 regulates TFEB has clinical implications. It may explain the expression of melanocyte lineage markers in tumours of patients with TSC (Martignoni et al, 2008
), a syndrome arising from mutations in either the TSC1
genes, resulting in constitutive mTORC1 activation (El-Hashemite et al, 2003
; Kenerson et al, 2007
). Patients with TSC are predisposed to develop renal angiomyolipomas, which express melanocytic markers such as HMB45 and Melan-A (Martignoni et al, 2008
), and since expression of these proteins in melanomas is thought to be driven by TFEB (or related family members with whom TFEB can heterodimerize) (Steingrimsson et al, 2004
), these markers may be similarly driven by TFEB in renal angiomyolipomas.
Interestingly, TFEB functions as an oncogene in the kidney. The TFEB
gene is translocated in a subset of renal tumours where it is subjected to a robust heterologous promoter (Davis et al, 2003
; Kuiper et al, 2003
). Translocation carcinomas tend also to aberrantly express melanocytic markers (Srigley and Delahunt, 2009
), which may be similarly driven by TFEB. TFEB activation may also account for the expression of cathepsin-K, a lysosomal protease that has been recently proposed as a marker for this tumour type (Martignoni et al, 2009
). Interestingly, translocation carcinomas are often recognized by immunohistochemical studies showing high levels of nuclear TFEB (Srigley and Delahunt, 2009
). Because TFEB nuclear localization is regulated by mTORC1 and as we show, sirolimus excludes TFEB from the nucleus, sirolimus, or its derivatives, temsirolimus and everolimus, may be effective against this tumour type.
While this study focused on a small subset of genes, many genes were identified whose expression was regulated by mTORC1 not only positively but also negatively (Supplementary Figures S8–S10
; Supplementary Table S1
). Moreover, genes were also found whose expression was regulated by Tsc2 independently of mTORC1 (LLHH and HHLL patterns). As previously reported (Brugarolas et al, 2003
), this would point towards mTORC1-independent functions of the Tsc1/Tsc2 complex (Supplementary Figure S8
; Supplementary Table S1
). In addition, the expression of some genes appeared to be regulated by rapamycin to a significantly greater extent than by Tsc2 (HLHL and LHLH patterns) (Supplementary Figure S8
; Supplementary Table S1
). Importantly, in all these settings, analyses of alternative patterns showed the associations to be statistically significant (Supplementary Figure S8
). Incidentally, among the genes with an HLHL pattern, there was enrichment for genes implicated in glycolysis, pentose phosphate pathway and fatty acid biosynthesis (Supplementary Table S5
). These pathways were recently reported to be regulated by mTORC1 (Duvel et al, 2010
) and our results suggest that they are affected by rapamycin to a greater extent than by Tsc1/Tsc2.
In summary, our work uncovered a novel regulatory network connecting mTORC1 to a master regulator of lysosome biogenesis, TFEB, which is essential for mTORC1-induced endocytosis.