Jose A. Martina

MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB

The mammalian target of rapamycin (MTOR) protein kinase complex is a key component of a pathway that regulates cell growth and proliferation in response to energy levels, hypoxia, nutrients and insulin. Inhibition of MTORC1 strongly induces autophagy by regulating the activity of the ULK protein kinase complex that is required for the formation of autophagosomes. However, the participation of MTORC1 in the expression of autophagy genes has not been characterized. Here we show that MTORC1 regulates nuclear localization and activity of the transcription factor EB (TFEB), a member of the bHLH leucine-zipper family of transcription factors that drives expression of autophagy and lysosomal genes. Under normal nutrient conditions, TFEB is phosphorylated in Ser211 in an MTORC1-dependent manner. This phosphorylation promotes association of TFEB with members of the YWHA (14-3-3) family of proteins and retention of the transcription factor in the cytosol. Pharmacological or genetic inhibition of MTORC1 causes dissociation of the TFEB/YWHA complex and rapid transport of TFEB to the nucleus where it increases transcription of multiple genes implicated in autophagy and lysosomal function. Active TFEB also associates with late endosomal/lysosomal membranes through interaction with the LAMTOR/RRAG/MTORC1 complex. Our results unveil a novel role for MTORC1 in the maintenance of cellular homeostasis by regulating autophagy at the transcriptional level.

Transcriptional Activation of Lysosomal Exocytosis Promotes Cellular Clearance

Summary Lysosomes are cellular organelles primarily involved in degradation and recycling processes. During lysosomal exocytosis, a Ca2+-regulated process, lysosomes are docked to the cell surface and fuse with the plasma membrane (PM), emptying their content outside the cell. This process has an important role in secretion and PM repair. Here we show that the transcription factor EB (TFEB) regulates lysosomal exocytosis. TFEB increases the pool of lysosomes in the proximity of the PM and promotes their fusion with PM by raising intracellular Ca2+ levels through the activation of the lysosomal Ca2+ channel MCOLN1. Induction of lysosomal exocytosis by TFEB overexpression rescued pathologic storage and restored normal cellular morphology both in vitro and in vivo in lysosomal storage diseases (LSDs). Our data indicate that lysosomal exocytosis may directly modulate cellular clearance and suggest an alternative therapeutic strategy for disorders associated with intracellular storage.

Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 γ–σ1 and AP-3 δ–σ3 hemicomplexes

The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present in the cytosolic tails of the proteins. A subset of these signals conform to the [DE]XXXL[LI] consensus motif and mediate sorting via interactions with heterotetrameric adaptor protein (AP) complexes. However, the identity of the AP subunits that recognize these signals remains controversial. We have used a yeast three-hybrid assay to demonstrate that [DE]XXXL[LI]-type signals from the human immunodeficiency virus negative factor protein and the lysosomal integral membrane protein II interact with combinations of the γ and σ1 subunits of AP-1 and the δ and σ3 subunits of AP-3, but not the analogous combinations of AP-2 and AP-4 subunits. The sequence requirements for these interactions are similar to those for binding to the whole AP complexes in vitro and for function of the signals in vivo. These observations reveal a novel mode of recognition of sorting signals involving the γ/δ and σ subunits of AP-1 and AP-3.

The Nutrient-Responsive Transcription Factor TFE3 Promotes Autophagy, Lysosomal Biogenesis, and Clearance of Cellular Debris

Enhancing TFE3 activity to promote expression of lysosome-associated genes may be beneficial in treating lysosomal storage disorders. Regulating Lysosomes and Autophagy When deprived of nutrients, cells inhibit anabolic processes, such as protein production, and promote catabolic processes, such as those mediated by lysosomes and autophagosomes. Disruption in lysosomal function causes lysosomal storage disorders. Martina et al. discovered that TFE3, like TFEB, another member of the MiTF/TFE (microphthalmia-associated transcription factor and transcription factor E) family, was inhibited at the lysosome under nutrient-replete conditions and translocated to the nucleus to stimulate genes involved in lysosome biogenesis and function and autophagy in response to nutrient deprivation. Data from various tissues and cell lines indicated that TFE3 and TFEB may be cell-specific mediators of lysosomal homeostasis. Overexpression of TFE3 stimulated lysosomal exocytosis and release of debris in a cellular model of a lysosomal storage disorder, thereby providing a potential therapeutic target. The discovery of a gene network regulating lysosomal biogenesis and its transcriptional regulator transcription factor EB (TFEB) revealed that cells monitor lysosomal function and respond to degradation requirements and environmental cues. We report the identification of transcription factor E3 (TFE3) as another regulator of lysosomal homeostasis that induced expression of genes encoding proteins involved in autophagy and lysosomal biogenesis in ARPE-19 cells in response to starvation and lysosomal stress. We found that in nutrient-replete cells, TFE3 was recruited to lysosomes through interaction with active Rag guanosine triphosphatases (GTPases) and exhibited mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1)–dependent phosphorylation. Phosphorylated TFE3 was retained in the cytosol through its interaction with the cytosolic chaperone 14-3-3. After starvation, TFE3 rapidly translocated to the nucleus and bound to the CLEAR elements present in the promoter region of many lysosomal genes, thereby inducing lysosomal biogenesis. Depletion of endogenous TFE3 entirely abolished the response of ARPE-19 cells to starvation, suggesting that TFE3 plays a critical role in nutrient sensing and regulation of energy metabolism. Furthermore, overexpression of TFE3 triggered lysosomal exocytosis and resulted in efficient cellular clearance in a cellular model of a lysosomal storage disorder, Pompe disease, thus identifying TFE3 as a potential therapeutic target for the treatment of lysosomal disorders.

Rag GTPases mediate amino acid–dependent recruitment of TFEB and MITF to lysosomes

Active Rag GTPases are required for recruitment of TFEB to lysosomes and its phosphorylation by mTORC1, inhibiting its function under nutrient-rich conditions.

Involvement of clathrin and AP-2 in the trafficking of MHC class II molecules to antigen-processing compartments

Major histocompatibility complex class II (MHC-II) molecules are composed of two polymorphic chains, α and β, which assemble with an invariant chain, Ii, in the endoplasmic reticulum. The assembled MHC-II complexes are transported to the Golgi complex and then to late endosomes/lysosomes, where Ii is degraded and αβ dimers bind peptides derived from exogenous antigens. Targeting of MHC-II molecules to these compartments is mediated by two dileucine-based signals in the cytoplasmic domain of Ii. These signals bind in vitro to two adaptor protein (AP) complexes, AP-1 and AP-2, which are components of clathrin coats involved in vesicle formation and cargo sorting. The physiological roles of these proteins in MHC-II molecule trafficking, however, remain to be addressed. Here, we report the use of RNA interference to examine the involvement of clathrin and four AP complexes (AP-1, AP-2, AP-3, and AP-4) in MHC-II molecule trafficking in vivo. We found that depletion of clathrin or AP-2 caused >10-fold increases in Ii expression on the cell surface and a concomitant decrease in Ii localization to endosomal/lysosomal vesicles. In addition, depletion of clathrin or AP-2 delayed the degradation of Ii and reduced the surface expression of peptide-loaded αβ dimers. In contrast, depletion of AP-1, AP-3, or AP-4 had little or no effect. These findings demonstrate that clathrin and AP-2 participate in MHC-II molecule trafficking in vivo. Because AP-2 is only associated with the plasma membrane, these results also indicate that a significant pool of MHC-II molecules traffic to the endosomal–lysosomal system by means of the cell surface.

Novel roles for the MiTF/TFE family of transcription factors in organelle biogenesis, nutrient sensing, and energy homeostasis

The MiTF/TFE family of basic helix–loop–helix leucine zipper transcription factors includes MITF, TFEB, TFE3, and TFEC. The involvement of some family members in the development and proliferation of specific cell types, such as mast cells, osteoclasts, and melanocytes, is well established. Notably, recent evidence suggests that the MiTF/TFE family plays a critical role in organelle biogenesis, nutrient sensing, and energy metabolism. The MiTF/TFE family is also implicated in human disease. Mutations or aberrant expression of most MiTF/TFE family members has been linked to different types of cancer. At the same time, they have recently emerged as novel and very promising targets for the treatment of neurological and lysosomal diseases. The characterization of this fascinating family of transcription factors is greatly expanding our understanding of how cells synchronize environmental signals, such as nutrient availability, with gene expression, energy production, and cellular homeostasis.

The calcium channel mucolipin-3 is a novel regulator of trafficking along the endosomal pathway.

The varitint‐waddler phenotype in mice is caused by gain‐of‐function mutations in mucolipin‐3 (MCOLN3), a member of the mucolipin family of ion channels. These mice are characterized by defects in pigmentation, hearing loss and vestibular defects, suggesting that MCOLN3 might play a role in melanosome trafficking and hair cell maturation. Recent evidence has shown that MCOLN3 is a Ca2+–permeable channel and its activity is regulated by pH. Here we show that MCOLN3 primarily localizes to early and late endosomes in human epithelial cells. This distribution at the less acidic portions of the endocytic pathway is consistent with the reported inactivation of the channel by low pH. Furthermore, overexpression of MCOLN3 causes dramatic alterations in the endosomal pathway, including enlargement of Hrs‐positive endosomes, delayed degradation of epidermal growth factor (EGF) and EGF receptor (EGFR) and defective autophagosome maturation, whereas depletion of endogenous MCOLN3 enhances EGFR degradation. Finally, we found that endosomal pH is higher in cells overexpressing MCOLN3 and propose a model in which Ca2+ release from endosomes mediated by MCOLN3 might be important for efficient endosomal acidification. Therefore, MCOLN3 is a novel Ca2+ channel that plays a crucial role in the regulation of cargo trafficking along the endosomal pathway.

Identification of the penta-EF-hand protein ALG-2 as a CA2+-dependent interactor of mucolipin-1

Loss of function mutations in mucolipin-1 (MCOLN1) have been linked to mucolipidosis type IV (MLIV), a recessive lysosomal storage disease characterized by severe neurological and ophthalmological abnormalities. MCOLN1 is an ion channel that regulates membrane transport along the endolysosomal pathway. It has been suggested that MCOLN1 participates in several Ca2+-dependent processes, including fusion of lysosomes with the plasma membrane, fusion of late endosomes and autophagosomes with lysosomes, and lysosomal biogenesis. Here, we searched for proteins that interact with MCOLN1 in a Ca2+-dependent manner. We found that the penta-EF-hand protein ALG-2 binds to the NH-terminal cytosolic tail of MCOLN1. The interaction is direct, strictly dependent on Ca2+, and mediated by a patch of charged and hydrophobic residues located between MCOLN1 residues 37 and 49. We further show that MCOLN1 and ALG-2 co-localize to enlarged endosomes induced by overexpression of an ATPase-defective dominant-negative form of Vps4B (Vps4BE235Q). In agreement with the proposed role of MCOLN1 in the regulation of fusion/fission events, we found that overexpression of MCOLN1 caused accumulation of enlarged, aberrant endosomes that contain both early and late endosome markers. Interestingly, aggregation of abnormal endosomes was greatly reduced when the ALG-2-binding domain in MCOLN1 was mutated, suggesting that ALG-2 regulates MCOLN1 function. Overall, our data provide new insight into the molecular mechanisms that regulate MCOLN1 activity. We propose that ALG-2 acts as a Ca2+ sensor that modulates the function of MCOLN1 along the late endosomal-lysosomal pathway.

TFEB and TFE3 are novel components of the integrated stress response

To reestablish homeostasis and mitigate stress, cells must activate a series of adaptive intracellular signaling pathways. The participation of the transcription factors TFEB and TFE3 in cellular adaptation to starvation is well established. Here, we show that TFEB and TFE3 also play an important role in the cellular response to ER stress. Treatment with ER stressors causes translocation of TFEB and TFE3 to the nucleus in a process that is dependent on PERK and calcineurin but not on mTORC1. Activated TFEB and TFE3 enhance cellular response to stress by inducing direct transcriptional upregulation of ATF4 and other UPR genes. Under conditions of prolonged ER stress, TFEB and TFE3 contribute to cell death, thus revealing an unexpected role for these proteins in controlling cell fate. This work evidences a broader role of TFEB and TFE3 in the cellular response to stress than previously anticipated and reveals an integrated cooperation between different cellular stress pathways.