bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2019‒11‒17
nineteen papers selected by
Viktor Korolchuk
Newcastle University

  1. J Cell Sci. 2019 Nov 13. pii: jcs222570. [Epub ahead of print]132(21):
      The mechanistic target of rapamycin (mTOR) signaling pathway coordinates environmental and intracellular cues to control eukaryotic cell growth. As a pivot point between anabolic and catabolic processes, mTOR complex 1 (mTORC1) signaling has established roles in regulating metabolism, translation and autophagy. Hyperactivity of the mTOR pathway is associated with numerous human diseases, including diabetes, cancer and epilepsy. Pharmacological inhibition of the mTOR pathway can extend lifespan in a variety of model organisms. Given its broad control of essential cellular processes and clear relevance to human health, there is extensive interest in elucidating how upstream inputs regulate mTORC1 activation. In this Cell Science at a Glance article and accompanying poster, we summarize our understanding of how extracellular and intracellular signals feed into the mTOR pathway, how the lysosome acts as an mTOR signaling hub, and how downstream signaling controls autophagy and lysosome biogenesis.
    Keywords:  Amino acids; Autophagy; Cell growth; Glucose; Lysosome; Nutrients; Signaling; mTORC1
  2. Cell Mol Life Sci. 2019 Nov 12.
      We investigated the role of autophagy, a controlled lysosomal degradation of cellular macromolecules and organelles, in glutamate excitotoxicity during nutrient deprivation in vitro. The incubation in low-glucose serum/amino acid-free cell culture medium synergized with glutamate in increasing AMP/ATP ratio and causing excitotoxic necrosis in SH-SY5Y human neuroblastoma cells. Glutamate suppressed starvation-triggered autophagy, as confirmed by diminished intracellular acidification, lower LC3 punctuation and LC3-I conversion to autophagosome-associated LC3-II, reduced expression of proautophagic beclin-1 and ATG5, increase of the selective autophagic target NBR1, and decreased number of autophagic vesicles. Similar results were observed in PC12 rat pheochromocytoma cells. Both glutamate-mediated excitotoxicity and autophagy inhibition in starved SH-SY5Y cells were reverted by NMDA antagonist memantine and mimicked by NMDA agonists D-aspartate and ibotenate. Glutamate reduced starvation-triggered phosphorylation of the energy sensor AMP-activated protein kinase (AMPK) without affecting the activity of mammalian target of rapamycin complex 1, a major negative regulator of autophagy. This was associated with reduced mRNA levels of autophagy transcriptional activators (FOXO3, ATF4) and molecules involved in autophagy initiation (ULK1, ATG13, FIP200), autophagosome nucleation/elongation (ATG14, beclin-1, ATG5), and autophagic cargo delivery to autophagosomes (SQSTM1). Glutamate-mediated transcriptional repression of autophagy was alleviated by overexpression of constitutively active AMPK. Genetic or pharmacological AMPK activation by AMPK overexpression or metformin, as well as genetic or pharmacological autophagy induction by TFEB overexpression or lithium chloride, reduced the sensitivity of nutrient-deprived SH-SY5Y cells to glutamate excitotoxicity. These data indicate that transcriptional inhibition of AMPK-dependent cytoprotective autophagy is involved in glutamate-mediated excitotoxicity during nutrient deprivation in vitro.
    Keywords:  Brain; Energy stress; Ischemia; Neurodegeneration; Neurotoxicity
  3. Sci Rep. 2019 Nov 12. 9(1): 16593
      Xenophagy is a selective lysosomal degradation pathway for invading pathogens in host cells. However, invading bacteria also develop survival mechanisms to inhibit host autophagy. RavZ is a protein secreted by Legionella that irreversibly delipidates mammalian autophagy-related protein 8 (mATG8) on autophagic membranes in host cells via efficient autophagic membrane targeting. In this study, we leveraged the autophagic membrane-targeting mechanism of RavZ and generated a new autophagosome probe by replacing the catalytic domain of RavZ with GFP. This probe is efficiently localized to mATG8-positive autophagic membranes via a synergistic combination between mATG8 protein-binding mediated by the LC3-interacting region (LIR) motifs and phosphoinositide-3-phosphate (PI3P) binding mediated by the membrane-targeting (MT) domain. Furthermore, the membrane association activity of this new probe with an MT domain was more efficient than that of probes with a hydrophobic domain that were previously used in LIR-based autophagosome sensors. Finally, by substituting the LIR motifs of RavZ with selective LIR motifs from Fyco1 or ULK2, we developed new probes for detecting LC3A/B- or GABARAP subfamily-positive autophagic membranes, respectively. We propose that these new RavZ-based sensors will be useful for monitoring and studying the function of mATG8-positive autophagic membranes in different cellular contexts for autophagy research.
  4. EMBO Rep. 2019 Nov 11. e48317
      Autophagy is a highly conserved catabolic process through which defective or otherwise harmful cellular components are targeted for degradation via the lysosomal route. Regulatory pathways, involving post-translational modifications such as phosphorylation, play a critical role in controlling this tightly orchestrated process. Here, we demonstrate that TBK1 regulates autophagy by phosphorylating autophagy modifiers LC3C and GABARAP-L2 on surface-exposed serine residues (LC3C S93 and S96; GABARAP-L2 S87 and S88). This phosphorylation event impedes their binding to the processing enzyme ATG4 by destabilizing the complex. Phosphorylated LC3C/GABARAP-L2 cannot be removed from liposomes by ATG4 and are thus protected from ATG4-mediated premature removal from nascent autophagosomes. This ensures a steady coat of lipidated LC3C/GABARAP-L2 throughout the early steps in autophagosome formation and aids in maintaining a unidirectional flow of the autophagosome to the lysosome. Taken together, we present a new regulatory mechanism of autophagy, which influences the conjugation and de-conjugation of LC3C and GABARAP-L2 to autophagosomes by TBK1-mediated phosphorylation.
    Keywords:  ATG4; ATG8; TBK1; autophagy; phosphorylation
  5. J Mol Biol. 2019 Nov 06. pii: S0022-2836(19)30623-0. [Epub ahead of print]
      We review current knowledge of the process of autophagosome formation with special emphasis on the very early steps: turning on the autophagy pathway, assembling the autophagy machinery, and building the autophagosome. The pathway is remarkably well co-ordinated spatially and temporally, and it shows broad conservation across species and cell types, including neurons. In addition, although much current knowledge derives mostly from settings of non-selective autophagy, recent work also indicates that selective autophagy, and more specifically mitophagy, shows similar dynamics. Having an understanding of this remarkable process may help the design of novel therapeutics for neurodegeneration and other pathologies.
    Keywords:  Autophagosomes; ULK complex; VPS34 complex; endoplasmic reticulum; mTOR; mitophagy
  6. Aging Cell. 2019 Nov 13. e13043
      Reduced insulin/IGF signaling (IIS) extends lifespan in multiple organisms. Different processes in different tissues mediate this lifespan extension, with a set of interplays that remain unclear. We here show that, in Drosophila, reduced IIS activity modulates methionine metabolism, through tissue-specific regulation of glycine N-methyltransferase (Gnmt), and that this regulation is required for full IIS-mediated longevity. Furthermore, fat body-specific expression of Gnmt was sufficient to extend lifespan. Targeted metabolomics showed that reducing IIS activity led to a Gnmt-dependent increase in spermidine levels. We also show that both spermidine treatment and reduced IIS activity are sufficient to extend the lifespan of Drosophila, but only in the presence of Gnmt. This extension of lifespan was associated with increased levels of autophagy. Finally, we found that increased expression of Gnmt occurs in the liver of liver-specific IRS1 KO mice and is thus an evolutionarily conserved response to reduced IIS. The discovery of Gnmt and spermidine as tissue-specific modulators of IIS-mediated longevity may aid in developing future therapeutic treatments to ameliorate aging and prevent disease.
    Keywords:  IGF; aging; autophagy; insulin; lifespan; metabolism; polyamine
  7. Histochem Cell Biol. 2019 Nov 12.
      Autophagy is a degradative cellular process that can be both non-selective and selective and begins with the formation of a unique smooth double-membrane phagophore which wraps around a portion of the cytoplasm. Excess and damaged organelles and cytoplasmic protein aggregates are degraded by selective autophagy. Previously, we reported that in fed HepG2 cells, cytoplasmic aggregates of EDEM1 and surplus fibrinogen Aα-γ assembly intermediates are targets of selective autophagy receptors and become degraded by a selective autophagy called aggrephagy. Here, we show by multiple confocal immunofluorescence and colocalization panels the codistribution of cytoplasmic protein aggregates with the selective autophagy receptors p62/SQSTM1 and NBR1 and with the phagophore marker LC3, and that phagophores induced by vinblastine treatment contain complexes of protein aggregates and selective autophagy receptors. By combined serial ultrathin section analysis and immunoelectron microscopy, we found that in fed HepG2 cells, a basically ribosome-free subdomain of rough endoplasmic reticulum (RER) cisternae forms a cradle that engulfs the cytoplasmic protein aggregates. This RER subdomain appears structurally different from omegasomes formed by the RER, which were suggested to provide a membrane platform from which the phagophore is derived in starvation-induced autophagy. Taken together, our observations provide further evidence for the importance of RER subdomains as a site and membrane source for phagophore formation and show their involvement in selective autophagy.
    Keywords:  Aggrephagy; EDEM1; Fibrinogen Aα–γ assembly intermediates; NBR1; Rough endoplasmic reticulum; Selective autophagy; p62/SQSTM1
  8. Autophagy. 2019 Nov 12. 1-3
      VCP (valosin containing protein) recognizes a wide variety of substrates and mediates their degradation via the ubiquitin-proteasome system and macroautophagy/autophagy. The substrate repertoire of VCP, however, is not fully understood. In our recent study, we found that Drosophila TER94/VCP mediates autophagic degradation of an Argonaute subfamily protein (AGO1), which binds microRNAs (miRNAs) and silences the expression of thousands of target genes. In the absence of TER94/VCP, miRNA-mediated gene silencing is globally impaired. Our findings reveal an unexpected connection between VCP and AGO, which may dramatically expand the biological significance of VCP.
    Keywords:  Argonaute; Iruka; Ufd1–Npl4; VCP; gene silencing; microRNA; ubiquitin
  9. Sci Adv. 2019 Nov;5(11): eaay4624
      The PINK1 protein kinase activates the PARK2 ubiquitin ligase to promote mitochondrial ubiquitylation and recruitment of ubiquitin-binding mitophagy receptors typified by OPTN and TAX1BP1. Here, we combine proximity biotinylation of OPTN and TAX1BP1 with CRISPR-Cas9-based screens for mitophagic flux to develop a spatial proteogenetic map of PARK2-dependent mitophagy. Proximity labeling of OPTN allowed visualization of a "mitochondrial-autophagosome synapse" upon mitochondrial depolarization. Proximity proteomics of OPTN and TAX1BP1 revealed numerous proteins at the synapse, including both PARK2 substrates and autophagy components. Parallel mitophagic flux screens identified proteins with roles in autophagy, vesicle formation and fusion, as well as PARK2 targets, many of which were also identified via proximity proteomics. One protein identified in both approaches, HK2, promotes assembly of a high-molecular weight complex of PINK1 and phosphorylation of ubiquitin in response to mitochondrial damage. This work provides a resource for understanding the spatial and molecular landscape of PARK2-dependent mitophagy.
  10. Elife. 2019 Nov 14. pii: e51401. [Epub ahead of print]8
      Cholesterol is a major structural component of the plasma membrane (PM). The majority of PM cholesterol forms complexes with other PM lipids, making it inaccessible for intracellular transport. Transition of PM cholesterol between accessible and inaccessible pools maintains cellular homeostasis, but how cells monitor PM cholesterol accessibility remains unclear. We show that endoplasmic reticulum (ER)-anchored lipid transfer proteins, the GRAMD1s, sense and transport accessible PM cholesterol to the ER. GRAMD1s bind one another and populate at ER-PM contacts by sensing a transient expansion of the accessible pool of PM cholesterol via GRAM domains and facilitate its transport via StART-like domains. Cells lacking all three GRAMD1s exhibit striking expansion of the accessible pool of PM cholesterol due to less efficient PM to ER transport of accessible cholesterol. Thus, GRAMD1s facilitate movement of accessible PM cholesterol to the ER in order to counteract acute increase of PM cholesterol, activating non-vesicular cholesterol transport.
    Keywords:  biochemistry; cell biology; chemical biology; human
  11. Mol Cell. 2019 Nov 05. pii: S1097-2765(19)30797-X. [Epub ahead of print]
      The family of bacterial SidE enzymes catalyzes non-canonical phosphoribosyl-linked (PR) serine ubiquitination and promotes infectivity of Legionella pneumophila. Here, we describe identification of two bacterial effectors that reverse PR ubiquitination and are thus named deubiquitinases for PR ubiquitination (DUPs; DupA and DupB). Structural analyses revealed that DupA and SidE ubiquitin ligases harbor a highly homologous catalytic phosphodiesterase (PDE) domain. However, unlike SidE ubiquitin ligases, DupA displays increased affinity to PR-ubiquitinated substrates, which allows DupA to cleave PR ubiquitin from substrates. Interfering with DupA-ubiquitin binding switches its activity toward SidE-type ligase. Given the high affinity of DupA to PR-ubiquitinated substrates, we exploited a catalytically inactive DupA mutant to trap and identify more than 180 PR-ubiquitinated host proteins in Legionella-infected cells. Proteins involved in endoplasmic reticulum (ER) fragmentation and membrane recruitment to Legionella-containing vacuoles (LCV) emerged as major SidE targets. The global map of PR-ubiquitinated substrates provides critical insights into host-pathogen interactions during Legionella infection.
    Keywords:  ADP-ribosylation; ER-fragmentation; Legionella pneumophila; SdeA; deubiquitinase; deubiquitination; endoplasmic reticulum; phosphodiesterase; phosphoribosyl serine ubiquitination
  12. Genes Cells. 2019 Nov 12.
      Atg2 is one of the essential factors for autophagy. Recent advance of structural and biochemical study on yeast Atg2 proposed that Atg2 tethers the edge of the isolation membrane (IM) to the endoplasmic reticulum and mediates direct lipid transfer from ER to IM for IM expansion. In mammals, two Atg2 orthologs, ATG2A and ATG2B, participate in autophagic process. Here we showed that human ATG2B possesses the membrane tethering (MT) and lipid transfer (LT) activity that was promoted by negatively charged membranes and an Atg18 ortholog WIPI4. By contrast, negatively charged membranes reduced the yeast Atg2 activities in the absence of Atg18. These results suggest that the MT/LT activity of Atg2 is evolutionally conserved although their regulation differs among species.
    Keywords:  ATG2B; Atg18; Atg2; Autophagy; WIPI4; lipid transfer
  13. Proc Natl Acad Sci U S A. 2019 Nov 15. pii: 201911393. [Epub ahead of print]
      The mammalian target of rapamycin (mTOR) pathway, which plays a critical role in regulating cellular growth and metabolism, is aberrantly regulated in the pathogenesis of a variety of neoplasms. Here we demonstrate that dual mTORC1/mTORC2 inhibitors OSI-027 and PP242 cause catastrophic macropinocytosis in rhabdomyosarcoma (RMS) cells and cancers of the skin, breast, lung, and cervix, whereas the effects are much less pronounced in immortalized human keratinocytes. Using RMS as a model, we characterize in detail the mechanism of macropinocytosis induction. Macropinosomes are distinct from endocytic vesicles and autophagosomes in that they are single-membrane bound vacuoles formed by projection, ruffling, and contraction of plasma membranes. They are positive for EEA-1 and LAMP-1 and contain watery fluid but not organelles. The vacuoles then merge and rupture, killing the cells. We confirmed the inhibition of mTORC1/mTORC2 as the underpinning mechanism for macropinocytosis. Exposure to rapamycin, an mTORC1 inhibitor, or mTORC2 knockdown alone had little or reduced effect relative to the combination. We further demonstrate that macropinocytosis depends on MKK4 activated by elevated reactive oxygen species. In a murine xenograft model, OSI-027 reduced RMS tumor growth. Molecular characterization of the residual tumors was consistent with the induction of macropinocytosis. Furthermore, relative to the control xenograft tumors, the residual tumors manifested reduced expression of cell proliferation markers and proteins that drive the epithelial mesenchymal transition. These data indicate a role of mTORC2 in regulating tumor growth by macropinocytosis and suggest that dual inhibitors could help block refractory or recurrent RMS and perhaps other neoplasms and other cancer as well.
    Keywords:  EMT; RMS xenografts; mTORC1/2 inhibitors; macropinocytosis; rhabdomyosarcoma cell lines
  14. Autophagy. 2019 Nov 11. 1-15
      BAG2 (BCL2 associated athanogene 2) is associated with cell fate determination in response to various pathological conditions. However, the effects of BAG2 on M. tuberculosis-induced endoplasmic reticulum (ER) stress remain elusive. Herein, we report that M. tuberculosis infection of macrophages triggered ER stress and downregulated BAG2 expression. Overexpression of BAG2 enhanced autophagic flux and activated macroautophagy/autophagy targeted to the ER (reticulophagy). In addition, through increasingly localizing SQSTM1 to the ER in BAG2-overexpressing macrophages, we found that the autophagy receptor protein SQSTM1/p62 (sequestosome 1) is associated with the BAG2-induced reticulophagy. Our data also confirmed that BAG2 could render cells resistant to M. tuberculosis-induced cellular damage, and the anti-apoptotic effects of BAG2 in M. tuberculosis-treated macrophages were partially abolished by the autophagic flux inhibitor bafilomycin A1. Furthermore, the dissociation of BECN1 and BCL2 mediated by activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) was responsible for BAG2-activated autophagy. In addition, XBP1 downstream of the ERN1/IRE1 signaling pathway was bound to the Bag2 promoter region and transcriptionally inhibited BAG2 expression. Collectively, these results indicated that BAG2 has anti-apoptotic effects on M. tuberculosis-induced ER stress, which is dependent on the promotion of autophagic flux and the induction of selective autophagy. We revealed a potential host defense mechanism that links BAG2 to ER stress and autophagy during M. tuberculosis infection.Abbreviations: ATF6: activating transcription factor 6; BECN1: beclin 1; Baf A1: bafilomycin A1; CASP3: caspase 3; DDIT3/CHOP/GADD153: DNA damage inducible transcript 3; DAPI: 4',6-diamidino-2-phenylindole; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; HSPA5/GRP78/BiP: heat shock protein 5; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPK/ERK: mitogen-activated protein kinase; SQSTM1/p62: sequestosome 1; UPR: unfolded protein response; XBP1: x-box binding protein 1.
    Keywords:  Apoptosis; BCL2 associated athanogene 2; M. tuberculosis; autophagy; endoplasmic reticulum stress; reticulophagy
  15. PLoS One. 2019 ;14(11): e0225214
      Dysfunctional mitochondria have been implicated in aging and age-related disorders such as Parkinson's diseases (PD). We previously showed that pink1 and parkin, two familial PD genes, function in a linear pathway to maintain mitochondrial integrity and function. Studies of mammalian cell lines also suggest that these genes regulate mitochondrial autophagy(mitophagy). Overexpressing Parkin promotes proteostasis and function of aged muscles both in fruit flies and mice, and recent studies also indicated that mitochondrial ubiquitination are accumulated in aged muscles. However, the underlying mechanisms for pink1 and parkin mediated mitophagy on longevity is not fully understood. Here, we found that mitochondrial ubiquitination increased in indirect flight muscles (IFMs) in an age-dependent manner. Overexpression of pink1 or parkin in IFMs can abolish mitochondrial ubiquitination, restore ATP level and extend lifespan, while blocking autophagy via ATG1 knock-down suppress these effects in aged IFMs. Taken together, these results show that pink1/parkin promotes mitophagy of mitochondrial ubiquitination in aged muscles and extend lifespan in an Atg1-dependent manner. Our study provides physiological evidence that mitophagy of mitochondrial ubiquitination mediated by PINK1/ Parkin is crucial for muscle function and highlights the role of mitophagy in the pathogenesis of chronic diseases like PD.
  16. Adv Exp Med Biol. 2019 ;1209 145-166
      Autophagy, an evolutionarily conserved cargo degradation process, is responsible to remove superfluous and unwanted cytoplasmic materials and maintain cellular homeostasis. Autophagy can be highly selective and target specific cargoes by utilizing multiple cargo receptors, which bind both ubiquitinated cargoes and autophagosomes. Mounting evidence has revealed the deep involvement of selective autophagy in innate immunity upon pathogen invasion, including eliminating microbial pathogens, initiating the anti-microbe responses, and inhibiting excessive immune responses. Given the importance of selective autophagy in innate immunity, how cargo receptors deliver pathogens and intracellular host constitutes to autophagosomes during infection remains to be elucidated. In this review, we summarize current evidence for the regulation of innate immunity by selective autophagy and try to elucidate the mechanisms employed by cargo receptor network in mediating diverse innate immune responses.
    Keywords:  Cargo receptor; Immune response; Selective autophagy; Ubiquitination
  17. Cell Death Dis. 2019 Nov 14. 10(11): 865
      Evolving concepts on Parkinson's disease (PD) pathology suggest that α-synuclein (aSYN) promote dopaminergic neuron dysfunction and death through accumulating in the mitochondria. However, the consequence of mitochondrial aSYN localisation on mitochondrial structure and bioenergetic functions in neuronal cells are poorly understood. Therefore, we investigated deleterious effects of mitochondria-targeted aSYN in differentiated human dopaminergic neurons in comparison with wild-type (WT) aSYN overexpression and corresponding EGFP (enhanced green fluorescent protein)-expressing controls. Mitochondria-targeted aSYN enhanced mitochondrial reactive oxygen species (ROS) formation, reduced ATP levels and showed severely disrupted structure and function of the dendritic neural network, preceding neuronal death. Transmission electron microscopy illustrated distorted cristae and many fragmented mitochondria in response to WT-aSYN overexpression, and a complete loss of cristae structure and massively swollen mitochondria in neurons expressing mitochondria-targeted aSYN. Further, the analysis of mitochondrial bioenergetics in differentiated dopaminergic neurons, expressing WT or mitochondria-targeted aSYN, elicited a pronounced impairment of mitochondrial respiration. In a pharmacological compound screening, we found that the pan-caspase inhibitors QVD and zVAD-FMK, and a specific caspase-1 inhibitor significantly prevented aSYN-induced cell death. In addition, the caspase inhibitor QVD preserved mitochondrial function and neuronal network activity in the human dopaminergic neurons overexpressing aSYN. Overall, our findings indicated therapeutic effects by caspase-1 inhibition despite aSYN-mediated alterations in mitochondrial morphology and function.
  18. Science. 2019 11 15. 366(6467): 818-822
      To achieve homeostasis, cells evolved dynamic and self-regulating quality control processes to adapt to new environmental conditions and to prevent prolonged damage. We discuss the importance of two major quality control systems responsible for degradation of proteins and organelles in eukaryotic cells: the ubiquitin-proteasome system (UPS) and autophagy. The UPS and autophagy form an interconnected quality control network where decision-making is self-organized on the basis of biophysical parameters (binding affinities, local concentrations, and avidity) and compartmentalization (through membranes, liquid-liquid phase separation, or the formation of aggregates). We highlight cellular quality control factors that delineate their differential deployment toward macromolecular complexes, liquid-liquid phase-separated subcellular structures, or membrane-bound organelles. Finally, we emphasize the need for continuous promotion of quantitative and mechanistic research into the roles of the UPS and autophagy in human pathophysiology.
  19. Sci Rep. 2019 Nov 14. 9(1): 16760
      The human L-type amino acid transporter 1 (LAT1), also known as SLC7A5, catalyzes the transport of large neutral amino acids across the plasma membrane. As the main transporter of several essential amino acids, notably leucine, LAT1 plays an important role in mTORC1 activation. Furthermore, it is overexpressed in various types of cancer cells, where it contributes importantly to sustained growth. Despite the importance of LAT1 in normal and tumor cells, little is known about the mechanisms that might control its activity, for example by promoting its downregulation via endocytosis. Here we report that in HeLa cells, activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) triggers efficient endocytosis and degradation of LAT1. Under these conditions we found LAT1 downregulation to correlate with increased LAT1 ubiquitylation. This modification was considerably reduced in cells depleted of the Nedd4-2 ubiquitin ligase. By systematically mutagenizing the residues of the LAT1 cytosolic tails, we identified a group of three close lysines (K19, K25, K30) in the N-terminal tail that are important for PMA-induced ubiquitylation and downregulation. Our study thus unravels a mechanism of induced endocytosis of LAT1 elicited by Nedd4-2-mediated ubiquitylation of the transporter's N-terminal tail.