TXNIP mediates LAT1/SLC7A5 endocytosis to reduce amino acid uptake in cells entering quiescence

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   Review Commons

   Mar 10, 2025

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   Reply to the reviewers

   *Reviewer # 1: *The study is well-executed, and the claims are supported by appropriate experiments. As introduced by the authors in their introduction, ubiquitin-dependent endocytosis of AA transporters has been previously shown in S. cerevisiae and TXNIP has previously been identified as a regulator of glucose uptake by promoting endocytosis of GLUT1 and GLUT4. Here, the authors identify the molecular mechanism by which TXNIP promotes the endocytosis, and degradation of amino acid transporters (SLC7A5-SLC7A3) through its interaction with HECT-type ubiquitin ligases. This is an advance in the field and will be of interest for researchers in the fields …

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   Reply to the reviewers

   *Reviewer # 1: *The study is well-executed, and the claims are supported by appropriate experiments. As introduced by the authors in their introduction, ubiquitin-dependent endocytosis of AA transporters has been previously shown in S. cerevisiae and TXNIP has previously been identified as a regulator of glucose uptake by promoting endocytosis of GLUT1 and GLUT4. Here, the authors identify the molecular mechanism by which TXNIP promotes the endocytosis, and degradation of amino acid transporters (SLC7A5-SLC7A3) through its interaction with HECT-type ubiquitin ligases. This is an advance in the field and will be of interest for researchers in the fields of quiescence, metabolism and cell biology. Experiments are well designed and important controls have been performed. Overall, the claims and the conclusions are supported by the data.* *

   Response: We thank the reviewer for the thorough evaluation of our manuscript and for the insightful, constructive comments. Reviewer 1 had five minor comments, and we have addressed them all.

   Minor comment 1:* The authors should indicate how often western blot experiments were repeated with similar results. Ideally band quantification (as in Fig. 2b) for the most relevant proteins should be provided for all shown Western blots. *

   Response: Each Western Blot (WB) experiment has been performed at least 3 times and each WB result for SLC7A5 is complemented by immunofluorescence and/or additionally by FACS analysis, across the manuscript.

   In the partially revised version of the manuscript, we already__ incorporated WB quantifications of SLC7A5 protein levels__ for Figures 1c, f, h, Figure 3b, Figure 4b, and Figure 5a, c in Supplementary Figure 1b, Supplementary Figure 2c, f, Supplementary Figure 4a, e, and in Supplementary Figure 5a, c, respectively.

   Minor comment 2: For confocal images no n number of experiments/analyzed cells is stated. Often only 2-3 cells are shown in these images. In some figures, conclusions from these confocal images are additionally supported by cell surface FACS.

   Response: Each immunofluorescence experiment has been performed at least 3 times.

   Minor ____comment 3:* For panels with missing cell surface FACS quantifications, the authors should consider using the existing imaging data to perform quantifications of the membrane signal. In this way the reader can get the right impression of the reproducibility of the phenotype described.** *

   Response: Each immunofluorescence experiment has been performed at least 3 times. In the partially revised version of the manuscript, line-scan quantification of immunofluorescence (IF) of SLC3A2 at the plasma membrane (PM) is now provided for immunofluorescence experiments in Figure 1e, g, Figure 3c, e in Supplementary Figure 2b, e, Supplementary Figure 4b, c, and for SLC2A1 in Supplementary Figure 3i, were FACS data was missing. In addition, WB experiments complement the results of each IF experiment.

   Minor comment 4: I appreciate that the authors have also investigated SLC2A1 endocytosis in their experimental setup. Interestingly, they found that TXNIP mediated downregulation of SLC7A5-SLC3A2 was not linked to TXNIP mediated SLC2A1 endocytosis. Since the role of TXNIP in glucose metabolism has been studied in more detail in the past, it would be interesting if the authors could further comment on the differences/similarities in the molecular mechanism of glucose and AA transporter downregulation in the discussion.* *

   Response: Thank you for bringing up this point. We now have added the following paragraph to the discussion to speculate about the differences/similarities in the molecular mechanism of glucose and AA transporter downregulation in the discussion:

   ‘Moreover, in RPE1 cells entering quiescence, GLUT1/4 was not downregulated. Hence, it seems that TXNIP can discriminate, in a context dependent manner, between targeting SLC7A5-SLC3A2 or GLUT1/4 for endocytosis. Since AKT mediated phosphorylation invariably appeared to inactivate TXNIP, and dephosphorylation re-activated it, additional mechanism must confer TXNIP selectivity towards SLC7A5-SLC3A2 or GLUT1/4. We consider it likely, that the exposure of sorting motifs in cytosolic tails of SLC7A5 or GLUT1/4 could regulate the binding of activated TXNIP and thus controls selective endocytosis to adapt nutrient uptake. The exposure of these sorting motifs could be dependent on the metabolic context / state of the cell. Indeed, yeast a-arrestins can detect n- or c-terminal acidic sorting motifs in amino acid transporters, respectively, that are alternatively exposed in response to amino acid excess or starvation (Ivashov* et al., 2020a) (Guiney et al, 2016). Inspection of the SLC7A5 sequence indicates a possible n-terminal acidic sorting motif (17EEKEEAREK25). Two lysine residues (K19, K25) in this sequence have been found to be ubiquitinated in an earlier study upon protein kinase C (PKC) activation and mTORC1 inhibition (Barthelemy & Andre, 2019; Rosario et al*, 2016).’

   Minor ____comment 5:* I would recommend a colour blind-friendly colour palette for the confocal images** *

   Response: Thank you for pointing this out – we have changed the color palette accordingly.

   *Reviewer # 2: *This study establishes TXNIP as a regulator of LAT1 endocytosis and metabolic homeostasis in quiescence. The integration of KO models and a TXNIP-deficient patient strengthens the findings, though clinical characterization remains underdeveloped relative to the mechanism reported, and biochemical interactions require endogenous validation. The work expands our understanding of TXNIP beyond association studies, positioning it as a key player in nutrient sensing and metabolic regulation. Addressing the concerns will enhance its relevance across fields - particularly metabolism, cell biology, and disease research. Overall, this is a very interesting study indeed. The use of TXNIP knockout models and a loss-of-function patient variant strengthens the conclusion that TXNIP is required for LAT1 degradation. The functional consequences of TXNIP deficiency (elevated intracellular aa, sustained mTORC1 activation, and accelerated quiescence exit) are well-supported by the data. The major concerns are as follows:

   Response: We thank the reviewer for the thorough evaluation of our manuscript and for the insightful, constructive comments. Reviewer 2 had three major concerns and one minor comment.

   Major concern 1.* The identification of a biallelic TXNIP loss-of-function variant in a patient with metabolic disease and neurological dysfunction is highly significant. However, it is problematic that the manuscript effectively presents a case report but does not explicitly frame it as such, and the clinical details are very superficial (lack of pedigree, genetics, structured disease timeline, differential diagnosis, any histology/scans/photography and broader metabolic profiling - please see best practices for case reports). Although whole-exome sequencing identified the TXNIP variant, it remains unclear whether other genetic or metabolic contributors were systematically excluded. At first glance, the clinical discovery strengthens the physiological significance of the cell biology. However, a discrepancy remains between the clear neurological presentation of the patient (intellectual disability, autism and epilepsy) and the fibroblast-based TXNIP-LAT1 mechanism described in the study. Furthermore, the metabolic phenotype described in this manuscript is significantly more severe than that reported in a previous Swedish study of TXNIP deficiency in humans, where the clinical presentation was milder. This discrepancy suggests that different TXNIP mutations may lead to a spectrum of clinical outcomes, which is highly novel (i.e. metabolic and neurological in terms of loss of function, and carcinogenesis with respect to association studies, reviewed in PMID: 37794178). Of course, this could be influenced by mutation type, genetic background, compensatory mechanisms or environmental factors - it is noteworthy that the previous siblings had mitochondrial dysfunction, and this remains unknown in the present individual. Addressing this variability and discussing potential reasons for the pronounced phenotype observed in this patient would strengthen the manuscript overall. It is noteworthy that LAT1 is highly expressed in brain endothelial cells, which can also adopt a quiescent state (PMID: 33627876), and the authors should expand beyond the single sentence in their discussion. In the absence of the above details, the title and conclusions of Figure 3 and in the discussion greatly overstate causality, implying a direct relationship between TXNIP loss and metabolic dysfunction, despite data from only one patient. his may indeed be the case, but the claims should be carefully revised to reflect an association rather than definitive causation until additional patients are identified. Additionally, while it is assumed that the authors have obtained ethical approval and informed consent, this needs to be explicitly stated for transparency, with dedicated details in the methods sections. Addressing these issues will improve the rigor and mechanistic coherence of the study - otherwise it is quite disjointed.*

   Response: We have addressed many these valid concerns and provide a detailed description of the patient in the partially revised manuscript (please see below).

   ‘The patient is a boy, born in 2014 as the first child of healthy, consanguineous parents of Turkish origin. During pregnancy, the mother was diagnosed with polyhydramnios. At 38 + 6 weeks of gestation, the baby was in a breech position, leading to a cesarean section. At birth, he weighed 3880 g (P90), measured 55 cm in length, and had a head circumference of 38 cm.

   On the seventh day of life, he exhibited floppiness, recurrent hypoglycaemia, and lactic acidosis, prompting his transfer from the birth hospital to a tertiary care centre. During the first three days there, his lowest recorded blood glucose level was 30 mg/dl, lactate levels were approximately 6.5 mmol/l, and pH was 7.11. Subsequently, he developed hypertriglyceridemia, with triglyceride levels reaching 364 mg/dl. Initially stable, he began experiencing elevated pCO2 levels (up to 70 mmHg due to bradypnea) and metabolic acidosis on day 10. A glucose infusion (10 mg/kg/min) stabilized his glucose and lactate concentrations, though lactate remained elevated at around 3-4 mmol/l. Regardless, his muscular hypotonia persisted. On day 12, a skin punch biopsy for a fibroblast culture was performed.

   By day 20, glucose and lactate levels had stabilized with regular feeding, allowing his transfer back to a peripheral hospital. During infancy, his blood glucose concentrations were within standard range (Supplementary Table 1), but the boy experienced recurrent hypoglycaemia in response to metabolic stress, e.g., infections. He exhibited psychomotor developmental delays and, from 18 months of age, experienced increasing epileptic seizures (up to 3-4 per month), which were managed with levetiracetam, topiramate, and lamotrigine. Currently, he remains metabolically stable but presents with significant developmental delay, muscular hypotonia, and autistic features.

   Whole-exome sequencing from peripheral blood of the patient detected a homozygous single nucleotide insertion c.642_643insT in exon 5 of 8 of the TXNIP gene. This variant was not recorded in the population genetic variant database gnomAD that lists TXNIP as likely haplosufficient (pLI = 0, LOEUF = 0,709: https://gnomad.broadinstitute.org accessed Sept. 10, 2024). No other (likely) pathogenic variant in any other gene, with known function in metabolism was identified as explanation of the clinical features in the child. Potential pathogenic variants in genes required for mitochondrial functions were also not detected, although they were initially expected to cause the phenotype of the boy.

   The TXNIP variant c.642_643insT caused a frameshift and a premature stop codon after 59 AA (denoted p.Ile215TyrfsTer59), likely causing nonsense-mediated decay (NMD) or the synthesis of a severely truncated TXNIP protein (Figure 3a). Both parents are healthy heterozygous carriers for the TXNIP variant. Serendipitously, this TXNIP variant was similar to the gene-edited version in the RPE1 TXNIPKO cells (p.I215TfsX11).

   The patient showed consistent metabolic alterations compatible with an AA transporter deficiency. Blood plasma concentrations of several large neutral amino acids (LNAAs, including L, I, V) were elevated throughout the years 2014 – 2022 (Supplementary Table 1). The increased molar ratio of the LNAAs (L, I, V) to aromatic AAs (F, Y), resulted in an elevated Fischer’s ratio (FR, 2014: FR = 4.46; 2016: FR = 5.38, 2018: FR = 5.90; 2021; FR= 6.98; 2022: FR = 4.23; FR reference range = 2.10 - 4). The methionine levels are not dramatically altered (Supplementary Table 1).’

   We also provide the following ethical statement:

   __‘Ethical statement __

   All patients’ data were extracted from the medical routine records. Written informed consent for molecular genetic studies and publication of data was obtained from the legal guardians of the patient. This approach was approved by the ethics committee of the Medical University of Innsbruck (UN4501-MUI). The study was conducted in accordance with the principles of the Declaration of Helsinki.’

   During the revision, we will additionally address how the other known TXNIP variant (TXNIP p.Gln58His; p.Gly59*; PMID: 30755400) affects nutrient transporter endocytosis. This TXNIP variant will be expressed in TXNIPKO RPE1 cells to analyze its effect on quiescence induced SLC7A5 downregulation. The results of this experiment will allow comparing directly the effect of both known TXNIP variants (p.Gln58His; p.Gly59* and p.Ile215TyrfsTer59) on SLC7A5 downregulation in an identical genetic background. In addition, we will compare how both TXNIP variants affect mitochondrial function (using Seahorse technology).

   Major concern 2.* The authors report that TXNIP interacts with HECT E3 ligases to regulate substrate degradation, yet this conclusion is drawn from overexpression-based immunoprecipitation studies, which do not confirm interaction under endogenous conditions. Without direct evidence of TXNIP-HECT E3 binding at native expression levels, this mechanistic link remains unresolved. Given that the authors have already generated antibody-validated TXNIP KO models, endogenous validation should be feasible if the interactions are not super-transient.*

   Response: While the manuscript was under review, we have improved the stringency of our TXNIP-HECT type ubiquitin ligase interaction experiments and developed additional biochemical experiments that strengthen our original conclusions. In the course of these experiments, we found that the interaction of TXNIP with NEDD4, WWP2 and HECW1/2 (but not with WWP1 or ITCH) were particularly dependent on the PPxY331 motif.

   During the revision, we will conduct additional experiments to substantiate these findings and to narrow down the list possible ubiquitin ligases that are required for the downregulation of SLC7A5. In particular, we will test if endogenous TXNIP co-immunoprecipitates (in a PPxY motif dependent manner) NEDD4, HECW1/2 or another HECT type ubiquitin ligase.

   Furthermore, we will include a newly developed ‘Bead-Immobilized Prey Assay (BIPA)’, were protein-protein interactions can be analyzed by microscopy in a fast in straight forward manner. In the BIPA, ALFA-TXNIP (or mutant variants) are first captured on ALFA-beads (Bead immobilized). These TXNIP beads are then incubated with cell lysates from HEK293 expressing GFP-tagged HECT type ubiquitin ligases (Prey). The binding of the GFP-tagged ubiquitin ligases to the TXNIP beads is analyzed by fluorescence microscopy and quantified (Figure 1b, a BIPA with YFP-NEDD4). This efficient assay will also be conducted with NEDD4, WWP1, WWP2, HECW2, and ITCH to analyze how they bind to TXNIP, TXNIP-PPxY331 and the PPxY double mutant.

   Together we are confident that our experiments establish that TXNIP must interact with a specific subset of HECT type ubiquitin ligase (our prime candidate are NEDD4 and HECW1/2) to trigger SLC7A5-SLC3A2 ubiquitination, endocytosis and lysosomal degradation.

   *Major concern 3. *What are the temporal dynamics of TXNIP-associated degradation, and is this process distinct from endosomal microautophagy (as reported in PMID: 30018090)? The authors present convincing, high-quality FACS-based data supporting TXNIP-mediated turnover. If this pathway is mechanistically separate from endosomal microautophagy, it suggests a hierarchy of degradation pathways leading to quiescence. Live cell imaging studies that define the temporal dynamics of this process using the tools the authors have created may reveal the relationship between these processes and refine the broader implications of TXNIP in homeostatic adaptation.

   Response: Thank you for this interesting suggestion.

   During the revision, we will first investigate a potential temporal correlation of endosomal micro-autophagy of p62/SQSTM1, NBR1, TAX1BP1, NDP52, and NCOA4 (PMID: 30018090) and the downregulation of SLC7A5 as cells enter quiescence. For these experiments, we will follow the turn-over of the above-mentioned autophagy adaptors and compare it to the turnover of SLC7A5, using either WB analysis, or microscopy or both.

   Next, we will test if SLC7A5-SLC3A2 endocytosis and lysosomal degradation is required to initiate endosomal micro-autophagy of p62/SQSTM1, NBR1, TAX1BP1, NDP52, and NCOA4 in TXNIPKO cells.

   Together, these experiments will address if the endosomal micro-autophagy and TXNIP mediated downregulation of SLC7A5 are mechanistically linked during entry into quiescence.

   Minor comment 1.* In the discussion, the authors might briefly speculate on the implications of any functional redundancy that might exist between other arrestins.*

   We will provide this information in the fully revised version of the manuscript.

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2. 

   Review Commons

   Mar 10, 2025

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   Referee #2

   Evidence, reproducibility and clarity

   Summary

   Cells entering quiescence must recalibrate metabolism to match lower energy demands, yet the role of endocytosis in this process remains poorly defined. In yeast, amino acid transporters undergo rapid endocytic degradation upon entry into quiescence, but whether a similar mechanism exists in human cells is unknown. Kahlhofer and colleagues demonstrate that human quiescent cells selectively degrade plasma membrane-resident amino acid (AA) transporters, particularly SLC7A5-SLC3A2 (LAT1-4F2hc) and SLC1A5 (ASCT2). TXNIP facilitates LAT1 endocytosis and lysosomal degradation, thereby limiting AA uptake and intracellular AA levels …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   Referee #2

   Evidence, reproducibility and clarity

   Summary

   Cells entering quiescence must recalibrate metabolism to match lower energy demands, yet the role of endocytosis in this process remains poorly defined. In yeast, amino acid transporters undergo rapid endocytic degradation upon entry into quiescence, but whether a similar mechanism exists in human cells is unknown. Kahlhofer and colleagues demonstrate that human quiescent cells selectively degrade plasma membrane-resident amino acid (AA) transporters, particularly SLC7A5-SLC3A2 (LAT1-4F2hc) and SLC1A5 (ASCT2). TXNIP facilitates LAT1 endocytosis and lysosomal degradation, thereby limiting AA uptake and intracellular AA levels to attenuate mTORC1 signaling and protein translation. In TXNIP-deficient cells, LAT1 remains at the plasma membrane, leading to persistent AA uptake, sustained mTORC1 activation, and accelerated proliferation upon exiting quiescence. In proliferating cells, AKT phosphorylates TXNIP at Ser308, inactivating it and preventing LAT1 degradation, a process that is reversed upon entering quiescence. Notably, the authors identify a biallelic TXNIP loss-of-function variant in a patient with severe metabolic disease, recurrent hypoglycemia, and amino acid imbalances. Patient-derived fibroblasts exhibit defective LAT1 internalization, a phenotype that cannot be rescued by complementation with the pathogenic TXNIP variant, supporting an important role in disease pathology. Functionally, TXNIP-deficient cells have elevated AA levels that sustain mTORC1 activation, enhancing translation, and accelerate exit from quiescence. This study establishes TXNIP as a key regulator of amino acid transporter endocytosis in quiescent cells, linking metabolic adaptation, mTORC1 signaling, and cell cycle control through a previously unrecognized mechanism.

   Major comments

   Overall, this is a very interesting study indeed. The use of TXNIP knockout models and a loss-of-function patient variant strengthens the conclusion that TXNIP is required for LAT1 degradation. The functional consequences of TXNIP deficiency (elevated intracellular aa, sustained mTORC1 activation, and accelerated quiescence exit) are well-supported by the data. The major concerns are as follows:

   1. The identification of a biallelic TXNIP loss-of-function variant in a patient with metabolic disease and neurological dysfunction is highly significant. However, it is problematic that the manuscript effectively presents a case report but does not explicitly frame it as such, and the clinical details are very superficial (lack of pedigree, genetics, structured disease timeline, differential diagnosis, any histology/scans/photography and broader metabolic profiling - please see best practices for case reports). Although whole-exome sequencing identified the TXNIP variant, it remains unclear whether other genetic or metabolic contributors were systematically excluded. At first glance, the clinical discovery strengthens the physiological significance of the cell biology. However, a discrepancy remains between the clear neurological presentation of the patient (intellectual disability, autism and epilepsy) and the fibroblast-based TXNIP-LAT1 mechanism described in the study. Furthermore, the metabolic phenotype described in this manuscript is significantly more severe than that reported in a previous Swedish study of TXNIP deficiency in humans, where the clinical presentation was milder. This discrepancy suggests that different TXNIP mutations may lead to a spectrum of clinical outcomes, which is highly novel (i.e. metabolic and neurological in terms of loss of function, and carcinogenesis with respect to association studies, reviewed in PMID: 37794178). Of course, this could be influenced by mutation type, genetic background, compensatory mechanisms or environmental factors - it is noteworthy that the previous siblings had mitochondrial dysfunction, and this remains unknown in the present individual. Addressing this variability and discussing potential reasons for the pronounced phenotype observed in this patient would strengthen the manuscript overall. It is noteworthy that LAT1 is highly expressed in brain endothelial cells, which can also adopt a quiescent state (PMID: 33627876), and the authors should expand beyond the single sentence in their discussion. In the absence of the above details, the title and conclusions of Figure 3 and in the discussion greatly overstate causality, implying a direct relationship between TXNIP loss and metabolic dysfunction, despite data from only one patient. his may indeed be the case, but the claims should be carefully revised to reflect an association rather than definitive causation until additional patients are identified. Additionally, while it is assumed that the authors have obtained ethical approval and informed consent, this needs to be explicitly stated for transparency, with dedicated details in the methods sections. Addressing these issues will improve the rigor and mechanistic coherence of the study - otherwise it is quite disjointed.
   2. The authors report that TXNIP interacts with HECT E3 ligases to regulate substrate degradation, yet this conclusion is drawn from overexpression-based immunoprecipitation studies, which do not confirm interaction under endogenous conditions. Without direct evidence of TXNIP-HECT E3 binding at native expression levels, this mechanistic link remains unresolved. Given that the authors have already generated antibody-validated TXNIP KO models, endogenous validation should be feasible if the interactions are not super-transient.
   3. What are the temporal dynamics of TXNIP-associated degradation, and is this process distinct from endosomal microautophagy (as reported in PMID: 30018090)? The authors present convincing, high-quality FACS-based data supporting TXNIP-mediated turnover. If this pathway is mechanistically separate from endosomal microautophagy, it suggests a hierarchy of degradation pathways leading to quiescence. Live cell imaging studies that define the temporal dynamics of this process using the tools the authors have created may reveal the relationship between these processes and refine the broader implications of TXNIP in homeostatic adaptation.

   Minor comments

   In the discussion, the authors might briefly speculate on the implications of any functional redundancy that might exist between other arrestins.

   Significance

   This study establishes TXNIP as a regulator of LAT1 endocytosis and metabolic homeostasis in quiescence. The integration of KO models and a TXNIP-deficient patient strengthens the findings, though clinical characterization remains underdeveloped relative to the mechanism reported, and biochemical interactions require endogenous validation. The work expands our understanding of TXNIP beyond association studies, positioning it as a key player in nutrient sensing and metabolic regulation. Addressing the concerns will enhance its relevance across fields - particularly metabolism, cell biology, and disease research.

   Referees cross-commenting

   The comments raised by Reviewer #1 are reasonable, well-founded and align well with the concerns I have raised.

   Expertise: Organelle dynamics/degradation, metabolism, biochemistry, tissue homeostasis/disease.

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3. 

   Review Commons

   Mar 10, 2025

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   Referee #1

   Evidence, reproducibility and clarity

   Summary:

   In their study, Kahlhofer et al. investigate the mechanism by which cells downregulate amino acid (AA) uptake while entering quiescence. Using western blotting, immunohistochemistry and KO cell lines, the authors show that the α-arrestin family protein TXNIP acts as a regulator of specific membrane-resident AA transporters. They demonstrate that TXNIP promotes the endocytosis and degradation of SLC7A5-SLC7A3 in serum-starved cells as a result of reduced AKT signalling. They further show that the molecular mechanism involves a direct interaction between a PPCY motif in TXNIP and HECT-type ubiquitin ligases which promote AA …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   Summary:

   In their study, Kahlhofer et al. investigate the mechanism by which cells downregulate amino acid (AA) uptake while entering quiescence. Using western blotting, immunohistochemistry and KO cell lines, the authors show that the α-arrestin family protein TXNIP acts as a regulator of specific membrane-resident AA transporters. They demonstrate that TXNIP promotes the endocytosis and degradation of SLC7A5-SLC7A3 in serum-starved cells as a result of reduced AKT signalling. They further show that the molecular mechanism involves a direct interaction between a PPCY motif in TXNIP and HECT-type ubiquitin ligases which promote AA transporter ubiquitination. Additionally, they identify a novel TXNIP loss-of-function in a patient and show that patient-derived fibroblasts fail to downregulate SLC7A5-SLC7A3 upon starvation. This dysregulation likely contributes to persistent alterations in serum AA levels observed in the patient.

   Experiments are well designed and important controls have been performed. Overall, the claims and the conclusions are supported by the data.

   Minor comments:

   Authors should indicate how often western blot experiments were repeated with similar results. Ideally band quantification (as in Fig. 2b) for the most relevant proteins should be provided for all shown Western blots.

   For confocal images no n number of experiments/analysed cells is stated. Often only 2-3 cells are shown in these images. In some figures, conclusions from these confocal images are additionally supported by cell surface FACS. For panels with missing cell surface FACS quantifications, the authors should consider using the existing imaging data to perform quantifications of the membrane signal. In this way the reader can get the right impression of the reproducibility of the phenotype described.

   I appreciate that the authors have also investigated SLC2A1 endocytosis in their experimental setup. Interestingly, they found that TXNIP mediated downregulation of SLC7A5-SLC3A2 was not linked to TXNIP mediated SLC2A1 endocytosis. Since the role of TXNIP in glucose metabolism has been studied in more detail in the past, it would be interesting if the authors could further comment on the differences/similarities in the molecular mechanism of glucose and AA transporter downregulation in the discussion.

   I would recommend a colour blind-friendly colour palette for the confocal images

   Significance

   The study is well-executed, and the claims are supported by appropriate experiments. As introduced by the authors in their introduction, ubiquitin-dependent endocytosis of AA transporters has been previously shown in S. cerevisiae and TXNIP has previously been identified as a regulator of glucose uptake by promoting endocytosis of GLUT1 and GLUT4. Here, the authors identify the molecular mechanism by which TXNIP promotes the endocytosis, and degradation of amino acid transporters (SLC7A5-SLC7A3) through its interaction with HECT-type ubiquitin ligases. This is an advance in the field and will be of interest for researchers in the fields of quiescence, metabolism and cell biology.

   Read the original source
4. 

   Version published to 10.1101/2024.10.29.620655 on bioRxiv

   Oct 30, 2024