Inorganic phosphate uptake is a universal function accomplished by transporters that are present across the living world. In contrast, no phosphate exporter has ever been identified in metazoans. Here, we show that depletion of XPR1, a multipass membrane molecule initially identified as the cell-surface receptor for xenotropic and polytropic murine leukemia retroviruses (X- and P-MLV), induced a decrease in phosphate export and that reintroduction of various XPR1 proteins, from fruit fly to human, rescued this defect. Inhibition of phosphate export was also obtained with a soluble ligand generated from the envelope-receptor-binding domain of X-MLV in all human cell lines tested, as well as in diverse stem cells and epithelial cells derived from renal proximal tubules, the main site of phosphate homeostasis regulation. These results provide new insights on phosphate export in metazoans and the role of Xpr1 in this function.

The metabolic state of quiescent hematopoietic stem cells (HSCs) is an important regulator of self-renewal, but it is unclear whether or how metabolic parameters contribute to HSC lineage specification and commitment. Here, we show that the commitment of human and murine HSCs to the erythroid lineage is dependent upon glutamine metabolism. HSCs require the ASCT2 glutamine transporter and active glutamine metabolism for erythroid specification. Blocking this pathway diverts EPO-stimulated HSCs to differentiate into myelomonocytic fates, altering in vivo HSC responses and erythroid commitment under stress conditions such as hemolytic anemia. Mechanistically, erythroid specification of HSCs requires glutamine-dependent de novo nucleotide biosynthesis. Exogenous nucleosides rescue erythroid commitment of human HSCs under conditions of limited glutamine catabolism, and glucose-stimulated nucleotide biosynthesis further enhances erythroid specification. Thus, the availability of glutamine and glucose to provide fuel for nucleotide biosynthesis regulates HSC lineage commitment under conditions of metabolic stress.

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ARS-CoV-2 is the etiologic agent of the coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV-2 is the third highly pathogenic coronavirus to cross the species barrier in the 21st century after SARS-CoV-1 in 2002-2003 (refs. 1-3 ) and MERS-CoV in 2012 (ref. 4 ). Four additional HCoVs (HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1) are known to circulate seasonally in humans, contributing to approximately one-third of common cold infections 5 . Like SARS-CoV-1 and HCoV-NL63, SARS-CoV-2 entry into target cells is mediated by the angiotensin-converting enzyme 2 (ACE2) receptor [6][7][8][9][10] . The cellular serine protease transmembrane protease serine 2 (TMPRSS2) is used by both SARS-CoV-1 and SARS-CoV-2 for Spike protein priming at the plasma membrane 6,11 . Cathepsins are also involved in SARS-CoV spike protein cleavage and fusion peptide exposure upon entry via an endocytic route, in the absence of TMPRSS2 (refs. [12][13][14][15] ).Several whole-genome KO CRISPR screens for the identification of coronavirus regulators have been reported [16][17][18][19][20][21] . These screens used naturally permissive simian Vero E6 cells of kidney origin 20 ; human Huh7 cells (or derivatives) of liver origin (ectopically expressing ACE2 and TMPRSS2, or not) 16,18,19 ; and A549 cells of lung origin, ectopically expressing ACE2 17,21 . Here, we conducted genome-wide, loss-of-function CRISPR KO screens and gain-of-function CRISPRa screens in several cell lines, including physiologically relevant human Calu-3 cells and Caco-2 cells, of lung and colorectal adenocarcinoma origin, respectively, followed by secondary screens in these cell lines and in Huh7.5.1 and A549 cells. Well-known SARS-CoV-2 host-dependency factors were identified among top hits, such as ACE2 and either TMPRSS2 or cathepsin L (depending on the cell type). We characterized the mechanism of action of the top hits and assessed their effect on other coronaviruses and influenza A orthomyxovirus. Altogether, this study provides insights into the coronavirus life cycle by identifying host factors that modulate replication and might lead to pan-coronavirus strategies for host-directed therapies. ResultsMeta-analysis of CRISPR KO screens highlights the importance of multiple models. Vero E6 cells present high levels of cytopathic effects (CPEs) upon SARS-CoV-2 replication, making them ideal to perform whole-genome CRISPR screens for host factor identification. A Chlorocebus sabaeus single-guide RNA (sgRNA) library was previously successfully used to identify host factors regulating SARS-CoV-2 (isolate USA-WA1/2020) replication 20 . Therefore, we initially repeated whole-genome CRISPR KO screens in Vero E6 cells using the SARS-CoV-2 isolate BetaCoV/France/ IDF0372/2020 (Fig. 1a). Importantly, ACE2 was a top hit (Fig. 1b

Inflammatory conditions can profoundly alter human neutrophils, a leukocyte subset generally viewed as terminally differentiated and catabolic. In cystic fibrosis (CF) patients, neutrophils recruited to CF airways show active exocytosis and sustained phosphorylation of prosurvival, metabolic pathways. Because the CF airway lumen is also characterized by high levels of free glucose and amino acids, we compared surface expression of Glut1 (glucose) and ASCT2 (neutral amino acids) transporters, as well as that of PiT1 and PiT2 (inorganic phosphate transporters), in blood and airway neutrophils, using specific retroviral envelope-derived ligands. Neither nutrient transporter expression nor glucose uptake was altered on blood neutrophils from CF patients compared with healthy controls. Notably, however, airway neutrophils of CF patients had higher levels of PiT1 and Glut1 and increased glucose uptake compared with their blood counterparts. Based on primary granule exocytosis and scatter profiles, CF airway neutrophils could be divided into two subsets, with one of the subsets characterized by more salient increases in Glut1, ASCT2, PiT1, and PiT2 expression. Moreover, in vitro exocytosis assays of blood neutrophils suggest that surface nutrient transporter expression is not directly associated with primary (or secondary) granule exocytosis. Although expression of nutrient transporters on CF blood or airway neutrophils was not altered by genotype, age, gender, or Pseudomonas aeruginosa infection, oral steroid treatment decreased Glut1 and PiT2 levels in blood neutrophils. Thus, neutrophils recruited from blood into the CF airway lumen display augmented cell surface nutrient transporter expression and glucose uptake, consistent with metabolic adaptation.

Mutations in XPR1, a gene encoding an inorganic phosphate exporter, have recently been identified in patients with primary familial brain calcification (PFBC). Using Sanger sequencing, we screened XPR1 in 18 unrelated patients with PFBC and no SLC20A2, PDGFB, or PDGFRB mutation. XPR1 variants were tested in an in vitro physiological complementation assay and patient blood cells were assessed ex vivo for phosphate export. We identified a novel c.260T > C, p.(Leu87Pro) XPR1 variant in a 41-year-old man complaining of micrographia and dysarthria and demonstrating mild parkinsonism, cerebellar ataxia and executive dysfunction. Brain (123)I-Ioflupane scintigraphy showed marked dopaminergic neuron loss. Peripheral blood cells from the patient exhibited decreased phosphate export. XPR1 in which we introduced the mutation was not detectable at the cell surface and did not lead to phosphate export. These results confirm that loss of XPR1-mediated phosphate export function causes PFBC, occurring in less than 8 % of cases negative for the other genes, and may be responsible for parkinsonism.

Metabolic adaptations and changes in the expression of nutrient transporters are known to accompany tumorigenic processes. Nevertheless, in the context of solid tumors, studies of metabolism are hindered by a paucity of tools allowing the identification of cell surface transporters on individual cells. Here, we developed a method for the dissociation of human breast cancer tumor xenografts combined with quantification of cell surface markers, including metabolite transporters. The expression profiles of four relevant nutrient transporters for cancer cells' metabolism, Glut1, ASCT2, PiT1 and PiT2 (participating to glucose, glutamine and inorganic phosphate, respectively), as detected by new retroviral envelope glycoprotein-derived ligands, were distinctive of each tumor, unveiling underlying differences in metabolic pathways. Our tumor dissociation procedure and nutrient transporter profiling technology provides opportunities for future basic research, clinical diagnosis, prognosis and evaluation of therapeutic responses, as well as for drug discovery and development. Markers of tumor cell metabolism are of valuable importance for basic and clinical cancer research. The identification of metabolic alterations that accompany cancer processes is becoming critical for refining the clinical evaluation and treatment of patients, 1,2 as well as for further drug discovery and development. While metabolomics relies mainly on the quantification of anabolic and catabolic end products, 3 the bidirectional fluxes of nutrients and metabolites, as well as the efflux of toxins and pharmaceutical compounds, are ensured by nutrient transporters. 4 However, the monitoring of metabolite and nutrient transporters at the cell surface has been hampered by the paucity, if not total lack, of exofacial antibodies to such transporters. 5,6 Cell entry of gamma-and deltaretroviruses occurs via recognition of membrane metabolite transporters by retroviral envelope glycoproteins (Env). 7,8 Viral entry occurs following the specific binding of the amino-terminal receptor binding domain (RBD) of an individual Env with its cognate receptor. We designed RBD probes, derived from gamma-and deltaretroviral Env, as specific ligands that bind to these cell surface transporters. Binding of defined ligands allows the quantification of distinct sets of metabolite transporters at the cell surface. 9-13 Furthermore, while cell surface labeling is readily achieved on cultured cell lines and circulating hematopoietic cells, singlecell labeling of solid tumors has remained problematic. Indeed, such analyses require dissociation methods that yield

The multifunctional protein E4 transcription factor 1 (E4F1) is an essential regulator of epidermal stem cell (ESC) maintenance. Here, we found that E4F1 transcriptionally regulates a metabolic program involved in pyruvate metabolism that is required to maintain skin homeostasis. E4F1 deficiency in basal keratinocytes resulted in deregulated expression of dihydrolipoamide acetyltransferase (Dlat), a gene encoding the E2 subunit of the mitochondrial pyruvate dehydrogenase (PDH) complex. Accordingly, E4f1 knock-out (KO) keratinocytes exhibited impaired PDH activity and a redirection of the glycolytic flux toward lactate production. The metabolic reprogramming of E4f1 KO keratinocytes associated with remodeling of their microenvironment and alterations of the basement membrane, led to ESC mislocalization and exhaustion of the ESC pool. ShRNA-mediated depletion of Dlat in primary keratinocytes recapitulated defects observed upon E4f1 inactivation, including increased lactate secretion, enhanced activity of extracellular matrix remodeling enzymes, and impaired clonogenic potential. Altogether, our data reveal a central role for Dlat in the metabolic program regulated by E4F1 in basal keratinocytes and illustrate the importance of PDH activity in skin homeostasis.enewal and wound healing of the epidermis rely on a pool of epidermal stem cells (ESC) located in the basal layer of the interfollicular epithelium (IFE) and in the bulge region of the hair follicle (HF). In the IFE, these long-lived ESC give rise to progenitors with increased proliferative capacities that differentiate into keratinocytes as they migrate upward into suprabasal layers. Numerous studies have addressed the role of several key signaling pathways, such as those implicating bone morphogenetic proteins, TGF-β, Notch, Sonic Hedgehog, or Wnt in skin homeostasis, and how they control ESC maintenance (1-3). The role of these pathways in regulating stemness has been attributed to the regulation of cell proliferation, cell death, cellular senescence, cell adhesion, or differentiation. Although previous data indicate that some of these stem cell regulators also control energy metabolism in the hematopoietic or neuronal lineages (4), very few studies have yet addressed their metabolic functions in keratinocytes. In addition, the potential role of specific metabolic regulators in the control of skin homeostasis remains poorly documented. Nevertheless, previous observations indicate that deregulation of the nutrient-sensing mammalian target of rapamycin pathway in basal keratinocytes occurs as a consequence of prolonged Wnt signaling, leading to the progressive exhaustion of HF bulge stem cells (5). Recent data also indicate that genetic inactivation in mouse epidermis of mitochondrial transcription factor A (Tfam), a gene involved in mitochondrial DNA replication and transcription, impinges on keratinocyte differentiation but does not impair maintenance of basal keratinocytes (6). Although these results suggest that basal keratinocytes display a metabolic st...