Clotilde Laussel

Véronique Albanèse

Francisco Javier García-Rodríguez

Alberto Ballin

Quentin Defenouillère

Sébastien Léon

1 2-deoxyglucose inhibits yeast AMPK signaling and triggers glucose transporter endocytosis, potentiating the drug toxicity Clotilde Laussel #1 , Véronique Albanèse #1 , Francisco Javier García-Rodríguez 1 , Alberto Ballin 1 , Quentin Defenouillère 1 , Sébastien Léon 1,2 Affiliations:

# : Equal contributions 1 : Université Paris Cité, CNRS, Institut Jacques Monod, F-75013, Paris, France. email: sebastien.leon@ijm.fr ; tel. +33 (0)1 57 27 80 57.

Running title:

Effects of 2-deoxyglucose on yeast AMPK signaling and endocytosis, and contribution to 2DG tolerance

Introduction

2DG is a glucose analog which lacks a hydroxyl group in position 2, and mainly acts as a competitive inhibitor of glucose metabolism [START_REF] Xi | The wonders of 2-deoxy-D-glucose[END_REF]. As such, it competes with glucose for entry into cells and for its subsequent phosphorylation by hexokinase, but is almost not further metabolized. This leads to the intracellular accumulation of 2DG-6-phosphate, a toxic metabolite that inhibits glycolysis and thus leads to a rapid depletion of ATP stores, and the activation of nutrient starvation-activated pathways such as 5'-AMP activated protein kinase, AMPK [START_REF] Hardie | Regulation of AMP-activated protein kinase by natural and synthetic activators[END_REF]. Moreover, since mannose is the C2 epimer of glucose, 2-deoxyglucose is also 2deoxymannose and thus also interferes with mannose metabolism, notably inhibiting protein N-glycosylation [reviewed in 3]. This causes a series of cellular effects, and triggers the onset of the Unfolded Protein Response pathway to face the stress encountered by a defective glycosylation of proteins at the endoplasmic reticulum, which is reportedly the main toxic effect of 2DG in some conditions [START_REF] Kurtoglu | Under normoxia, 2-deoxy-D-glucose elicits cell death in select tumor types not by inhibition of glycolysis but by interfering with N-linked glycosylation[END_REF].

The realization that many cancer cells display an aberrantly high glucose uptake and metabolism has led to the idea that glycolysis inhibitors could be used to preferentially target and kill tumor cells [START_REF] Luengo | Targeting Metabolism for Cancer Therapy[END_REF]. This also allowed the use of radiolabeled glucose derivatives, such as 18-fluoro-2-deoxyglucose, to preferentially label tumors for cancer imaging (PET-scans) [START_REF] Pajak | 2-Deoxy-d-Glucose and Its Analogs: From Diagnostic to Therapeutic Agents[END_REF]. So far, trials using 2DG as the sole chemotherapeutic agent have failed because of the relative toxicity of this molecule, however strategies using 2DG or other 2-deoxy-substituted versions of glucose in combination with other drugs are under study and are promising [START_REF] Pajak | 2-Deoxy-d-Glucose and Its Analogs: From Diagnostic to Therapeutic Agents[END_REF].

2DG has also been extensively used to understand glucose signaling in micro-organisms such as the baker's yeast Saccharomyces cerevisiae, where it has been instrumental to delineate the molecular mechanisms by which glucose regulates gene expression [reviewed in 3]. The metabolism of S. cerevisiae is biased towards the preferential use of glucose as a carbon source. At the molecular level, glucose represses the expression of many genes involved in respiratory metabolism or in the use of other carbon sources, thereby favoring the preferential use of glucose by fermentation [START_REF] Kayikci | Glucose repression in Saccharomyces cerevisiae[END_REF]. Among the actors in charge of adapting the transcriptional program with respect to glucose availability is the yeast orthologue of AMPK, Snf1 [START_REF] Kayikci | Glucose repression in Saccharomyces cerevisiae[END_REF][START_REF] Coccetti | Conventional and emerging roles of the energy sensor Snf1/AMPK in Saccharomyces cerevisiae[END_REF]. Snf1 is the catalytic subunit of a heterotrimeric complex which is active in the absence of glucose in the medium. A canonical example of a Snf1-regulated transcription is that of the invertase SUC2, required for sucrose hydrolysis and metabolism. Transferring yeast cells to sucrose medium activates Snf1, causing the phosphorylation of the transcriptional repressor Mig1, its translocation out of the nucleus and the derepression of SUC2 [START_REF] Vit | Regulated nuclear translocation of the Mig1 glucose repressor[END_REF][START_REF] Papamichos-Chronakis | The Snf1 kinase controls glucose repression in yeast by modulating interactions between the Mig1 repressor and the Cyc8-Tup1 co-repressor[END_REF].

Snf1, like other kinases of the AMPK family, is activated by phosphorylation of a residue in its activation loop [START_REF] Mccartney | Regulation of Snf1 kinase. Activation requires phosphorylation of threonine 210 by an upstream kinase as well as a distinct step mediated by the Snf4 subunit[END_REF]. This involves one of 3 upstream kinases whose activity is, surprisingly, not regulated by glucose.

Rather, evidence suggests that Snf1 activity is regulated by its dephosphorylation by the Protein Phosphatase 1 (PP1) complex made of the catalytic subunit, Glc7, and the glucose-specific regulatory subunit Reg1 [START_REF] Rubenstein | Access denied: Snf1 activation loop phosphorylation is controlled by availability of the phosphorylated threonine 210 to the PP1 phosphatase[END_REF].

How PP1 activity is linked to glucose availability is not yet clear, but deletion of REG1 leads to constitutive Snf1 activity and to the expression of glucose-repressed genes even in the presence of glucose [START_REF] Tu | REG1 binds to protein phosphatase type 1 and regulates glucose repression in Saccharomyces cerevisiae[END_REF].

Another phenotype displayed by the reg1∆ strain is its ability to grow robustly in presence of 2DG [START_REF] Laussel | Cellular toxicity of the metabolic inhibitor 2-deoxyglucose and associated resistance mechanisms[END_REF][START_REF] Ralser | A catabolic block does not sufficiently explain how 2-deoxy-D-glucose inhibits cell growth[END_REF][START_REF] Schmidt | Sugarcoating' 2-deoxyglucose: mechanisms that suppress its toxic effects[END_REF]. This is attributed to the constitutive activation of Snf1 in this mutant, as mutations in SNF1 cause hypersensitivity to 2DG and yeast lacking both Reg1 and Snf1 are also hypersensitive [START_REF] Mccartney | Genetic analysis of resistance and sensitivity to 2deoxyglucose in Saccharomyces cerevisiae[END_REF].

Despite the multifaceted cellular effects of 2DG, cells can indeed overcome 2DG toxicity by mechanisms that are currently being unraveled [reviewed in [START_REF] Laussel | Cellular toxicity of the metabolic inhibitor 2-deoxyglucose and associated resistance mechanisms[END_REF][START_REF] Schmidt | Sugarcoating' 2-deoxyglucose: mechanisms that suppress its toxic effects[END_REF]. A prolonged exposure of HeLa cells in 2DG-containing medium gave rise to resistant clones [START_REF] Barban | Studies on the mechanism of resistance to 2-deoxy-D-glucose in mammalian cell cultures[END_REF]. These cells displayed a high phosphatase activity towards 2DG-6phosphate (2DG-6-P), suggesting the presence of an enzyme that could detoxify this metabolite back into 2DG

and thus prevent the metabolic blockade. Studies in yeast also identified phosphatases, named Dog1 and Dog2, with a similar activity in vitro and whose overexpression allows growth in 2DG-containing medium [START_REF] Sanz | Molecular characterization of a gene that confers 2-deoxyglucose resistance in yeast[END_REF][START_REF] Randez-Gil | DOGR1 and DOGR2: two genes from Saccharomyces cerevisiae that confer 2-deoxyglucose resistance when overexpressed[END_REF].

Recently, we found that the most abundant isoform, Dog2, is itself a glucose-repressed gene and thus is regulated by Snf1/AMPK function [START_REF] Defenouillėre | The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose[END_REF]. This partially explains why strains with high Snf1/AMPK activity are resistant to 2DG, such as reg1∆ [START_REF] Ralser | A catabolic block does not sufficiently explain how 2-deoxy-D-glucose inhibits cell growth[END_REF][START_REF] Mccartney | Genetic analysis of resistance and sensitivity to 2deoxyglucose in Saccharomyces cerevisiae[END_REF], other reg1 mutant alleles [START_REF] Defenouillėre | The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose[END_REF][START_REF] Soncini | Spontaneous mutations that confer resistance to 2-deoxyglucose act through Hxk2 and Snf1 pathways to regulate gene expression and HXT endocytosis[END_REF], a viable glc7 mutation (Q48K) [START_REF] Soncini | Spontaneous mutations that confer resistance to 2-deoxyglucose act through Hxk2 and Snf1 pathways to regulate gene expression and HXT endocytosis[END_REF] or gain-of-function mutations in components of the AMPK complex [START_REF] Mccartney | Genetic analysis of resistance and sensitivity to 2deoxyglucose in Saccharomyces cerevisiae[END_REF][START_REF] Soncini | Spontaneous mutations that confer resistance to 2-deoxyglucose act through Hxk2 and Snf1 pathways to regulate gene expression and HXT endocytosis[END_REF][START_REF] Barney | Drug resistance in diploid yeast is acquired through dominant alleles, haploinsufficiency, gene duplication and aneuploidy[END_REF]. However, additional resistance mechanisms independent of DOG2 expression are at stake in these strains [START_REF] Defenouillėre | The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose[END_REF][START_REF] Soncini | Spontaneous mutations that confer resistance to 2-deoxyglucose act through Hxk2 and Snf1 pathways to regulate gene expression and HXT endocytosis[END_REF]. This is suggested by the observation that DOG2 deletion does not completely re-sensitize reg1∆ mutants to 2DG, and a point mutation in REG1 (reg1-P231L) confers 2DG resistance but does not impact on the glucose-mediated repression of genes [START_REF] Soncini | Spontaneous mutations that confer resistance to 2-deoxyglucose act through Hxk2 and Snf1 pathways to regulate gene expression and HXT endocytosis[END_REF]. Thus, Snf1 likely controls additional factors participating in 2DG resistance.

One such candidate is the arrestin-related protein, Rod1. Arrestin-related proteins (ARTs) are important regulators of plasma membrane protein endocytosis in response to extracellular cues [START_REF] Piper | Ubiquitin-dependent sorting in endocytosis[END_REF][START_REF] Macgurn | Ubiquitin and membrane protein turnover: from cradle to grave[END_REF]. ARTs recruit the ubiquitin ligase Rsp5 to nutrient transporters at the plasma membrane, promoting their subsequent ubiquitylation and endocytosis [START_REF] Sardana | Membrane Protein Quality Control Mechanisms in the Endo-Lysosome System[END_REF]. ART activity is regulated by nutrient signaling pathways, allowing to remodel the landscape of transporters at the plasma membrane to meet the physiological needs of cells facing a nutrient challenge [START_REF] Babst | Regulation of nutrient transporters by metabolic and environmental stresses[END_REF][START_REF] Kahlhofer | The alpha-arrestin family of ubiquitin ligase adaptors links metabolism with selective endocytosis[END_REF]. Particularly, Rod1 regulates endocytosis in response to glucose availability, and its activity is oppositely regulated by Snf1 and PP1 [28][START_REF] Llopis-Torregrosa | Regulation of the Yeast Hxt6 Hexose Transporter by the Rod1 alpha-Arrestin, the Snf1 Protein Kinase, and the Bmh2 14-3-3 Protein[END_REF][START_REF] O'donnell | AMPK-Mediated Regulation of Alpha-Arrestins and Protein Trafficking[END_REF], being a direct target of Snf1 [START_REF] Shinoda | Rod1, an arrestin-related protein, is phosphorylated by Snf1-kinase in Saccharomyces cerevisiae[END_REF]. A genome-wide screen revealed that rod1∆ is indeed resistant to 2DG [START_REF] Ralser | A catabolic block does not sufficiently explain how 2-deoxy-D-glucose inhibits cell growth[END_REF], thus the Snf1-mediated inhibition of Rod1 might contribute to 2DG resistance.

Point mutations aimed at abolishing Rod1 interaction with the ubiquitin ligase Rsp5, thereby annihilating their function as adaptor proteins in endocytosis, increased resistance to 2DG suggesting that it mediates 2DG toxicity through its function in endocytosis [START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF]. 2DG resistance of the rod1∆ strain is further increased by the additional deletion of the ROD1 paralogue ROG3 [START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF]. Rod1 (and Rog3, to a lesser extent) controls the endocytosis of the glucose transporters Hxt1 and Hxt3 triggered in response to 2DG exposure, and overexpression of the same transporters in a snf1∆ mutant restored partial resistance to 2DG [START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF]. Deletion of ROD1 and ROG3 stabilized hexose transporters at the plasma membrane in snf1∆ and restored its 2DGsensitivity to WT levels [START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF]. However, whether the effects of 2DG on endocytosis extend beyond glucose transporters is unknown, and there is currently no molecular understanding of how transporter stability/localization contributes to 2DG resistance.

In this study, we examined the effects of 2DG on endocytosis and found that 2DG triggers the endocytosis of many plasma membrane proteins, most of which depend on Rod1. Mechanistically, we show that opposite to the situation in mammalian cells, 2DG treatment leads to the dephosphorylation of Snf1/AMPK and several targets including Rod1, likely through a transient surge in PP1 activity. This favors Rod1 function and thus provides a rationale as to why this arrestin is central to 2DG-induced endocytosis. Finally, we demonstrate that 2DG resistance of the rod1∆ strain can solely be attributed to the maintenance of active glucose transporters at the membrane, where they can increase glucose uptake and contribute to detoxifying 2DG out of the cells, thus terminating the detoxification process.

Results

2DG triggers the endocytosis of many plasma membrane proteins

We previously described the proteomic changes occurring in response to 2DG and focused on proteins upregulated in this condition [START_REF] Defenouillėre | The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose[END_REF]. In addition, a few transmembrane/PM proteins displayed a significantly decreased abundance after 2h30 2DG treatment, including the amino acid permease Tat1 and the hexose transporter Hxt2 [START_REF] Defenouillėre | The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose[END_REF]. This could either be caused by a decreased synthesis or an active degradation by endocytosis. We verified this by looking at the regulation of the endogenously GFP-tagged proteins. Treatment [START_REF] Yang | ESCRT, not intralumenal fragments, sorts ubiquitinated vacuole membrane proteins for degradation[END_REF]. This may be due to the fact that 2DG causes ER stress in yeast [START_REF] Defenouillėre | The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose[END_REF], which itself induces vacuolar fragmentation [START_REF] Stauffer | Target of rapamycin signaling mediates vacuolar fission caused by endoplasmic reticulum stress in Saccharomyces cerevisiae[END_REF].

Tat2 and Hxt2 endocytosis in response to 2DG is reminiscent of the behavior of the low affinity hexose transporters Hxt1 and Hxt3 [START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF]. To document the extent of plasma membrane remodeling caused by 2DG exposure, we studied the regulation of a representative subset of plasma membrane proteins tagged with GFP at their endogenous loci before and after 2DG treatment. This included transporters of various substrates or structural classes, as well as receptor/sensor proteins whose expression could be detected in glucose-grown cells. This showed that many of these membrane proteins are endocytosed in response to 2DG (Fig

1D, Fig S2),
implying that 2DG triggers a general endocytosis response.

In yeast, nutrient transporter endocytosis is driven by their ubiquitylation at the plasma membrane, which relies on the action of the ubiquitin ligase Rsp5 and adaptor proteins of the arrestin-like family (ARTs) [START_REF] Macgurn | Ubiquitin and membrane protein turnover: from cradle to grave[END_REF][START_REF] Kahlhofer | The alpha-arrestin family of ubiquitin ligase adaptors links metabolism with selective endocytosis[END_REF].

Previous work highlighted the importance of the arrestin-related protein Rod1 in the 2DG-induced endocytosis of Hxt1 and Hxt3 [START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF]. Indeed, we found that most of the cargoes studied here depended on Rod1 for their

2DG triggers endocytosis by inducing Rod1 dephosphorylation and ubiquitylation

We then focused on understanding why Rod1 function is so central for 2-deoxyglucose endocytosis. In the case of glucose-induced endocytosis, Rod1 activity is regulated through changes in its post-translational modifications [28,[START_REF] Llopis-Torregrosa | Regulation of the Yeast Hxt6 Hexose Transporter by the Rod1 alpha-Arrestin, the Snf1 Protein Kinase, and the Bmh2 14-3-3 Protein[END_REF]. In a glucose-deprived medium, AMPK/Snf1 is active and phosphorylates Rod1, inhibiting its function [28,[START_REF] Llopis-Torregrosa | Regulation of the Yeast Hxt6 Hexose Transporter by the Rod1 alpha-Arrestin, the Snf1 Protein Kinase, and the Bmh2 14-3-3 Protein[END_REF][START_REF] Shinoda | Rod1, an arrestin-related protein, is phosphorylated by Snf1-kinase in Saccharomyces cerevisiae[END_REF][START_REF] Becuwe | Integrated control of transporter endocytosis and recycling by the arrestin-related protein Rod1 and the ubiquitin ligase Rsp5[END_REF]. Conversely, glucose addition in the medium leads to Rod1 dephosphorylation in a 7 PP1-dependent manner, followed by its ubiquitylation by Rsp5 [28,[START_REF] Llopis-Torregrosa | Regulation of the Yeast Hxt6 Hexose Transporter by the Rod1 alpha-Arrestin, the Snf1 Protein Kinase, and the Bmh2 14-3-3 Protein[END_REF]. These modifications promote its activity as an Rsp5 adaptor, leading to transporter ubiquitylation, endocytosis and vacuolar sorting [28,[START_REF] Llopis-Torregrosa | Regulation of the Yeast Hxt6 Hexose Transporter by the Rod1 alpha-Arrestin, the Snf1 Protein Kinase, and the Bmh2 14-3-3 Protein[END_REF][START_REF] Becuwe | Integrated control of transporter endocytosis and recycling by the arrestin-related protein Rod1 and the ubiquitin ligase Rsp5[END_REF]. Similarly, 2DG addition to glucose-grown cells triggers Rod1 ubiquitylation in an Rsp5-and PP1-dependent manner, but paradoxically 2DG was also reported to increase Snf1 activity [START_REF] O'donnell | AMPK-Mediated Regulation of Alpha-Arrestins and Protein Trafficking[END_REF][START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF]. Thus, how 2DG regulates the relative activities of Snf1/PP1 and how this impacts on Rod1 activity are not fully understood.

Previously, the effect of 2DG on Rod1 post-translational modifications was evaluated using an 3HA-tagged construct [START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF]. Although Rod1-3HA could support the glucose-induced endocytosis of Jen1 [28], we realized that it was unable to restore the 2DG-induced endocytosis of Hxt1-GFP and Hxt3-GFP in a rod1∆ context , contrary to Flag-tagged Rod1 (Flag) which functionally behaved like the wild-type protein (Fig S4A andB). We then re-evaluated Rod1 post-translational modifications using this functional construct. As for Rod1-3HA [START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF],

we observed drastic changes in Rod1 mobility on gel after 2DG treatment (Fig 2A). In glucose-grown cells, Rod1-Flag migrated as a diffuse band resulting from a mild phosphorylation, as determined by phosphatase treatment, and similarly to previously published results using Rod1-3HA [28,[START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF] (Fig 2A). This pattern gave rise to two discrete bands upon 2DG exposure, with the upper band corresponding to ubiquitylated Rod1, as shown by its disappearance when mutating the known ubiquitylated lysine(s) on Rod1 as described previously [28,[START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF]. The lower band corresponded to a dephosphorylated species of Rod1, as demonstrated by the fact that it migrated similarly as Rod1-Flag from phosphatase-treated extracts (Fig 2B). This fits with previous results obtained in the context of glucose-induced endocytosis, in which Rod1 ubiquitylation was preceded by its dephosphorylation [28,[START_REF] Llopis-Torregrosa | Regulation of the Yeast Hxt6 Hexose Transporter by the Rod1 alpha-Arrestin, the Snf1 Protein Kinase, and the Bmh2 14-3-3 Protein[END_REF]. As expected, Rod1 was constitutively phosphorylated in the PP1 phosphatase mutant reg1∆ and this phosphorylation was no longer affected by 2DG treatment (Fig 2B). In contrast, mutations in the described Rod1 phosphatase, calcineurin (cnb1∆) [START_REF] Alvaro | Differential Phosphorylation Provides a Switch to Control How alpha-Arrestin Rod1 Down-regulates Mating Pheromone Response in Saccharomyces cerevisiae[END_REF][START_REF] Alvaro | Specific alpha-arrestins negatively regulate Saccharomyces cerevisiae pheromone response by down-modulating the G-protein-coupled receptor Ste2[END_REF] or in various signaling pathways whose activation is triggered by 2DG treatment [START_REF] Defenouillėre | The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose[END_REF] (MAPK= slt2∆, hog1∆; UPR: hac1∆) had no impact (Fig 2C).

This suggested that PP1 activity is required for the 2DG-induced dephosphorylation of Rod1. Moreover, deletion of SNF1 or mutation of 12 serine residues within potential Snf1 consensus sequences [START_REF] Alvaro | Differential Phosphorylation Provides a Switch to Control How alpha-Arrestin Rod1 Down-regulates Mating Pheromone Response in Saccharomyces cerevisiae[END_REF] led to a profile comparable to that obtained in 2DG-treated cells, suggesting that lack of Rod1 phosphorylation mimics

2DG-treatment (Fig 2D).

Having confirmed how Rod1-Flag is post-translationally modified in response to 2DG, we assessed the functional consequences abrogating these modifications on the function of Rod1 in endocytosis. Instead of focusing on Hxt1 or Hxt3, which are glucose-regulated [START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF], we used Ina1-GFP as a model cargo (see

Fig 1D).
Ina1 is a protein of the SUR7/PalI family, with carries 3 predicted transmembrane domains and localizes to the plasma membrane [START_REF] Germann | TopBP1/Dpb11 binds DNA anaphase bridges to prevent genome instability[END_REF]. Mutation of the ubiquitylation site(s) within Rod1 resulted in a slowdown of Ina1

2DG promotes the PP1-dependent dephosphorylation of Snf1 and several of its targets

The fact that 2DG triggers Rod1 dephosphorylation led us to think that this may occur through a transient activation of PP1. Instead, 2DG was previously described to induce a mild phosphorylation (activation) of the PP1 substrate Snf1 within 2h after treatment with 2DG [START_REF] Mccartney | Genetic analysis of resistance and sensitivity to 2deoxyglucose in Saccharomyces cerevisiae[END_REF], similar to observations in mammalian cells [START_REF] Laussel | Cellular toxicity of the metabolic inhibitor 2-deoxyglucose and associated resistance mechanisms[END_REF][START_REF] Vincent | Differential effects of AMPK agonists on cell growth and metabolism[END_REF].

To explain this discrepancy, we reexamined the effects of 2DG on Snf1 signaling. After 10 min 2DG treatment, we never observed Snf1 activation, even at higher 2DG concentration ( This was further confirmed by studying another Snf1 substrate, the transcriptional repressor Mig1 [START_REF] Smith | The SNF1 kinase complex from Saccharomyces cerevisiae phosphorylates the transcriptional repressor protein Mig1p in vitro at four sites within or near regulatory domain 1[END_REF][START_REF] Treitel | Snf1 protein kinase regulates phosphorylation of the Mig1 repressor in Saccharomyces cerevisiae[END_REF] which was also dephosphorylated after 2DG treatment in a PP1-dependent manner (Fig 3D). Again, this mimicked the situation described when glucose-starved cells are treated with glucose [START_REF] Shashkova | The yeast Mig1 transcriptional repressor is dephosphorylated by glucose-dependent and -independent mechanisms[END_REF]. Thus, 2DG triggers both an immediate and transitory PP1-dependent inhibition of Snf1, and the direct or indirect dephosphorylation of Snf1 substrates such as Mig1 or Rod1.

Snf1 inactivation by 2DG requires 2DG phosphorylation by Hxk2

We then investigated the origin of the signal triggering the PP1-dependent dephosphorylation of substrates. 2DG is taken up by hexose transporters and is then phosphorylated into 2DG6P [START_REF] Van Steveninck | Transport-associated phosphorylation of 2-deoxy-D-glucose in yeast[END_REF], which interferes with several metabolic pathways. Hxk2 is the main hexokinase isoform expressed in glucose-grown yeast cells [START_REF] Herrero | Transcriptional regulation of the Saccharomyces cerevisiae HXK1, HXK2 and GLK1 genes[END_REF] and we studied its contribution in 2DG phosphorylation by measuring 2DG6P appearance after 2DG treatment using an enzyme-based assay [START_REF] Saito | An enzymatic photometric assay for 2deoxyglucose uptake in insulin-responsive tissues and 3T3-L1 adipocytes[END_REF]. 2DG phosphorylation by hexokinase into the dead-end metabolite 2DG6P requires ATP and causes a strong ATP depletion [START_REF] Karczmar | Selective depletion of tumor ATP by 2-deoxyglucose and insulin, detected by 31P magnetic resonance spectroscopy[END_REF][START_REF] Ibanez | Mass spectrometry-based metabolomics of single yeast cells[END_REF]. This can be visualized within minutes after 2DG addition in WT cells using an optimized, pH-insensitive version of the AT1.03 ATP FRET biosensor [START_REF] Botman | An Improved ATP FRET Sensor For Yeast Shows Heterogeneity During Nutrient Transitions[END_REF] ( activation. ROD1 deletion leads to a partial 2DG resistance [START_REF] Ralser | A catabolic block does not sufficiently explain how 2-deoxy-D-glucose inhibits cell growth[END_REF], but the underlying mechanism remains unclear. We previously described that a frequent strategy leading to 2DG resistance involves an increased expression of the Dog2 phosphatase at the transcriptional level [START_REF] Defenouillėre | The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose[END_REF]. However, using a pDOG2:LacZ reporter, we found that this was not the case in the rod1∆ mutant, suggesting a distinct mechanism (Fig 5A).

The 2DG resistance of the rod1∆ strain is exacerbated upon the further deletion of its paralogue ROG3 [START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF], and other data indicate that 2DG resistance correlates with Rod1 activity in endocytosis [START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF] (see also Fig We postulated that 2DG resistance originates from the stabilization of one or several Rod1-regulated cargoes at the plasma membrane. We initially focused on the low affinity hexose transporters, Hxt1 and Hxt3, for the following reasons. First, the hypersensitivity of the snf1∆ mutant to 2DG is rescued by overexpression of Hxt1 and Hxt3 through an unknown mechanism [START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF]. Second, duplication of a chromosomal region containing hexose transporter genes (HXT3, HXT6 and HXT7) confers 2DG-resistance in a dominant manner [START_REF] Barney | Drug resistance in diploid yeast is acquired through dominant alleles, haploinsufficiency, gene duplication and aneuploidy[END_REF]. Since these transporters are not endocytosed in the rod1∆ mutant (Fig 1C andS2) [START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF], we thus considered that their stabilization could contribute to 2DG resistance. Indeed, deletion of either HXT3 or HXT1

abolished the 2DG resistance of the rod1∆ mutant strain (Fig 5C), showing that both transporters are required for this process. In contrast, deletion of the high-affinity hexose transporter gene HXT6 did not cause the same phenotype (Fig 5C). Although, it seems that only low-affinity hexose transporters promote 2DG resistance, this conclusion may be biased by the fact that low-and high-affinity hexose transporters are expressed to a lower level, the former contributing more to glucose uptake than the latter in glucose-rich conditions [START_REF] Ozcan | Function and regulation of yeast hexose transporters[END_REF].

The growth defect of the obtained rod1∆ hxt3∆ strain on 2DG was restored by expressing a GFP-tagged Structure/function studies of transporters [reviewed in 50] identified residues critical for hexose binding and translocation. In particular, residue N317 of the human glucose transporter GLUT1 binds glucose [START_REF] Mueckler | Model of the exofacial substrate-binding site and helical folding of the human Glut1 glucose transporter based on scanning mutagenesis[END_REF][START_REF] Deng | Crystal structure of the human glucose transporter GLUT1[END_REF] and mutation of the corresponding residue in yeast transporters alters substrate specificity and transport [START_REF] Farwick | Engineering of yeast hexose transporters to transport D-xylose without inhibition by D-glucose[END_REF][START_REF] Nijland | Engineering of an endogenous hexose transporter into a specific D-xylose transporter facilitates glucose-xylose co-consumption in Saccharomyces cerevisiae[END_REF][START_REF] Shin | An engineered cryptic Hxt11 sugar transporter facilitates glucose-xylose co-consumption in Saccharomyces cerevisiae[END_REF]. Particularly, mutation of this residue in Hxt1 (N370A) [START_REF] Roy | Assessing glucose uptake through the yeast hexose transporter 1 (Hxt1)[END_REF] and other yeast hexose transporters [START_REF] Farwick | Engineering of yeast hexose transporters to transport D-xylose without inhibition by D-glucose[END_REF][START_REF] Nijland | Efficient, D-glucose insensitive, growth on D-xylose by an evolutionary engineered Saccharomyces cerevisiae strain[END_REF] abolished glucose uptake. We mutated the corresponding residue in Hxt3 (N367A) and found that its overexpression no longer rescued growth on 2DG, further linking glucose transporter function with 2DG resistance (Fig 5D).

Stabilization of glucose transporters at the plasma membrane facilitates glucose uptake and 2DG export

The fact that glucose transport correlates with 2DG resistance was counterintuitive because 2DG enters into cells through glucose transporters [START_REF] Van Steveninck | Transport-associated phosphorylation of 2-deoxy-D-glucose in yeast[END_REF], thus their stabilization at the cell surface may increase 2DG uptake.

Indeed, deleting both HXT1 and HXT3 causes partial resistance to 2DG (Fig 6A), indicating that 2DG is mostly transported into cells through these transporters.

We considered two hypotheses to explain how glucose transporter accumulation at the plasma membrane results in 2DG resistance. First, by depleting glucose transporters from the plasma membrane, 2DG-induced endocytosis could lower glucose intake at a time where glycolysis is already impacted, resulting in energy shortage as previously proposed [START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF]. Preventing endocytosis may therefore attenuate the glycolysis blockade by maintaining glucose uptake. Alternatively, transporters present at the cell surface may instead favor 2DG detoxification and release into the medium, as previously observed [START_REF] Van Steveninck | Transport-associated phosphorylation of 2-deoxy-D-glucose in yeast[END_REF] since the directionality of transport (import or export) through hexose transporters is dictated by the concentration gradient.

We first tested whether rod1∆ cells could detoxify 2DG6P more efficiently than WT cells. Cells were treated for 2h with 2DG, and 2DG6P was generated at comparable levels in both strains (Fig 6B). Interestingly, the concentration of 2DG6P after 2h 2DG treatment was comparable to that observed after 15 min (Fig

6C),
showing that saturation is reached very early after treatment. Cells were then transferred to a 2DG-free medium for 30 min and 2DG6P was assayed again. In both strains, 2DG6P concentration decreased during these 30 min, revealing they could detoxify 2DG6P (Fig 6B). However, the 2DG6P content was much lower in rod1∆ than in WT cells, suggesting that 2DG6P is better detoxified in this strain. Therefore, the endocytosis blockade caused by the deletion of ROD1 allows a better detoxification of 2DG.

We also tested whether hexose transporters maintenance at the membrane impacts on glucose uptake, by using an intracellular glucose FRET sensor based on the bacterial glucose/galactose-binding transport protein MglB [START_REF] Bermejo | Dynamic analysis of cytosolic glucose and ATP levels in yeast using optical sensors[END_REF]. We assessed intracellular glucose following glucose exposure after a 4h-treatment with

Discussion

Several strategies are known to modulate 2DG sensitivity/resistance in yeast [START_REF] Laussel | Cellular toxicity of the metabolic inhibitor 2-deoxyglucose and associated resistance mechanisms[END_REF]. The most common mechanism is through the aberrant activation of the AMPK orthologue, Snf1, either by gain-of-function mutation in constituents of the AMPK heterotrimeric complex, or by loss-of-function mutations of its inhibitory phosphatase (PP1: Glc7/Reg1) [START_REF] Laussel | Cellular toxicity of the metabolic inhibitor 2-deoxyglucose and associated resistance mechanisms[END_REF][START_REF] Soncini | Spontaneous mutations that confer resistance to 2-deoxyglucose act through Hxk2 and Snf1 pathways to regulate gene expression and HXT endocytosis[END_REF]. How AMPK hyperactivation contributes to 2DG resistance is not fully understood, but we previously found that this partially occurs through the expression of the detoxification enzyme Dog2 [START_REF] Defenouillėre | The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose[END_REF]. Here, we provide important insights into the cellular effects that 2DG has on AMPK signaling, how this impacts Rod1 activity and subsequently, on endocytosis, and how endocytosis interference leads to 2DG resistance through the control of glucose transport activity.

We first report that 2DG acts as a strong endocytosis signal, leading to the endocytosis of many plasma membrane proteins. These include a GPCR (Ste2), various classes of transporters, such as permeases, solute carriers such as multidrug transporters of the major facilitator superfamily (MFS) or ATP-binding cassette (ABC)

types. This massive 2DG-induced endocytosis of membrane proteins is reminiscent of that observed in other situations of metabolic stresses such as acute glucose or nitrogen starvation [START_REF] Lang | Glucose starvation inhibits autophagy via vacuolar hydrolysis and induces plasma membrane internalization by down-regulating recycling[END_REF][START_REF] Müller | The coordinated action of the MVB pathway and autophagy ensures cell survival during starvation[END_REF][START_REF] Ivashov | Complementary alpha-arrestin-ubiquitin ligase complexes control nutrient transporter endocytosis in response to amino acids[END_REF], or upon treatment with other drugs, stressors or heat shock [START_REF] Babst | Regulation of nutrient transporters by metabolic and environmental stresses[END_REF][START_REF] Volland | Endocytosis and degradation of the yeast uracil permease under adverse conditions[END_REF][START_REF] Macgurn | TORC1 regulates endocytosis via Npr1-mediated phosphoinhibition of a ubiquitin ligase adaptor[END_REF][START_REF] Nikko | Arrestin-mediated endocytosis of yeast plasma membrane transporters[END_REF][START_REF] Megarioti | The Bul1/2 Alpha-Arrestins Promote Ubiquitylation and Endocytosis of the Can1 Permease upon Cycloheximide-Induced TORC1-Hyperactivation[END_REF][START_REF] Crapeau | Stress Conditions Promote Yeast Gap1 Permease Ubiquitylation and Down-regulation via the Arrestin-like Bul and Aly Proteins[END_REF][START_REF] Zhao | The ART-Rsp5 ubiquitin ligase network comprises a plasma membrane quality control system that protects yeast cells from proteotoxic stress[END_REF]. Because 2DG is an artificial molecule, the observed endocytosis is unlikely to originate from an adaptation strategy but rather informs us that 2DG-induced metabolic blockade triggers endocytosis through mechanisms that we further explored.

First, our work highlights the major role played by the arrestin Rod1 in 2DG-induced endocytosis. Rod1

participates in the endocytosis of the glucose transporters Hxt1 and Hxt3 upon 2DG treatment [START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF], and we extend these findings to many other membrane proteins. Using a tagged but fully functional protein, we confirm previous findings [START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF] that 2DG treatment strongly affects Rod1 post-translational modifications (phosphorylation and ubiquitylation), reminiscent of past studies focusing on the regulation of Rod1 by glucose availability [28,[START_REF] Llopis-Torregrosa | Regulation of the Yeast Hxt6 Hexose Transporter by the Rod1 alpha-Arrestin, the Snf1 Protein Kinase, and the Bmh2 14-3-3 Protein[END_REF][START_REF] Shinoda | Rod1, an arrestin-related protein, is phosphorylated by Snf1-kinase in Saccharomyces cerevisiae[END_REF][START_REF] Alvaro | Differential Phosphorylation Provides a Switch to Control How alpha-Arrestin Rod1 Down-regulates Mating Pheromone Response in Saccharomyces cerevisiae[END_REF]. Phosphorylation is a frequent mechanism for arrestin inhibition [28,[START_REF] Macgurn | TORC1 regulates endocytosis via Npr1-mediated phosphoinhibition of a ubiquitin ligase adaptor[END_REF][START_REF] Merhi | Internal amino acids promote Gap1 permease ubiquitylation via TORC1/Npr1/14-3-3-dependent control of the Bul arrestin-like adaptors[END_REF][START_REF] Lee | Methionine triggers Ppz-mediated dephosphorylation of Art1 to promote cargo-specific endocytosis[END_REF],

perhaps by introducing negative charges that may hinder transporter interactions [START_REF] Ivashov | Complementary alpha-arrestin-ubiquitin ligase complexes control nutrient transporter endocytosis in response to amino acids[END_REF] [reviewed in 27].

Accordingly, Rod1 dephosphorylation and its subsequent ubiquitylation are required for its activity in endocytosis [28,[START_REF] Llopis-Torregrosa | Regulation of the Yeast Hxt6 Hexose Transporter by the Rod1 alpha-Arrestin, the Snf1 Protein Kinase, and the Bmh2 14-3-3 Protein[END_REF]. Arrestin ubiquitylation is often required for their function [28,[START_REF] Merhi | Internal amino acids promote Gap1 permease ubiquitylation via TORC1/Npr1/14-3-3-dependent control of the Bul arrestin-like adaptors[END_REF][START_REF] Lin | Arrestin-related ubiquitin-ligase adaptors regulate endocytosis and protein turnover at the cell surface[END_REF], and this may favor interaction with Rsp5 and thus the Rsp5-mediated ubiquitylation of transporters [START_REF] Macdonald | A Cycle of Ubiquitination Regulates Adaptor Function of the Nedd4-Family Ubiquitin Ligase Rsp5[END_REF]. Overall, we propose that 2DG induces an aberrant activation of Rod1 which explains the increased endocytosis observed in response to 2DG.

Second, we gained insights into how 2DG triggers these changes on Rod1, given that the precise effect of 2DG on Rod1 regulatory kinase(s) or phosphatase(s) was unclear until now. 2DG is well known to trigger AMPK activation in mammalian cells even at short timepoints [START_REF] Laussel | Cellular toxicity of the metabolic inhibitor 2-deoxyglucose and associated resistance mechanisms[END_REF][START_REF] Vincent | Differential effects of AMPK agonists on cell growth and metabolism[END_REF][START_REF] Wu | AMPK-dependent degradation of TXNIP upon energy stress leads to enhanced glucose uptake via GLUT1[END_REF], and a similar but milder effect was reported for Snf1 in yeast after 2h treatment [START_REF] Mccartney | Genetic analysis of resistance and sensitivity to 2deoxyglucose in Saccharomyces cerevisiae[END_REF]. Yet, this apparent activation of Snf1 did not translate into an increased phosphorylation of its substrate Mig1 [START_REF] Mccartney | Genetic analysis of resistance and sensitivity to 2deoxyglucose in Saccharomyces cerevisiae[END_REF], and the reported dephosphorylation of Rod1 in these conditions was also not in line with an increased Snf1 activity. Rather, by focusing on shorter timepoints, we observed a sharp but transient decrease in Snf1 phosphorylation shortly after 2DG addition. Thus, 2DG treatment rapidly inhibits yeast Snf1 activity, contrary to observations on mammalian AMPK.

This inhibition likely occurred through an increased activity of its cognate phosphatase PP1 (Reg1/Glc7) [START_REF] Rubenstein | Access denied: Snf1 activation loop phosphorylation is controlled by availability of the phosphorylated threonine 210 to the PP1 phosphatase[END_REF][START_REF] Tu | The GLC7 type 1 protein phosphatase is required for glucose repression in Saccharomyces cerevisiae[END_REF] (or increased accessibility of Snf1 to PP1). Accordingly, phosphorylation of the Snf1/PP1 target Mig1 also decreased in response to 2DG within a few minutes. Since Rod1 requires PP1 for dephosphorylation in response to glucose [28,[START_REF] Llopis-Torregrosa | Regulation of the Yeast Hxt6 Hexose Transporter by the Rod1 alpha-Arrestin, the Snf1 Protein Kinase, and the Bmh2 14-3-3 Protein[END_REF] or 2DG [this study and 32], a sudden increase in PP1 activity in response to 2DG would also account for the changes in Rod1 post-translational modifications. The increased activity of PP1 in response to 2DG is reminiscent to that observed when glucose is added to cells deprived of glucose [START_REF] Castermans | Glucose-induced posttranslational activation of protein phosphatases PP2A and PP1 in yeast[END_REF]. Thus, shortly after treatment and even in glucose-grown cells, 2DG treatment mimics glucose-replete conditions rather than starvation, and part of the early 2DG response involves a boost in PP1 activity.

The molecular mechanism by which PP1 is activated in these conditions is not yet clear. Despite a basal level of phosphorylation of Snf1 and Mig1 in glucose-grown cells, PP1 activity is already high in these conditions [START_REF] Rubenstein | Access denied: Snf1 activation loop phosphorylation is controlled by availability of the phosphorylated threonine 210 to the PP1 phosphatase[END_REF][START_REF] Tu | The GLC7 type 1 protein phosphatase is required for glucose repression in Saccharomyces cerevisiae[END_REF], so how can 2DG further increase this activity? This apparent activation of PP1 by 2DG involves Hxk2, as 2DG was no longer able to trigger Snf1/Rod1 dephosphorylation or Ina1 endocytosis in the hxk2∆ strain.

Hxk2 may either have a unique function in 2DG toxicity beyond its enzymatic function, but we favor the hypothesis that Hxk2 mediates 2DG-induced signaling through its unique ability to generate 2DG6P. Despite the fact that 2DG6P can be generated by other sugar-phosphorylating enzymes (Hxk1 and glucokinase Glk1) in vitro [START_REF] Soncini | Spontaneous mutations that confer resistance to 2-deoxyglucose act through Hxk2 and Snf1 pathways to regulate gene expression and HXT endocytosis[END_REF], only HXK2 deletion causes 2DG resistance [START_REF] Ralser | A catabolic block does not sufficiently explain how 2-deoxy-D-glucose inhibits cell growth[END_REF][START_REF] Mccartney | Genetic analysis of resistance and sensitivity to 2deoxyglucose in Saccharomyces cerevisiae[END_REF][START_REF] Defenouillėre | The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose[END_REF][START_REF] Soncini | Spontaneous mutations that confer resistance to 2-deoxyglucose act through Hxk2 and Snf1 pathways to regulate gene expression and HXT endocytosis[END_REF]. Moreover, our in vivo data suggest that Hxk2 is the most prominent 2DG-phosphorylating enzyme, since hxk2∆ cells have a lower 2DG6P content and 2DG has a lower impact on the ATP status in these cells. The lack of effect of Hxk1 or Glk1 on 2DG toxicity could be explained by a lower expression level or other in vivo regulatory mechanisms that would prevent an efficient 2DG phosphorylation. Interestingly, both HXK2 deletion and overexpression of the 2DG-6-P phosphatase Dog2, which converts 2DG-6-P back into 2DG, protect snf1∆ against 2DG hypersensitivity [START_REF] Mccartney | Genetic analysis of resistance and sensitivity to 2deoxyglucose in Saccharomyces cerevisiae[END_REF][START_REF] Defenouillėre | The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose[END_REF], so both may act through a decreased 2DG6P content. More generally, 2DG has virtually no effect in strains overexpressing Dog2, further suggesting that 2DG6P (or a derived metabolite) contributes to the potential surge of PP1 activity. Further work should be aimed at understanding how the PP1/Snf1 balance is controlled by 2DG at the molecular level, which may help decipher the long-sought mechanism by which glucose-starved cells react to glucose addition [START_REF] Rubenstein | Access denied: Snf1 activation loop phosphorylation is controlled by availability of the phosphorylated threonine 210 to the PP1 phosphatase[END_REF][START_REF] Castermans | Glucose-induced posttranslational activation of protein phosphatases PP2A and PP1 in yeast[END_REF][START_REF] Randez-Gil | The expression of a specific 2-deoxyglucose-6P phosphatase prevents catabolite repression mediated by 2-deoxyglucose in yeast[END_REF][START_REF] Momcilovic | Alterations at dispersed sites cause phosphorylation and activation of SNF1 protein kinase during growth on high glucose[END_REF].

After delving into the mechanisms by which 2DG triggers endocytosis through the modulation of glucose signaling pathways and Rod1 activity, we also obtained insights into how ROD1 deletion results in 2DG

resistance. The study of Rod1 point mutants indicate a correlation between endocytosis and 2DG resistance [this work, and 32] and Hxt1 and Hxt3 stabilization at the plasma membrane was necessary for 2DG resistance, in line with the fact that their overexpression allows 2DG resistance [START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF]. Although Hxt1 and Hxt3 are the main transporters involved in 2DG uptake, their maintenance at the plasma membrane is required for long-term resistance through two possible pathways that possibly synergize (Fig 7). The first pathway involves 2DG detoxification. Indeed, 2DG triggers the induction of the Dog1 and Dog2 phosphatases which detoxicate 2DG6P

into 2DG [START_REF] Defenouillėre | The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose[END_REF], the latter of which could presumably be exported once its cytosolic concentration is higher than that of the medium and provided that hexose transporters be present at the plasma membrane. This is in line with observations showing that (i) transport across the membrane is required to confer resistance, (ii) the same transport systems are used both for 2DG and glucose [START_REF] Van Steveninck | Transport-associated phosphorylation of 2-deoxy-D-glucose in yeast[END_REF], and (iii In mammalian cells, 2DG causes a fast energy depletion and AMPK activation [START_REF] Vincent | Differential effects of AMPK agonists on cell growth and metabolism[END_REF]. This is exemplified by the opposite effect that 2DG has on the endocytosis of glucose transporters. In hepatocytes, the 2DG-induced activation of AMPK triggers the phosphorylation of the arrestin-like protein TXNIP, resulting in its degradation and stabilization of the glucose transporter GLUT1 [START_REF] Wu | AMPK-dependent degradation of TXNIP upon energy stress leads to enhanced glucose uptake via GLUT1[END_REF], a response which is consistent with energy depletion and starvation response. In contrast, 2DG inhibits yeast Snf1, thus promoting Rod1-mediated glucose transporter endocytosis [this work and 32]. This somewhat paradoxical response likely reveals that in yeast, 2DG causes energy deprivation but is also likely sensed as excess glucose (causing a decreased Snf1/AMPK activity). The differences in 2DG signaling between these two model systems may originate from evolutionary differences regarding their ability to detect glucose as a signaling molecule, in addition to being a source of energy. Glucose is the preferred carbon source for Saccharomyces cerevisiae and is mostly used by fermentation, implying a high glycolytic flux to sustain proliferation. Yeast has evolved various sensing systems at the plasma membrane to detect extracellular glucose, that are wired into various signaling pathways [START_REF] Santangelo | Glucose signaling in Saccharomyces cerevisiae[END_REF].

)
It is likely that Hxk2 and/or glucose phosphorylation itself acts as a glucose sensor within the Snf1/AMPK pathway [START_REF] Vega | Hexokinase 2 Is an Intracellular Glucose Sensor of Yeast Cells That Maintains the Structure and Activity of Mig1 Protein Repressor Complex[END_REF][START_REF] Moreno | The hexokinase 2-dependent glucose signal transduction pathway of Saccharomyces cerevisiae[END_REF][START_REF] Ma | The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae[END_REF], and 2DG phosphorylation by Hxk2 may produce a glucose-replete signal, leading to Snf1 inactivation despite changes in the energy load. This is probably only true shortly after 2DG treatment, before 2DG inhibits other aspects of cellular physiology (such as protein glycosylation or structural sugar metabolism) leading to a complex response through stress-signaling pathway (such as UPR or MAPK-based signaling) [START_REF] Laussel | Cellular toxicity of the metabolic inhibitor 2-deoxyglucose and associated resistance mechanisms[END_REF][START_REF] Defenouillėre | The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose[END_REF]. In conclusion, the characterization of 2DG resistance strategies led once more to a better understanding of the cellular effects of 2DG and the diversity of mechanisms that can be deployed to bypass 2DG-induced metabolic inhibition.

Materials and Methods

Yeast strain construction and growth conditions

All yeast strains used in this study derive from the Saccharomyces cerevisiae BY4741 or BY4742 background and are listed in table S1. Apart from the mutant strains obtained from the yeast deletion collection (Euroscarf)

and the fluorescent GFP-tagged strains originating from the yeast GFP clone collection {Huh, 2003 #123}, all yeast strains were constructed by transformation with the standard lithium acetate-polyethylene glycol protocol using homologous recombination and verified by polymerase chain reaction (PCR) on genomic DNA prepared with a lithium acetate (200 mM)/SDS (0.1%) method (104). Yeast cells were grown in YPD medium (2%) or in SC medium [containing yeast nitrogen base (1.7 g/liter; MP Biomedicals), ammonium sulfate (5 g/liter; Sigma-Aldrich), the appropriate drop-out amino acid preparations (MP Biomedicals), and 2% (w/v) glucose, unless otherwise indicated]. Precultures were incubated at 30°C for 8 hours and diluted in the evening to 20-ml cultures to reach mid-log phase the next morning. 2DG (Sigma) was added to mid-log phase yeast cultures grown overnight to final concentrations of 0.2% (w/v) and incubated for the indicated times.

Plasmid construction

All plasmids used in this study are listed in table S2.

Plasmid pSL553 (pRS313, pROD1:ROD1-NcoI-Flag) was generated by site-directed mutagenesis on pSL234 (pROD1:ROD1-3HA) [28], pSL119 (pROD1:ROD1-PYm-3HA) [28], pSL152 (pROD1:ROD1-S12A-3HA) (unpublished, below), and pSL147 (pROD1:ROD1-KR-3HA) [28] respectively. They were digested with SalI/NcoI and cloned at these sites into pSL553 (pROD1:ROD1-NcoI-Flag). Plasmid pSL152 (pRS415, pROD1-ROD1(S12A)-3HA) was made from a synthetic gene designed to mutate S125, S138, S315, S358, S447, S623, S641, S706, S720, S734, S781, and S821 residues into A (Eurofins), digested PacI/XmaI and cloned PacI/XmaI into pSL94 at the place of WT ROD1 (pRS415, pROD1:ROD1-3HA) [28].

To construct pSL589 (pUG35-based, pHXT3-Hxt3-GFP), the HXT3 promoter (1kb) was first cloned into pUG35 after amplification by PCR on WT gDNA (oSL1739/oSL1740) at BamHI/SpeI sites to give pSL592. The HXT3-GFP ORF was PCR-amplified (oSL1770/oSL1771) from gDNA of the Hxt3-GFP strain (ySL1027) using primers overlapping with the pSL592 sequence and cloned into pSL592 by gap-repair, and later rescued in bacteria to obtain pSL589. The N367A mutation was introduced using 2 overlapping PCR products amplified from gDNA of the HXT3-GFP strain (ySL1027) (oSL1730/oSL682 and oSL1731/oSL1563) and cloned by gap-repair into pSL592 to give pSL591. Plasmid pSL599 (pRS426, pHXT1:HXT1) was obtained by (1) amplifying the HXT1 gene (-515/+225) from WT genomic DNA (oSL435/oSL932) into a pCRII-blunt topo vector (pSL597), (2) cloning a BamHI/XhoI fragment of pSL597 into pRS425 at BamHI/XhoI sites. For plasmid pSL602 (pRS426, pHXT1:HXT1), A PCR amplifying the HXT3 gene (and 1kb of promoter) was obtained from WT genomic DNA (oSL1739/oSL1734), digested BamhI/SpeI and cloned BamHI/SpeI in pRS426.

All constructs were verified by sequencing.

Total protein extracts and immunoblotting

Yeast were always grown in SC medium. For each protein sample, 1.4 ml of culture was incubated with 140µl of 100% TCA for 10 min on ice to precipitate proteins, centrifuged at 16,000g at 4°C for 10 min, and broken for 10 min with glass beads. Lysates were transferred to another 1.5-ml tube to remove glass beads and centrifuged for 5 min at 16,000g at 4°C, supernatants were discarded, and protein pellets were resuspended in sample buffer [50 mM tris-HCl (pH 6.8), 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, and 10% glycerol, complemented with 50 mM tris-base (pH 8.8)] (50µl/initial OD). Protein samples were heated at 37°C for 5 min and 10 µl was loaded on SDS-polyacrylamide gel electrophoresis (PAGE) gels (4-20% Mini-PROTEAN TGX Stain-Free, Bio-Rad). After electrophoresis for 30 min at 200V, total proteins were visualized by in-gel fluorescence using a trihalo compound incorporated in SDS-PAGE gels (stain-free TGX gels, 4-20%; Bio-Rad) after 45 sec UV-induced photoactivation using a ChemiDoc MP imager (BioRad), serving as a loading control.

Gels were transferred on nitrocellulose membranes for 60 min (100V) in a liquid transfer system (Bio-Rad).

Membranes were blocked in Tris-buffered saline solution containing 0.5% Tween-20 (TBS-T) and 2% milk for 30 min and incubated for at least 2 hours with the corresponding primary antibodies. Membranes were washed 3x10 min in TBS-T and incubated for at least an hour with the corresponding secondary antibody (coupled with horseradish peroxidase). Membranes were then washed again 3x10 min in TBS-T and incubated with SuperSignal™ West Femto reagent (Thermo). Luminescence signals were acquired using a ChemiDoc MP (BioRad). Primary and secondary antibodies used in this study as well as their dilutions are listed in table S3.

Treatment of protein extracts with calf intestinal phosphatase (CIP)

Calf Intestine Phosphatase (CIP, Roche ref 11097075001) was used to dephosphorylate proteins prior to electrophoresis. Samples were prepared using 20 μL of TCA protein extract (see "Protein extracts and immunoblotting", above), to which 2 μL of 10X CIP buffer supplied with the enzyme, as well as 0.6 μL of 1.5M

Tris pH 8.8, 0.2 μL 0.1M MgSO4, and 17.2 μL of H2O were added. The extract was split in 2 and 0.6 U CIP was added to one of the samples. Samples were incubated at 37°C for 3 h, and loaded on SDS-PAGE.

Purification of Ina1-GFP for ubiquitylation analysis

Strains expressing Ina1-GFP were transformed with an empty plasmid or a plasmid containing 6xHis-tagged ubiquitin under the control of the promoter of the copper-inducible CUP1 gene (pSL205). Ubiquitylation analysis was performed as described before [START_REF] Hovsepian | The yeast arrestin-related protein Bul1 is a novel actor of glucose-induced endocytosis[END_REF]. Briefly, cells were grown to the exponential phase before induction with 100µM CuS04, with or without 2DG 0.2%, for 1h at 30°C. Around 20 OD of cells were centrifuged at 4,000 g for 5 minutes, the pellets were washed with cold water and resuspended with 200µl of RIPA buffer (50 mM TRIS /HCl pH 7.5. 150 mM NaCl. 0,1% SDS, 2 mM EDTA, 50 mM NaF, 1% NP-40, 0,5% Na-Deoxycholate, 1% Glycerol) supplemented with protease inhibitor (Complete EDTA-free, Roche), 1mM PMSF (Sigma), 10mM NEM (Sigma) and 0.1mM MG-132 (Enzo Life Sciences) and broken with glass beads for 8 min at 4°C. Lysates were then rotated at 4°C for 15 minutes and centrifuged at 10,000g for 10 min at 4°C. 10µl of GFP-Trap agarosemagnetic beads (Chromotek) were added to the lysates and rotated overnight at 4°C. After 2 washes with 1.5 mL of wash buffer (50 mM TRIS /HCl pH 7.5, 150 mM NaCl, 1% NP-40, 5% Glycerol) at 4°C and 1 wash with 1.5mL TBST 0.1% at RT, the beads were resuspended with 25µl of boiling buffer (50 mM TRIS, pH =7,5, 1 mM EDTA, 1% SDS, 20% Glycerol) and incubated at 37°C for 30 minutes. 25µl of Urea SDS buffer (150mM TRIS, pH =6.8, 6M Urea, 6% SDS, bromophenol blue) were added and the proteins were incubated for another 30 minutes at 37°C before loading on a 4-12% SDS-PAGE (Mini-PROTEAN TGX Stain-Free, Bio-Rad).

Beta-galactosidase assays

Beta-galactosidase assays were performed using 1 ml of mid-log phase yeast cultures carrying the pDOG1-LacZ (pSL409) or pDOG2-LacZ plasmids (pSL410) [START_REF] Defenouillėre | The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose[END_REF], grown overnight to the mid-log phase in SC medium without uracil with 2% glucose and switched to the specified conditions. The OD(600 nm) of the culture was measured, and samples were taken and centrifuged at 16,000g at 4°C for 10 min. Cell pellets were snap-frozen in liquid nitrogen and resuspended in 800 µl of buffer Z (pH 7, 50 mM NaH2PO4, 45 mM Na2HPO4, 10 mM MgSO4, 10 mM KCl, and 38 mM beta-mercaptoethanol). After addition of 160µl of ONPG (ortho-nitrophenyl-dgalactopyranoside, 4 mg/ml; Sigma-Aldrich), samples were incubated at 37°C. Enzymatic reactions were stopped in the linear phase (60-min incubation for pDOG2-LacZ and 120-min incubation for the pDOG1-LacZ plasmid, as per initial tests) [START_REF] Defenouillėre | The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose[END_REF] by addition of 400 µl of Na2CO3, and cell debris were discarded by centrifugation at 16,000g. The absorbance of clarified samples was measured with a spectrophotometer set at 420 nm.

Galactosidase activities (arbitrary units) were calculated using the formula 1000*[A420/(A600*t)], where A420 refers to the measured enzyme activity, A600 is the turbidity of the culture, and t is the incubation time. Each enzymatic assay was repeated independently at least three times.

2DG-6-phosphate assays

Cells were grown overnight in SC media until mid-log phase. 2DG was added into the culture to a final concentration of 0.2% for the indicated time. For the chase experiments, the cultures were centrifuged after 2 hours, washed with water and re-suspended in the same volume of SC medium without 2DG. For each time points, 1 OD equivalent of cells were taken, immediately placed on ice, centrifuged at 4°C for 4' at 16,000g, washed once with cold PBS and lysed using Y-PER™ Yeast Protein Extraction Reagent (ThermoFisher Scientific) as previously described [START_REF] Jayakody | SUMO expression shortens the lag phase of Saccharomyces cerevisiae yeast growth caused by complex interactive effects of major mixed fermentation inhibitors found in hot-compressed water-treated lignocellulosic hydrolysate[END_REF]. Briefly, 1 OD equivalent of cells was resuspended in 100µL of Y-PER buffer, incubated for 20' at 30°C, and centrifuged for 4' at 16100g at room temperature. The supernatant (between 0.0125 and 0.0625 OD equivalents) was used for the assay and diluted in Y-PER when needed. The assay was performed using the 2DG uptake measurement kit (Cosmo Bio USA, Carlsbad, CA, USA;, Ref. CSR-OKP-PMG-K01H) following the manufacturer's protocol adapted for a total reaction volume of 100 µL. Absorbance at 420 nm was measured every minute for 1 hour on a SpectraMax® M2 Microplate Reader (Molecular Devices).

Measures taken between 20 and 35 minutes (linear range) were taken into account to compare the slopes.

Each sample was assayed at least twice in each experiment and at least three independent experiments were performed. Mean values were calculated and were plotted with error bars representing SEM. Statistical significance was determined using a t-test for paired variables assuming a normal distribution of the values, as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; ns, P > 0.05 using GraphPad Prism7.

Glucose FRET sensor experiments

Yeast transformed with the plasmid FLII12Pglu-700μδ6 (pSL590, [a gift from Wolf Frommer: Addgene #28002, 58] were grown overnight in SC-URA medium to reach an OD600nm of 0.3-0.7 in the morning. Cells were centrifuged at 3000g for 5 minutes and the cell pellets were resuspended in 20mM MES pH=6 to a final OD of 0.6. 180µl of the cell suspension were distributed in 12 wells of a 96-well black PS microplate with flat bottom (Greiner). Fluorescence was measured with a plate reader (Spark, TECAN) set at 30°C before the experiment was started.

A first measurement for 3 cycles was performed to establish a baseline (each cycle is around 90 seconds with 10 seconds shaking before each measurement of the fluorescence). Cells were excited at CFP wavelength excitation (428 nm) and the emission intensities at 485 nm (CFP) and 528 nm (Citrine) were collected. The cycle was then paused to inject 20µl of the glucose solutions using a multichannel pipette, and the plate rapidly reloaded in the reader to restart measurements for another 17 cycles. Data were collected and analyzed in an Excel spreadsheet and in Prism 7.0 (GraphPad). Emission intensity values of yeast transformed with an empty plasmid were subtracted from emissions values at 485 nm and 528 nm for each measurement at the different glucose concentrations. The emission intensity ratio for Citrine (528 nm) over CFP (485 nm) was calculated. All analyses were repeated (with three technical replicates) at least three times independently. Emission ratios were normalized to the average of the three initial ratio values before glucose addition.

ATP FRET sensor experiments

Yeast transformed with the plasmid pDRF1-GW yAT1.03 (pSL608) [a gift from Bas Teusink: Addgene #132781, 48] were grown overnight in SC-URA medium to reach an OD600nm of 0.3-0.7 in the morning. FRET measurements were performed as above except cells were excited at 438 nm and the emission intensities at 483 nm (ymTq2∆11) and 593nm (tdTomato) were collected. A plasmid encoding a mutated sensor which does not bind ATP (pSL609) [a gift from Bas Teusink: Addgene #132782, 48] was used as a negative control.

Drop tests

Yeast cells grown in liquid-rich or SC medium for at least 6 hours at 30°C were adjusted to an optical density (600 nm) of 1. Serial 10-fold dilutions were prepared in 96-well plates and spotted on plates containing rich or SC medium containing 2% (w/v) agar and, when indicated, 2DG [0.05 or 0.2% (w/v)]. Plates were incubated at 30°C for 3 to 4 days before scanning on a desktop scanner (Epson).

Microscopy

Cells were mounted in synthetic complete medium with the appropriate carbon source and observed with a motorized Olympus BX-61 fluorescence microscope equipped with an Olympus PlanApo 100× oil-immersion objective (1.40 NA), a Spot 4.05 charge-coupled device camera and the MetaVue acquisition software (Molecular Devices; Sunnyvale, CA) and imaged at room temperature. GFP-tagged proteins were visualized using a Chroma GFP II filter (excitation 440-470 nm). mCherry-tagged proteins were visualized using an HcRed I filter (excitation 525-575 nm). Images were processed in ImageJ (NIH). Vacuolar staining was obtained by incubating cells grown in the desired condition with 100 µM CMAC (Life Technologies) for 10 min under agitation at 30°C. Cells are then washed twice with water before observations with a fluorescence microscope equipped with a DAPI filter.

Quantification of vacuolar fragmentation

Quantification of vacuolar fragmentation (Fig. S1B) was performed with ImageJ. For each experiment and each strain, at least 30 cells were observed and the quantification was done blindly. Cells with more than 3 vacuoles were considered as cells with highly fragmented vacuoles.

Quantification of Western-blots

Western blot quantifications were performed with ImageJ. The integrated density of proteins of interest was divided by the integrated density of total proteins. Results were normalized to the Glc ON condition of each

Statistical analysis

Mean values were calculated using a minimum of three independent measurements from three biological replicates and were plotted with error bars representing SEM. Statistical significance was determined using a t-test for paired variables assuming a normal distribution of the values, as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; ns, P > 0.05 using GraphPad Prism7.

Table S1: Yeast strains used in this study 883

Name

Genotype & Description Origin & ySL2221: Can1-GFP MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 CAN1::GFP-HIS3MX [88] ySL2222: Lyp1-GFP MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 LYP1::GFP-HIS3MX [88] ySL2223: Fui1-GFP MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 FUI1::GFP-HIS3MX [88] ySL2224: Itr1-GFP MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 ITR1::GFP-HIS3MX [88] ySL2237: Pdr12-GFP snf1Δ

MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 PDR12::GFP-HIS3MX snf1∆::NATNT2 Bas Teusink [START_REF] Botman | An Improved ATP FRET Sensor For Yeast Shows Heterogeneity During Nutrient Transitions[END_REF] pSL609 pDRF1-GW-derived (2µ, URA3), yAT1.03 ATP FRET sensor, mutated R122K,R126K (non-ATP binding), Addgene #132782

Bas Teusink [START_REF] Botman | An Improved ATP FRET Sensor For Yeast Shows Heterogeneity During Nutrient Transitions[END_REF] Table S3: Antibodies used this study Fluorescence at 485 and 528 nm was measured every 90 sec in a plate reader before and after glucose addition Laussel et al.

  of glucose-grown cells with 0.2% 2DG led to the degradation of PM-localized Tat1 and Hxt2 (Fig 1A), which was accompanied by their targeting to the vacuole (Fig 1B). Of note, we observed that the vacuole was strongly fragmented in response to 2DG (Fig S1), confirming recent findings

2DG-induced

  endocytosis (Fig 1D, Fig S2). Although the Rod1 paralogue Rog3 displays some level of functional redundancy with Rod1 [32], Rog3 was not responsible for the endocytosis of the Rod1-independent cargo Lyp1 (Fig S3), although this may be the case for other cargoes. Altogether, these results indicate a prominent role for the ART protein Rod1 in 2DG-induced endocytosis.

  Fig 3A). On the contrary, 2DG consistently decreased Snf1 phosphorylation within minutes after treatment (Fig 3B-C). A kinetic analysis of phospho-Snf1 response to 2DG revealed a drop in Snf1 phosphorylation that is followed by an increase at later time points (Fig 3B-C, 60' and 120'). Snf1 dephosphorylation by 2DG required the PP1 subunit Reg1, thus mimicking the effect that glucose has on glucose-starved cells. Altogether, we conclude that 2DG triggers a PP1-dependent dephosphorylation of Snf1, consistent with our (Fig 2) and others' [32] observations on Rod1.

  After 15 min exposure to 0.2% 2DG, 2DG6P was readily detectable in WT cells (Fig 4A). In hxk2∆, 2DG6P only reached 25% of the WT value (Fig 4A), suggesting that Hxk2 is the main 2DG-phosphorylating enzyme.

Fig

  4B). The decrease in FRET signal occurred only upon 2DG treatment and revealed changes in ATP content as demonstrated using a mutant version of the FRET sensor which does not bind ATP[START_REF] Botman | An Improved ATP FRET Sensor For Yeast Shows Heterogeneity During Nutrient Transitions[END_REF] (Fig4B). We found that comparatively to the WT, the drop in FRET was lower in hxk2∆ cells, again suggesting that Hxk2 is key for 2DG phosphorylation (Fig4C).In line with these findings, we found that neither Snf1 nor Rod1 were dephosphorylated in response to 2DG in the hxk2∆ mutant (Fig 4D). Similarly, 2DG no longer triggered Ina1-GFP endocytosis in these cells (Fig 4E). Overall, our data suggest that either Hxk2 itself or 2DG phosphorylation is required for the 2DG-mediated activation of PP1. Noteworthy, overexpression of the 2DG6P phosphatase Dog2, but not that of its catalyticdead mutant (Dog2-DDAA), also abolished Ina1-GFP endocytosis (Fig 4F), suggesting that 2DG-6-P levels directly or indirectly control PP1 activity. Altogether, we propose that 2DG phosphorylation by Hxk2 triggers a transient activation of PP1, leading to Snf1 dephosphorylation, Rod1 dephosphorylation and endocytosis. Stabilization of glucose transporters at the plasma membrane confers 2DG resistance to the rod1∆ mutant So far, our data provide mechanistic insights as to how 2DG triggers endocytosis through PP1 and Rod1

2F) .

 . Since Rod1 has a central role in plasma membrane protein remodeling by 2DG (Fig 1D and Fig S2), we first hypothesized that the 2DG-mediated activation of Rod1 might trigger excessive endocytosis that would be deleterious for the cell, and could be counteracted by ROD1 deletion. Importantly, deletion of the gene encoding the amphiphysin orthologue RVS167, involved in endocytosis, did not result in 2DG resistance (Fig 5B). Thus, it seems that only a partial endocytosis defect such as that observed in the rod1∆ (or rod1∆ rog3∆) mutant causes 2DG resistance.

  version of Hxt3 driven by its own promoter on a low-copy (centromeric) plasmid (Fig5D). This mild overexpression of Hxt3 also further increased 2DG resistance of the already resistant rod1∆ strain but had no effect on WT cells. Only a stronger expression of Hxt3 from a multicopy (2µ) plasmid conferred resistance to WT cells (Fig5E), whereas that of Hxt1 had little or no effect as found previously[START_REF] O'donnell | 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and alpha-arrestin-mediated trafficking of hexose transporters 1 and 3[END_REF]. Hxt1 and Hxt3 may have different functions regarding 2DG resistance, since Hxt1 overexpression did not compensate for the loss of Hxt3 in the rod1∆ hxt3∆ background (Fig5E). Overall, we conclude that a high expression of glucose transporters generally correlates with 2DG resistance.

  2DG, and found that glucose accumulated slightly more in rod1∆ than in WT cells (Fig 6E). This depended on the presence of the glucose transporter Hxt3, since the additional deletion of HXT3 in the context of rod1∆ restored glucose uptake to WT levels (Fig 6E) and 2DG sensitivity (Fig 5C-E). Finally, deletion of both HXT1 and HXT3 strongly impaired glucose uptake, as expected. Thus, ROD1 deletion could facilitate cell survival independently of 2DG detoxification by allowing a sustained glucose uptake. This was further illustrated by the fact that deletion of ROD1 conferred 2DG resistance to a strain lacking both DOG1 and DOG2, that are central to 2DG detoxification (Fig 6F). Therefore, the lack of endocytosis of hexose transporters protects cells from 2DG toxicity by several mechanisms.

  proteins of the GLUT/HXT family support bidirectional fluxes across the membrane [eg. GLUT2 in hepatocytes, reviewed in 77]. Accordingly, we found that ROD1 deletion increases the ability to detoxify 2DG6P. The second pathway posits that transporter stabilization at the plasma membrane allows a sustained glucose import, and thus may help bypass the 2DGinduced glycolytic blockade. Over time, 2DG6P synthesis and accumulation should inhibit hexokinase and thus blocks glucose phosphorylation, which itself drives glucose import[START_REF] Bisson | Involvement of kinases in glucose and fructose uptake by Saccharomyces cerevisiae[END_REF] [reviewed in 79], but given the existence of detoxification mechanisms, the glycolytic blockade may be only temporary. Increasing transporter availability should in principle boost glucose import and help glycolysis recover, and accordingly, rod1∆ cells contain more glucose than WT cells when exposed to glucose after 2DG treatment. Altogether, we propose that 2DG-induced endocytosis is detrimental to cells, and that deletion of ROD1 partially corrects this by stabilizing glucose transporters, leading to a sustained glycolysis and 2DG export (Fig7).

(

  pRS313-based, pROD1:ROD1-Flag, a kind gift of O. Vincent, CSIC, Madrid, Spain) to integrate a NcoI site between ROD1 and the Flag-tag, allowing to substitute the WT ORF of ROD1 by mutated versions at SalI-NcoI sites as follows. The inserts pROD1:ROD1-WT-Flag (pSL559), pROD1:ROD1-PYm-Flag (pSL560) pROD1:ROD1-S12A-Flag (pSL561) and pROD1:ROD1-KR-Flag (pSL563) were PCR amplified (oSL1662/oSL1663) using as templates pSL94

  protein. The ratio of undegraded protein is shown. The experiments were repeated three times. Datas were analyzed and processed with Prism7 software (GraphPad). The statistical test performed was a one-way analysis of variance (ANOVA) test. *: 0.01<P-value<0.05; **: 0.001<P-value<0.01; ***: 0.0001<P-value<0.001; ****: P-value<0.0001.

  pSL412 pRS426-derived (2μ, URA3) pGPD:DOG2 [20] pSL436 pRS426-derived (2μ, URA3) pGPD:DOG2(DD>AA) [20] pSL559 pRS313-derived (CEN, HIS3) pROD1:ROD1-Flag This study pSL560 pRS313-derived (CEN, HIS3) pROD1:ROD1-PYm-Flag This study pSL561 pRS313-derived (CEN, HIS3) pROD1:ROD1-S12A-Flag This study pSL563 pRS313-derived (CEN, HIS3) pROD1:ROD1-KR-Flag This study pSL589 pUG35-derived (ARS/CEN, URA3) pHXT3:HXT3-GFP This study pSL590 pDRf1GW-ura3-derived (2µ, URA3) FLII12Pglu-700μδ6 Glucose FRET sensor, Addgene #28002 Wolf Frommer [58] pSL591 pUG35-derived (ARS/CEN, URA3) pHXT3:HXT3(N370A)-GFP This study pSL599 pRS426-derived (2μ, URA3) pHXT1:HXT1 This study pSL602 pRS426-derived (2μ, URA3) pHXT3:HXT3 This study pSL608 pDRF1-GW-derived (2µ, URA3), yAT1.03 ATP FRET sensor, Addgene #132781

Figure 1 -

 1 Figure 1 -2DG treatment induces the endocytosis of many plasma membrane proteins, that is mostly Rod1-dependent. (A) Western blot on total protein extracts from Hxt2-GFP-and Tat1-GFP-expressing cells before and after 2DG addition for 2h30, using anti-GFP antibodies. (B) Cells expressing Hxt2-GFP and Tat1-GFP cells were grown overnight in a glucose-containing medium (exponential phase) and treated with 2DG for 2h30 and observed by fluorescence microscopy. PM: plasma membrane ; V: vacuole. Scale bar: 5 µm. (C) Plasma membrane proteins whose localization was examined by fluorescence microscopy before and after 2h30 2DG.When endocytosed, the same proteins were also observed in a rod1∆ context to check for Rod1 dependence (NT, not tested). (D) Fluorescence microscopy of WT and rod1∆ cells expressing the indicated transporters tagged with GFP before and after 2DG treatment for 4h. Scale bar: 5 µm.

Figure 2 -

 2 Figure 2 -Following 2DG treatment, Rod1 is dephosphorylated in a PP1-dependent manner. (A) Total protein extracts of WT cells expressing either Rod1-Flag or Rod1-KR-Flag were prepared before and after 2DG addition for 10', and immunoblotted using an anti-Flag antibody. (B) Total protein extracts of WT or reg1∆ cells expressing Rod1-Flag were prepared before and after 2DG addition for 10', and immunoblotted using an anti-

Figure 3 -Figure 4 -Figure 5 -Figure 6 -

 3456 Figure 3 -2DG causes a fast and temporary dephosphorylation of Snf1 and its substrates through PP1 activation (A) Total protein extracts of WT, reg1∆ and snf1∆ cells expressing Mig1-Flag were prepared before and after 2DG addition at the indicated concentrations for 10 min and immunoblotted using anti-phospho-AMPK and anti-polyHistidine antibodies. (B) Left, Total protein extracts of WT, reg1∆ and snf1∆ cells were prepared before and after 2DG addition for the indicated time and immunoblotted using anti-phospho-AMPK and anti-polyHistidine antibodies. Right, quantification of the signals. **: p-value £ 0,01 (C) Total protein extracts of WT, reg1∆ and snf1∆ cells were prepared before and after 2DG addition for the indicated time and immunoblotted using anti-phospho-AMPK and anti-polyHistidine antibodies. (D) Total protein extracts of WT, reg1∆ and snf1∆ cells expressing Mig1-Flag were prepared before or after 2DG addition for 10' and 2h and immunoblotted using anti-Flag antibodies.

Figure 7 -Figure S2 -

 7S2 Figure 7 -Hypothetical model for a role of hexose transporters in 2DG detoxification. Treatment of cells with 2DG triggers the accumulation of intracellular 2DG6P. This activates stress-signaling pathways such as cell-wall integrity signaling (CWI) or the unfolded protein response (UPR), converging onto DOG2 expression(1)[START_REF] Defenouillėre | The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose[END_REF]. On the other hand, 2DG6P elicits a fast PP1 activation (2) which causes AMPK inhibition and, either directly or indirectly, Rod1 dephosphorylation. By the time Dog2 is induced and begins to dephosphorylate 2DG6P (3), Rod1 has already triggered the endocytosis of many plasma membrane proteins (4), including that of low affinity glucose transporters. This may prevent 2DG exit and detoxification but also affect cell survival by reducing glucose import[START_REF] Luengo | Targeting Metabolism for Cancer Therapy[END_REF].

Figure S3 -

 S3 Figure S3 -Rog3 is not responsible for the observed Rod1-independent endocytosis of Lyp1-GFP in response to 2DG. WT, rod1∆ and rod1∆ rog3∆ cells expressing Lyp1-GFP tagged at its endogenous genomic locus were observed by fluorescence microscopy before and after treatment with 2DG for 4h. Scale bar, 5 µm.

Figure S4 -

 S4 Figure S4-Expression of Rod1-3HA does not restore endocytosis nor sensitivity to 2DG in a rod1∆ context, contrary to Rod1-Flag. (A) The indicated strains were observed by fluorescence microscopy after growth in a glucose-containing medium and after 2DG treatment for 4h. Scale bar, 5 µm. (B) Serial dilutions of cultures of the indicated mutants were spotted on dropout SC medium (SC) or SC + 0,2% 2DG and grown for 3 days at 30°C.

Table S2 : Plasmids used in this study 885

 S2

		ySL2580: Vht1-GFP ySL3099: rod1∆ dog1∆	MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 VHT1::GFP-HIS3MX MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 rod1∆::KANMX	This study This study
		rod1Δ dog2∆	rod1∆::URA3 dog1∆dog2∆::LEU2
		ySL2591: Ste2-GFP ySL3118 : Hxt4-GFP	MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 STE2::GFP-HIS3MX MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 HXT4::GFP-HIS3MX	This study This study
		rod1Δ rod1∆	rod1∆::URA3 rod1∆::KANMX
		ySL2592: Can1-GFP ySL3124: Hxt2-GFP	MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 CAN1::GFP-HIS3MX MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 HXT2::GFP-HIS3MX	This study This study
		rod1Δ rod1Δ	rod1∆::URA3 rod1∆::HPHNT1	This study
	884	ySL2623: Wsc3-GFP	MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 WSC3::GFP-HIS3MX	[88]
		ySL2288: Ina1-GFP ySL2628: Wsc2-GFP	MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 INA1::GFP-HIS3MX MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 WSC2::GFP-HIS3MX	[88] [88]
		ySL2315: hog1Δ ySL2671: Ina1-GFP hxk2Δ Name Description	MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 hog1∆::KANMX HIS3MX hxk2∆::KANMX MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 ; INA1::GFP-Origin & Reference	Euroscarf This study
		ySL2325: Ina1-GFP npi1 MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 INA1::GFP-HIS3MX pSL093 pRS416-derived (CEN, URA3) pROD1:ROD1-GFP [35] ySL2724: Hxt1-GFP MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 HXT1::GFP-HIS3MX pRSP5::KANMX rod1Δ rod1∆::HPHNT1 pSL094 pRS415-derived (CEN, LEU2) pROD1:ROD1-3HA [28]	This study This study
		ySL2395: Ina1-GFP snf1Δ rod1Δ ySL2725: Hxt3-GFP pSL205 pCUP1:6xHis-Ub (2µ, LEU2) (pJD421) MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 snf1∆::HIS3MX INA1::GFP-rod1∆::HPHNT1 HPHNT1 MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 HXT3::GFP-HIS3MX [89]	This study This study
		ySL2406: Ina1-GFP rod1Δ pSL237 pRS316-derived (CEN, URA3) pRod1:Rod1-3Flag MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 INA1::GFP-HPHNT1 Olivier Vincent ySL2726: Itr1-GFP rod1Δ MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 ITR1::GFP-HIS3MX rod1∆::KANMX rod1∆::HPHNT1 pSL409 Yep358-based (2μ, URA3) pDOG1(1000bp):LacZ [20]	This study This study
		ySL2412: Ina1-GFP rvs167Δ rod1Δ ySL2727: Pdr12-GFP pSL410 Yep358-based (2μ, URA3) pDOG2(1000bp):LacZ MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 rvs167∆KANMX4 INA1::GFP-rod1∆::HPHNT1 HPHNT1 MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 PDR12::GFP-HIS3MX [20]	This study This study
		ySL2434: Ina1-Δ5-GFP ySL2728: Lyp1-GFP	MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 Ina1-∆5::GFP-KANMX MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 LYP1::GFP-HIS3MX	This study This study
		ySL2443: Pil1-GFP rod1Δ rog3Δ	MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 PIL1::GFP-HIS3MX rod1∆::KANMX; rog3∆::NATNT2	[88]
		ySL2444: Sur7-GFP ySL2739: Vph1-mCherry MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 ; Vph1-mCherry::NATNT2 MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 SUR7::GFP-HIS3MX	[88] This study
		ySL2445: Lsp1-GFP ySL2746: rod1∆ hxt6∆	MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 LSP1::GFP-HIS3MX MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 rod1∆::KANMX hxt6∆::NATNT2 This study [88]
		ySL2446: Seg1-GFP ySL2880: Ina1-GFP Pil1-	MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 SEG1::GFP-HIS3MX MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 INA1::GFP-HIS3MX	[88] This study
		mCherry	PIL1::mCherry-NATNT2
		ySL2451: Lyp1-GFP rod1Δ ySL3003: rod1Δ Hxt3-	MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 LYP1::GFP-HIS3MX rod1∆::KANMX MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 rod1∆::KANMX Hxt3::GFP-	This study This study
		GFP	HPHNT1
		ySL2452: Qdr3-GFP rod1Δ ySL3026: hxt3Δ	MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 QDR3::GFP-HIS3MX rod1∆::KANMX MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 hxt3∆::KANMX	This study Euroscarf
		ySL2545: Tat1-GFP ySL3027: rod1Δhxt3Δ	MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 TAT1::GFP-HIS3MX MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 hxt3∆::KANMX rod1∆::HPHNT1 This study This study
		rod1Δ ySL3028: hxt1Δ	rod1∆::KANMX MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 hxt1∆::KANMX	Euroscarf
		ySL2546: Fui1-GFP rod1Δ ySL3029: rod1Δhxt1Δ	MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 FUI1::GFP-HIS3MX rod1∆::KANMX MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 hxt1∆::KANMX rod1∆::HPHNT1 This study This study
		ySL2548: rvs167Δ ySL3084: hxt1Δhxt3∆	MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 rvs167∆::KANMX MAT a; ura3Δ0, his3Δ1, leu2Δ0, met15Δ0 hxt1∆::KANMX hxt3∆::HPHNT1 This study Euroscarf

Acknowledgments

We thank Agathe Verraes for technical assistance, Olivier Vincent (CSIC, Madrid, Spain) and Michel Becuwe (Harvard University, Cambridge, MA, USA) for sharing reagents and Alexandre Soulard (Université Claude Bernard Lyon 1, Lyon, France) for helpful discussions and critical reading of the manuscript. We also acknowledge the IJM ImagoSeine facility, member of IBiSA and the France-BioImaging infrastructure (https://anr.fr/ProjetIA-10-INBS-0004; ANR-10-INBS-04). This work was supported by grants from the Fondation ARC pour la recherche sur le cancer (https://www.fondation-arc.org, ARCPJA32020060002096 to SL), Université de Paris IDEx (https://u-paris.fr; ANR-18-IDEX-0001, "Emergence en Recherche" RS30J20IDXA1_EMERLEON) and individual fellowships from the Ligue contre le cancer (https://www.liguecancer.net; TAZK20115 to CL), the Fondation pour la Recherche Médicale (https://www.frm.org; SPF20150934065 to QD) and the French Ministry for Research and Education (https://www.enseignementsuprecherche.gouv.fr; to AB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests:

The authors have declared that no competing interests exist. Laussel et al. Figure S5

Supplementary materials

D.