2-deoxyglucose transiently inhibits yeast AMPK signaling and triggers glucose transporter endocytosis,potentiating the drug toxicity

2-deoxyglucose is a glucose analog that impacts many aspects of cellular physiology. After its uptake and its phosphorylation into 2-deoxyglucose-6-phosphate (2DG6P), it interferes with several metabolic pathways including glycolysis and protein N-glycosylation. Despite this systemic effect, resistance can arise through strategies that are only partially understood. In yeast, 2DG resistance is often associated with mutations causing increased activity of the yeast 5’-AMP activated protein kinase (AMPK), Snf1. Here we focus on the contribution of a Snf1 substrate in 2DG resistance, namely the alpha-arrestin Rod1 involved in nutrient transporter endocytosis. We report that 2DG triggers the endocytosis of many plasma membrane proteins, mostly in a Rod1-dependent manner. Rod1 participates in 2DG-induced endocytosis because 2DG, following its phosphorylation by hexokinase Hxk2, triggers changes in Rod1 post-translational modifications and promotes its function in endocytosis. Mechanistically, this is explained by a transient, 2DG-induced inactivation of Snf1/AMPK by protein phosphatase 1 (PP1). We show that 2DG-induced endocytosis is detrimental to cells, and the lack of Rod1 counteracts this process by stabilizing glucose transporters at the plasma membrane. This facilitates glucose uptake, which may help override the metabolic blockade caused by 2DG, and 2DG export—thus terminating the process of 2DG detoxification. Altogether, these results shed a new light on the regulation of AMPK signaling in yeast and highlight a remarkable strategy to bypass 2DG toxicity involving glucose transporter regulation.     

Identification of a key residue determining substrate affinity in the human glucose transporter GLUT1

Asn³³¹ in transmembrane segment 7 of the yeast Saccharomyces cerevisiae transporter Hxt2 has been identified as a single key residue for high-affinity glucose transport by comprehensive chimera approach. The glucose transporter GLUT1 of mammals belongs to the same major facilitator superfamily as Hxt2 and may therefore show a similar mechanism of substrate recognition. The functional role of Ile²⁸⁷ in human GLUT1, which corresponds to Asn³³¹ in Hxt2, was studied by its replacement with each of the other 19 amino acids. The mutant transporters were individually expressed in a recently developed yeast expression system for GLUT1. Replacement of Ile²⁸⁷ generated transporters with various affinities for glucose that correlated well with those of the corresponding mutants of the yeast transporter. Residues exhibiting high affinity for glucose were medium-sized, non-aromatic, uncharged and irrelevant to hydrogen-bond capability, suggesting an important role of van der Waals interaction. Sensitivity to phloretin, a specific inhibitor for the presumed exofacial glucose binding site, was decreased in two mutants, whereas that to cytochalasin B, a specific inhibitor for the presumed endofacial glucose binding site, was unchanged in the mutants. These results suggest that Ile²⁸⁷ is a key residue for maintaining high glucose affinity in GLUT1 as revealed in Hxt2 and is located at or near the exofacial glucose binding site.     

Genome-wide screen reveals novel mechanisms for regulating cobalt uptake and detoxification in fission yeast

Cobalt is an essential micronutrient but is toxic when present in excess. To study cobalt homeostasis we performed a genome-wide screen for deletion strains that show sensitivity or resistance to CoCl(2). Among 54 cobalt-sensitive strains, 18 are supersensitive strains, which are involved in histidine biosynthetic process, ubiquitination, mitochondria function, membrane trafficking, transporter and a variety of other known functions or still unknown functions. Furthermore, we identified 56 cobalt-resistant deletion strains, which are mainly involved in mitochondria function, signal transduction, ubiquitination, and gene expression and chromatin remodeling. Notably, deletion of the zhf1 (+) gene, encoding a zinc ion transporter, confers supersensitivity to cobalt and overexpression of the zhf1 (+) gene confers marked tolerance to cobalt, indicating that Zhf1 play key roles in cobalt detoxification. Interestingly, all the histidine-auxotrophic mutants displayed cobalt sensitivity and deletion of cationic amino acid transporter Cat1, which was shown to be involved in histidine uptake, suppressed the CoCl(2)-sensitive growth defect of the his2 mutants, suggesting that CoCl(2) may be transported into the cell together with histidine via histidine transporters including Cat1. In addition, we obtained results suggesting that the E2 ubiquitin conjugating enzyme Rhp6 and Sty1 stress MAP kinase pathway are involved in the regulation of cobalt homeostasis. Altogether, our genome-wide study demonstrates for the first time the mechanisms of cobalt homeostasis, particularly its uptake and detoxification in fission yeast.     

A molecular switch on an arrestin-like protein relays glucose signaling to transporter endocytosis

Endocytosis regulates the plasma membrane protein landscape in response to environmental cues. In yeast, the endocytosis of transporters depends on their ubiquitylation by the Nedd4-like ubiquitin ligase Rsp5, but how extracellular signals trigger this ubiquitylation is unknown. Various carbon source transporters are known to be ubiquitylated and endocytosed when glucose-starved cells are exposed to glucose. We show that this required the conserved arrestin-related protein Rod1/Art4, which was activated in response to glucose addition. Indeed, Rod1 was a direct target of the glucose signaling pathway composed of the AMPK homologue Snf1 and the PP1 phosphatase Glc7/Reg1. Glucose promoted Rod1 dephosphorylation and its subsequent release from a phospho-dependent interaction with 14-3-3 proteins. Consequently, this allowed Rod1 ubiquitylation by Rsp5, which was a prerequisite for transporter endocytosis. This paper therefore demonstrates that the arrestin-related protein Rod1 relays glucose signaling to transporter endocytosis and provides the first molecular insights into the nutrient-induced activation of an arrestin-related protein through a switch in post-translational modifications.     

Isolation and characterization of two distinct myo-inositol transporter genes of Saccharomyces cerevisiae

By the complementation of a yeast mutant defective in myo-inositol transport (Nikawa, J., Nagumo, T., and Yamashita, S. (1982) J. Bacteriol. 150, 441-446), we isolated two myo-inositol transporter genes, ITR1 and ITR2, from a yeast gene library. The ITR1 and ITR2 genes contained long open reading frames capable of encoding 584 and 612 amino acids with calculated relative molecular masses of 63,605 and 67,041, respectively. The sequence similarity between the ITR1 and ITR2 products was extremely high, suggesting that the two genes arose from a common ancestor. Both gene products show significant sequence homology with a superfamily of sugar transporters, including human HepG2 hepatoma/erythrocyte glucose transporter and Escherichia coli xylose transporter. Hydropathy analysis indicated that the ITR1 and ITR2 products are both hydrophobic and contain 12 putative membrane-spanning regions. Thus, yeast myo-inositol transporters could be classified into the sugar transporter superfamily. Gene disruption and tetrad analysis showed that yeast cells contain two separate myoinositol transporters. The ITR1 product was the major transporter and the ITR2 product the minor one in cells grown in minimum medium containing glucose. Northern blot analysis showed that ITR1 mRNA was much more abundant than ITR2 mRNA. The previously isolated myo-inositol transport mutant was determined to be defective in ITR1.     

Characterisation of glucose transport in Saccharomyces cerevisiae with plasma membrane vesicles (countertransport) and intact cells (initial uptake) with single Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 or Gal2 transporters

The yeast glucose transporters Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 and Gal2, individually expressed in an hxt1-7 null mutant strain, demonstrate the phenomenon of countertransport. Thus, these transporters, which are the most important glucose transporters in Saccharomyces cerevisiae, are facilitated diffusion transporters. Apparent K(m)-values from high to low affinity, determined from countertransport and initial-uptake experiments, respectively, are: Hxt6 0.9+/-0.2 and 1.4+/-0.1 mM, Hxt7 1.3+/-0.3 and 1.9+/-0.1 mM, Gal2 1.5 and 1.6+/-0.1 mM, Hxt2 2.9+/-0.3 and 4.6+/-0.3 mM, Hxt4 6.2+/-0.5 and 6.2+/-0.3 mM, Hxt3 28.6+/-6.8 and 34.2+/-3.2 mM, and Hxt1 107+/-49 and 129+/-9 mM. From both independent methods, countertransport and initial uptake, the same range of apparent K(m)-values was obtained for each transporter. In contrast to that in human erythrocytes, the facilitated diffusion transport mechanism of glucose in yeast was symmetric. Besides facilitated diffusion there existed in all single glucose transport mutants, except for the HXT1 strain, significant first-order behaviour.     

Constitutive Tor2 Activity Promotes Retention of the Amino Acid Transporter Agp3 at Trans-Golgi/Endosomes in Fission Yeast

Amino acid transporters are located at specific subcellular compartments, and their localizations are regulated by the extracellular availability of amino acids. In yeast, target of rapamycin (TOR) activation induces the internalization of amino acid transporters located at the plasma membrane. However, whether and how TOR signaling regulates other amino acid transporters located at intracellular compartments remains unknown. Here, we demonstrate that in the fission yeast, the TOR inhibitor Torin–1 induces the transfer of several yellow fluorescent protein (YFP)-fused intracellular amino acid transporters, including Agp3, Isp5, Aat1, and Put4, from trans-Golgi/endosomes into the vacuoles. By contrast, the localizations of YFP-fused Can1, Fnx1, and Fnx2 transporter proteins were unaffected upon Torin–1 treatment. There are two TOR isoforms in fission yeast, Tor1 and Tor2. Whereas tor1 deletion did not affect the Torin-1-induced transfer of Agp3-YFP, Tor2 inhibition using a temperature-sensitive mutant induced the transfer of Agp3-YFP to the vacuolar lumen, similar to the effects of Torin–1 treatment. Tor2 inhibition also induced the transfer of the YFP-fused Isp5, Aat1, and Put4 transporter proteins to the vacuoles, although only partial transfer of the latter two transporters was observed. Under nitrogen depletion accompanied by reduced Tor2 activity, Agp3-YFP was transferred from the trans-Golgi/endosomes to the plasma membrane and then to the vacuoles, where it was degraded by the vacuolar proteases Isp6 and Psp3. Mutants with constitutively active Tor2 showed delayed transfer of Agp3-YFP to the plasma membrane upon nitrogen depletion. Cells lacking Tsc2, a negative regulator of Tor2, also showed a delay in this process in a Tor2-dependent manner. Taken together, these findings suggest that constitutive Tor2 activity is critical for the retention of amino acid transporters at trans-Golgi/endosomes. Moreover, nitrogen depletion suppresses Tor2 activity through Tsc2, thereby promoting the surface expression of these transporters.     

Functional expression and characterisation of a gut facilitative glucose transporter, NlHT1, from the phloem-feeding insect Nilaparvata lugens (rice brown planthopper)

Phloem-sap feeding Hemipteran insects have access to a sucrose-rich diet but are dependent on sucrose hydrolysis and hexose transport for carbon nutrition. A cDNA library from Nilaparvata lugens (rice brown planthopper) was screened for clones encoding potential transmembrane transporters. A selected cDNA, NlHT1, encodes a 53kDa polypeptide with sequence similarity to facilitative hexose transporters of eukaryotes and prokaryotes, including GLUT1, the human erythrocyte hexose transporter. NlHT1 was expressed as a recombinant protein in the methylotropic yeast Pichia pastoris, and was identified in a membrane fraction isolated from transformed yeast cells. Transport experiments using membrane vesicles containing NlHT1 showed that the protein is a saturable, sodium independent transporter, with a relatively low affinity for glucose (K(m) 3.0mM), which can be inhibited by cytochalasin B. Competition experiments with fructose demonstrate NlHT1 is glucose specific. In situ localisation studies revealed that NlHT1 mRNA is expressed in N. lugens gut tissue, mainly in midgut regions, and that expression is absent in hindgut and Malpighian tubules. NlHT1 is therefore likely to play an important role in glucose transport from the gut, and in carbon nutrition in vivo. This is the first report of a facilitative glucose transporter from a phloem-feeding insect pest.     

IFN‐β rescues neurodegeneration by regulating mitochondrial fission via STAT5, PGAM5, and Drp1

Mitochondrial homeostasis is essential for providing cellular energy, particularly in resource‐demanding neurons, defects in which cause neurodegeneration, but the function of interferons (IFNs) in regulating neuronal mitochondrial homeostasis is unknown. We found that neuronal IFN‐β is indispensable for mitochondrial homeostasis and metabolism, sustaining ATP levels and preventing excessive ROS by controlling mitochondrial fission. IFN‐β induces events that are required for mitochondrial fission, phosphorylating STAT5 and upregulating PGAM5, which phosphorylates serine 622 of Drp1. IFN‐β signaling then recruits Drp1 to mitochondria, oligomerizes it, and engages INF2 to stabilize mitochondria–endoplasmic reticulum (ER) platforms. This process tethers damaged mitochondria to the ER to separate them via fission. Lack of neuronal IFN‐β in the Ifnb ^–/– model of Parkinson disease (PD) disrupts STAT5‐PGAM5‐Drp1 signaling, impairing fission and causing large multibranched, damaged mitochondria with insufficient ATP production and excessive oxidative stress to accumulate. In other PD models, IFN‐β rescues dopaminergic neuronal cell death and pathology, associated with preserved mitochondrial homeostasis. Thus, IFN‐β activates mitochondrial fission in neurons through the pSTAT5/PGAM5/^S622Drp1 pathway to stabilize mitochondria/ER platforms, constituting an essential neuroprotective mechanism.     

Amino acid neurotransmitter transporters: Structure,function, and molecular diversity

Many biologically active compounds including neurotransmitters, metabolic precursors, and certain drugs are accumulated intracellularly by transporters that are coupled to the transmembrane Na⁺ gradient. Amino acid neurotransmitter transporters play a key role in the regulation of extracellular amino acid concentrations and termination of neurotransmission in the CNS

1 Abbreviations: CNS, central nervous system; GABA, γ-aminobutyric acid; cDNA, complementary deoxyribonucleic acid; mRNA, messenger ribonucleic acid; NMDA, N-methyl-D-aspartate; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; DAG, diacyl glycerol; R59022, DAG kinase inhibitor; AA, arachidonic acid; ACHC, cis-3-aminocyclohexanecarboxylic acid; GAT-A, ACHC-sensitive GABA transporter; GAT-B, β-alanine-sensitive GABA transporter; GLY-1 and GLYT-1, glycine transporters; PROT-1, proline transporter; BGT-1, betaine transporter.

. Transporters for the major amino acid neurotransmitters glutamate, GABA, and glycine are found in both neurons and glial cells. Recent work has resulted in the identification of cDNAs encoding several amino acid neurotransmitter transport proteins, all of which belong to the Na⁺-and Cl^?-dependent transporter gene family. The diversity of this family suggests a degree of transporter heterogeneity that is greater than that indicated by biochemical and pharmacological studies.     

Expression of human glucose transporters in Xenopus oocytes: kinetic characterization and substrate specificities of the erythrocyte, liver, and brain isoforms

We describe the functional expression of three members of the family of human facilitative glucose transporters, the erythrocyte-type transporter (GLUT 1), the liver-type transporter (GLUT 2), and the brain-type transporter (GLUT 3), by microinjection of their corresponding mRNAs into Xenopus oocytes. Expression was determined by the appearance of transport activity, as measured by the transport of 3-O-methyl-D-glucose or 2-deoxy-D-glucose. We have measured the Km for 3-O-methyl-D-glucose of GLUTs 1, 2, and 3, and the results are discussed in light of the possible roles for these different transporters in the regulation of blood glucose. The substrate specificity of these transporter isoforms has also been examined. We show that, for all transporters, the transport of 2-deoxy-D-glucose is inhibited by D-but not by L-glucose. In addition, both D-galactose and D-mannose are transported by GLUTs 1-3 at significant rates; furthermore, GLUT 2 is capable of transporting D-fructose. The nature of the glucose binding sites of GLUTs 1-3 was investigated by using hexose inhibition of 2-deoxy-D-glucose uptake. We show that the characteristics of this inhibition are different for each transporter isoform.     

Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway

The target of rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol 3-kinase-related kinases. TOR proteins are found at the core of two evolutionary conserved complexes, known as TORC1 and TORC2. In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. TORC2 has been implicated in a wide variety of functions; however, the signals that regulate TORC2 activity have so far remained obscure. TORC2 has one known direct substrate, the AGC kinase Gad8, which is related to AKT in human cells. Gad8 is phosphorylated by TORC2 at Ser-546 (equivalent to AKT Ser-473), leading to its activation. Here, we show that glucose is necessary and sufficient to induce Gad8 Ser-546 phosphorylation in vivo and Gad8 kinase activity in vitro. The glucose signal that activates TORC2-Gad8 is mediated via the cAMP/PKA pathway, a major glucose-sensing pathway. By contrast, Pmk1, similar to human extracellular signal-regulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad8 phosphorylation and activation. Inhibition of TORC2-Gad8 also occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Our findings highlight the significance of glucose availability in regulation of TORC2-Gad8 and indicate a novel link between the cAMP/PKA, Pmk1/MAPK, and TORC2-Gad8 signaling.