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Hereditary folate malabsorption (HFM) patients harbor inactivating mutations including R113S in the proton-coupled folate transporter (PCFT), an intestinal folate transporter with optimal activity at acidic pH. Here we identified and characterized a novel R113C mutation residing in the highly conserved first intracellular loop of PCFT. Stable transfectants overexpressing a Myc-tagged wild-type (WT) and mutant R113C PCFT displayed similar transporter targeting to the plasma membrane. However, whereas WT PCFT transfectants showed a 22-fold increase in [ 3 H]folic acid influx at pH 5.5, R113C or mock transfectants showed no increase. Moreover, WT PCFT transfectants displayed a 50% folic acid growth requirement concentration of 7 nM, whereas mock and R113C transfectants revealed 24-to 27-fold higher values. Consistently, upon fluorescein-methotrexate labeling, WT PCFT transfectants displayed a 50% methotrexate displacement concentration of 50 nM, whereas mock and R113C transfectants exhibited 12-to 14-fold higher values. Based on the crystal structure of the homologous Escherichia coli glycerol-3-phosphate transporter, we propose that the cationic R113 residue of PCFT is embedded in a hydrophobic pocket formed by several transmembrane helices that may be part of a folate translocation pore. These findings establish a novel loss of function mutation in HFM residing in an intracellular loop of PCFT crucial for folate transport. IntroductionFolates are essential micronutrients that serve as one-carbon donors and acceptors in a multitude of biosynthetic reactions including de novo biosynthesis of purines, thymidylate, methionine, glycine and methylation reactions. 1,2 Hence, folate vitamins are essential for DNA replication and cellular proliferation. However, mammalian organisms are devoid of folate biosynthesis and thus dietary sources must meet their metabolic requirement for folate cofactors. As such, mammalians rely on an efficient intestinal transport of folates that proceeds via a carrier-mediated system displaying optimal transport activity at acidic pH occurring primarily in the acidic microenvironment of the upper intestinal mucosal epithelium including that of the duodenum and the upper jejunum. [3][4][5] Recently, the human proton-coupled folate transporter (PCFT) gene, PCFT/SLC46A1, was identified that encodes for an intestinal folate transporter displaying optimal transport activity at acidic pH. 6 PCFT exhibits high affinities (Kt ϭ 0.5-1 M) for oxidized folates (folic acid) and reduced folates [eg, (6S)5-methylTHF and 5-formylTHF], as opposed to the reduced folate carrier (RFC/ SLC19A1) that has high affinity for reduced folates (Kt ϭ 2-4 M) but low affinity for folic acid (Ki ϭ 200-400 M). [6][7][8] Recently, several loss-of-function mutations were identified in the PCFT gene from patients with hereditary folate malabsorption (HFM), a congenital disease (OMIM 229050). 6,9 HFM is a rare autosomal recessive disorder caused by impaired intestinal folate absorption. 6,7,[9][10][11] Patients with HFM presen...

Background: Zinc transporters (ZnTs) presumably form dimers, thereby modulating zinc transport activity. Results: Bimolecular fluorescence complementation (BiFC) revealed ZnT1-4, ZnT7 homodimers and ZnT5-ZnT6 heterodimers. Conclusion: BiFC pinpointed WT and mutant ZnT2 dimerization in live cells. Significance: BiFC provides the first in situ evidence for the subcellular localization of WT and mutant ZnT2 dimers in live cells, thereby establishing the molecular basis underlying zinc deficiency.

PI3K‐Akt‐FoxO‐mTOR signaling is the central pathway controlling growth and metabolism in all cells. Ubiquitination of the protein kinase Akt prior to its phosphorylation is required for PI3K‐Akt activity. Here, we found that the deubiquitinating (DUB) enzyme USP1 removes K63‐linked polyubiquitin chains on Akt to restrict PI3K‐Akt‐FoxO signaling in mouse muscle during prolonged starvation. DUB screening platform identified USP1 as a direct DUB for Akt, and USP1 depletion in mouse muscle increased Akt ubiquitination, PI3K‐Akt‐FoxO signaling, and glucose uptake during fasting. Co‐immunoprecipitation and mass spectrometry identified disabled homolog‐2 (Dab2), the tuberous sclerosis complex TSC1/TSC2, and PHLPP1 as USP1 bound proteins. During starvation, Dab2 is essential for Akt recruitment to USP1‐TSC1‐PHLPP1 complex, and for PI3K‐Akt‐FoxO inhibition. Surprisingly, USP1 limits TSC1 levels to sustain mTOR‐mediated basal protein synthesis rates and maintain its own protein levels. We propose that Dab2 recruits Akt to USP1‐TSC1‐PHLPP1 complex to efficiently terminate the transmission of growth signals when cellular energy level is low.

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