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Oxygen is critical for a multitude of metabolic processes that are essential for human life. Biological processes can be identified by treating cells with 18O2or other isotopically labelled gases and systematically identifying biomolecules incorporating labeled atoms. Here we labelled cell lines of distinct tissue origins with 18O2to identify the polar oxy-metabolome, defined as polar metabolites labelled with 18O under different physiological O2tensions. The most highly 18O-labelled feature was 4-hydroxymandelate (4-HMA). We demonstrate that 4-HMA is produced by hydroxyphenylpyruvate dioxygenase-like (HPDL), a protein of previously unknown function in human cells. We identify 4-HMA as an intermediate involved in the biosynthesis of the coenzyme Q10 (CoQ10) headgroup in human cells. The connection of HPDL to CoQ10 biosynthesis provides crucial insights into the mechanisms underlying recently described neurological diseases related to HPDL deficiencies1–4and cancers with HPDL overexpression5.

Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.

N6-methyladenosine (m6A) is a common modification of mRNA with potential roles in fine-tuning the RNA life cycle. Here, we identify a dense network of proteins interacting with METTL3, a component of the methyltransferase complex, and show that three of them (WTAP, METTL14, and KIAA1429) are required for methylation. Monitoring m6A levels upon WTAP depletion allowed the definition of accurate and near single-nucleotide resolution methylation maps and their classification into WTAP-dependent and -independent sites. WTAP-dependent sites are located at internal positions in transcripts, topologically static across a variety of systems we surveyed, and inversely correlated with mRNA stability, consistent with a role in establishing “basal” degradation rates. WTAP-independent sites form at the first transcribed base as part of the cap structure and are present at thousands of sites, forming a previously unappreciated layer of transcriptome complexity. Our data shed light on the proteomic and transcriptional underpinnings of this RNA modification.

Glucose 6-phosphate dehydrogenase (G6PD) stands at the head of the pentose phosphate pathway, which is responsible for nucleotide synthesis. The identification and thorough validation of an improved G6PD inhibitor provide a valuable new tool compound for studying metabolism.

During clathrin-mediated endocytosis Hsc70, supported by the J-domain protein auxilin, uncoats clathrin-coated vesicles. Auxilin contains both a clathrin-binding domain and a J-domain that binds Hsc70, and it has been suggested that these two domains are both necessary and sufficient for auxilin activity. To test this hypothesis, we created a chimeric protein consisting of the J-domain of auxilin linked to the clathrin-binding domain of the assembly protein AP180. This chimera supported uncoating, but unlike auxilin it acted stoichiometrically rather than catalytically because, like Hsc70, it remained associated with the uncoated clathrin. This observation supports our proposal that Hsc70 chaperones uncoated clathrin by inducing formation of a stable Hsc70-clathrin-AP complex. It also shows that Hsc70 acts by dissociating individual clathrin triskelions rather than cooperatively destabilizing clathrin-coated vesicles. Because the chimera lacks the C-terminal subdomain of the auxilin clathrin-binding domain, it seemed possible that this subdomain is required for auxilin to act catalytically, and indeed its deletion caused auxilin to act stoichiometrically. In contrast, deletion of the N-terminal subdomain weakened auxilin-clathrin binding and prevented auxilin from polymerizing clathrin. Therefore the C-terminal subdomain of the clathrin-binding domain of auxilin is required for auxilin to act catalytically, whereas the N-terminal subdomain strengthens auxilin-clathrin binding.

Light activation of NADP-linked glyceraldehyde-3-P dehydrogenase involves reductive cleavage of a disulfide bond. We have proposed that the inactivating disulfide locks the two domains of the enzyme, preventing catalysis, and we have tentatively identified the two critical cysteine residues in the chloroplast enzyme (D. Li, F.J. Stevens, M. Schiffer and L.E. Anderson (1994) Biophys J. 67: 29–35). We reasoned that if activation of this enzyme involves these cysteines that enzymes lacking one or both should be active in the dark and insensitive to reductants. One of these cysteines is present in the enzymes from Anabaena variabilis and Synechocystis PCC 6803 but the other is not. Consistent with the proposed mechanism, glyceraldehyde-3-P dehydrogenase is not affected by DTT-treatment in extracts of either of these cyanobacteria. Fructosebisphosphatase is DTT-activated in extracts of both of these cyanobacteria and glucose-6-P dehydrogenase is inactivated in Synechocystis, as in higher plant chloroplasts. Apparently reductive modulation is possible in these cyanobacteria but glyceraldehyde-3-P dehydrogenase is not light activated.