Cancer cells reprogram their metabolism to meet elevated energy demands and favor glycolysis for energy production. This boost in glycolytic flux supports proliferation, but also generates acid in the form of hydrogen ions that must be eliminated from the cytoplasm to maintain the alkaline intracellular pH (pHi) associated with transformation. To cope with acid production, tumor cells employ ion transport systems, including the family of sodium–hydrogen exchangers (NHE). Here, we identify NHE7 as a novel regulator of pHi in pancreatic ductal adenocarcinoma (PDAC). We determine that NHE7 suppression causes alkalinization of the Golgi, leading to a buildup of cytosolic acid that diminishes tumor cell fitness mainly through the dysregulation of actin. Importantly, NHE7 knockdown in vivo leads to the abrogation of tumor growth. These results identify Golgi acidification as a mechanism to control pHi and point to the regulation of pHi as a possible therapeutic vulnerability in PDAC. Significance: NHE7 regulates cytosolic pH through Golgi acidification, which points to the Golgi as a “proton sink” for metabolic acid. Disruption of cytosolic pH homeostasis via NHE7 suppression compromises PDAC cell viability and tumor growth. See related commentary by Ward and DeNicola, p. 768. This article is highlighted in the In This Issue feature, p. 747

Biosynthesis of macromolecules requires precursors such as sugars or amino acids, originating from exogenous/dietary sources, reutilization/salvage of degraded molecules, or de novo synthesis. Since these sources are assumed to contribute to one homogenous pool, their individual contributions are often overlooked. Protein glycosylation uses monosaccharides from all the above sources to produce nucleotide sugars required to assemble hundreds of distinct glycans. Here, we demonstrate that cells identify the origin/heritage of the monosaccharide, fucose, for glycosylation. We measured the contribution of GDP-fucose from each of these sources for glycan synthesis and found that different fucosyltransferases, individual glycoproteins, and linkage-specific fucose residues identify and select different GDP-fucose pools dependent on their heritage. This supports the hypothesis that GDP-fucose exists in multiple, distinct pools, not as a single homogenous pool. The selection is tightly regulated since the overall pool size remains constant. We present novel perspectives on monosaccharide metabolism, which may have a general applicability.

Solute carrier family 35 member A5 (SLC35A5) is a member of the SLC35A protein subfamily comprising nucleotide sugar transporters. However, the function of SLC35A5 is yet to be experimentally determined. In this study, we inactivated the SLC35A5 gene in the HepG2 cell line to study a potential role of this protein in glycosylation. Introduced modification affected neither N- nor O-glycans. There was also no influence of the gene knock-out on glycolipid synthesis. However, inactivation of the SLC35A5 gene caused a slight increase in the level of chondroitin sulfate proteoglycans. Moreover, inactivation of the SLC35A5 gene resulted in the decrease of the uridine diphosphate (UDP)-glucuronic acid, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine Golgi uptake, with no influence on the UDP-galactose transport activity. Further studies demonstrated that SLC35A5 localized exclusively to the Golgi apparatus. Careful insight into the protein sequence revealed that the C-terminus of this protein is extremely acidic and contains distinctive motifs, namely DXEE, DXD, and DXXD. Our studies show that the C-terminus is directed toward the cytosol. We also demonstrated that SLC35A5 formed homomers, as well as heteromers with other members of the SLC35A protein subfamily. In conclusion, the SLC35A5 protein might be a Golgi-resident multiprotein complex member engaged in nucleotide sugar transport.

GDP‐Fucose used for glycan fucosylation is synthesized de novo from either glucose (Glc) or mannose (Man) or utilized directly from salvaged/exogenous fucose (Fuc). Meticulous radioisotope dilution studies performed in the 1970’s only in HeLa cells using 0.3 μM Fuc showed that the de novo pathway provides >90% of GDP‐Fuc and salvage <10%. Employing different Fuc‐specific lectins, we showed that at low concentrations (150 fucosylated N‐glycans containing 1–4 Fuc residues by comparing the proportion of ¹³ C Fuc incorporation at various concentrations for 24h. Over the labeling period, cells had to choose between GDP‐Fuc synthesized de novo or from exogenous fucose. N‐glycans with a single Fuc show a proportional increase in ¹³ C‐labeled species with increasing concentration of label. To our surprise, glycans with 2, 3, and 4 Fuc each required progressively higher exogenous ¹³ C Fuc to become fully labeled. Based on LC‐MS/MS and lectin staining, 1–6αFuc labels more efficiently with exogenous Fuc than 1–2α, 1–3α and 1–4αFuc, suggesting differential access to exogenous sugar that depends partially on Fuc linkage. K _m differences between fucosyltransferases cannot explain preferential use of GDP‐Fuc synthesized from exogenous monosaccharide over de novo produced substrate. Rather, it suggests the existence of separate, ill‐defined pools of GDP‐Fuc. Altogether it raises the question whether salvaged fucose equivalent to exogenous Fuc? Their behavior clearly differs from de novo synthesized GDP‐Fuc. Support or Funding Information: The Rocket Fund and R01DK099551.