Oxygen levels are critical for cellular physiology. Under low oxygen (hypoxia), cells increase glycolysis while limiting mitochondrial oxidative metabolism. Such adaptation is largely mediated by the transcription factors HIF, which accumulate under hypoxia [1, 2]. Prolyl hydroxylase domain proteins (PHD) function as cellular oxygen sensors and target HIF for subsequent degradation under normoxia [2]. The PHD/HIF pathway is conserved in Drosophila, where a single prolyl hydroxylase, Fatiga, has been characterized. We have previously reported that Fatiga regulates cellular growth rates in a HIF-independent manner, yet mechanisms remained elusive [3]. To characterize novel mechanisms controlling growth and metabolism in response to oxygen sensing, we immunoprecipitated HA-tagged Fatiga from Drosophila S2 cells and detected interacting proteins by mass spectrometry (Supplementary information, Table S1 and Data S1). We identified Sima, the fly HIF-α ortholog, as well as several components of the T-complex chaperon proteins (TriC), whose mammalian homologs interact with PHD3 [4]. In addition, we identified PyK, encoding the glycolytic enzyme pyruvate kinase (PK; EC 2.7. 1.40), which produces pyruvate from phosphoenolpyruvate (PEP)(Figure 1A). Importantly, a Fatiga hydroxylase-deficient mutant still interacted with PyK, but lost its interaction to Sima (Figure 1B). To test whether the PyK-Fatiga interaction would be conserved in mammalian proteins, we used the M2 isoform of human PK (PK-M2) in pulldown assays. Indeed, PK-M2 interacted with PHD3, but not PHD1 or PHD2 (Figure 1C). As proof of this interaction in vivo, endogenous PK-M2 and PHD3 did co-immunoprecipitate in mouse embryonic fibroblasts (MEFs; Figure 1D).

In mammals, four isoenzymes of PK are found: type L in liver, R in erythrocytes, M1 in adult and M2 in embryonic cells. During tumorigenesis, tissue-specific isoforms of PK are replaced by PK-M2, and this isoform plays an important role in the metabolic shift of cancer cells [5, 6]. PK-M2 is a tetramer in the presence of FBP (fructose