Sensing and responding to changes in environmental O2 is critical for aerobic organisms. Metazoans accomplish this by destabilizing a transcription factor called hypoxia inducible factor (HIF) in O2 sufficiency. This is achieved by a O2-dependent prolyl 4-hydroxylase (PHD) that modifies proline residues in the HIF-1? subunit making it the target of an E3 polyubiquitin ligase and ultimately dispatching it to the 26S-proteasome for degradation. PHDs are non-heme dioxygenases that use O2 and ketoglutarate to hydroxylate a proline residue and form succinate and CO2. Protozoans have PHDs but lack HIF and thus sense oxygen differently. In the social amoeba Dictyostelium, we discovered that its PHD modifies a proline in Skp1, which is a component of the SCF (Skp1/Cullin1/F-box protein/Rbx1) polyubiquitin ligase complex. Skp1 prolylhydroxylation does not affect its stability, but allows it to be modified by a series of glycosyltransferases. In turn glycosylation stabilizes the binding of Skp1 to F-box proteins essential for assembly of the SCF polyubiquitin ligases. Genome analysis and biochemical assays demonstrated that this Skp1 modification pathway is conserved in the protozoan parasite Toxoplasma gondii, but not in its human host. Moreover, loss of Toxoplasma PHD, or either of two glycosyltransferases, leads to a defect in parasite replication at low but physiological oxygen levels, and to changes in its Skp1 interactome. Because of its medical importance, we will focus on Toxoplasma and pursue three specific aims: i) Is Skp1 the key substrate for the Toxoplasma PHD and what is the contribution of novel glycosylation to PhyA activation? ii) How is Skp1 and polyubiquitin ligase assembly affected by prolyl hydroxylation? iii) How is the parasite PHD regulated and how can these data be used to develop parasite-specific pharmacological PHD inhibitors? To maximize progress in this renewal application, we have pooled the expertise of 3 independent investigators: i) a glycobiologist specialized in protozoan Skp1 and polyubiquitin ligases, ii) a Toxoplasma specialist experienced in hypoxia research, and iii) an organic chemist specializing in non-heme ? ketoglutarate-dependent dioxygenases and their inhibition.