Updated project metadata. Since the discovery of eukaryotes without mitochondria, intestinal flagellates oxymonads, it was clear that the loss of mitochondria was facilitated by the remodelling of the FeS cluster synthesis. Yet, this remodelling is common for a broader linage including free-living species with reduced mitochondria and so question remains what the functions of their organelles are. We resolved to a high precision the proteome of one of them using Localisation of Organelle Proteins by Isotope Tagging (LOPIT) and demonstrate that it performs the synthesis of folate derivates bearing one carbon units. This pathway links to a previously localised glycine cleavage system (GCS) and its only conceivable client seems cytosolic methionine cycle recycling S-adenosine methionine. This observation gives a sense for the consistent presence of GCS in reduced mitochondria of free-living anaerobes and its absence in endobionts, which typically lose the methionine cycle and in the case of oxymonads also mitochondria.