A high-confidence inventory of the proteins in the mitochondrion-related organelle of Giardia

Giardia lamblia is a microaerophilic protozoan parasite that infects nearly 280 million people worldwide each year. Giardia lacks a mitochondrial genome and mitochondrial enzymes for oxidative phosphorylation. However, it retains mitochondrion-related organelles (MROs) that house an iron-sulfur cluster (ISC) assembly pathway. To date, 32 MRO proteins have been identified with high confidence. Purifying MROs is extremely challenging due to their small size and low abundance. Here, as part of the MitoCarta Tree of Life Consortium, we used large-scale culture, density gradient fractionation, affinity purification, data-independent acquisition mass spectrometry (DIA-MS), protein-correlation profiling, and confocal microscopy to generate the Giardia MitoCarta (GlMitoCarta) inventory. GlMitoCarta consists of 59 proteins, 27 of which are new and 34 with unknown function. Metronidazole unexpectedly upregulates many components of the MRO. Immunoprecipitation (IP)-MS analysis reveals that MROX6 and MROX13, proteins of unknown function, reciprocally interact and share an overlapping interactome related to ISC biology. Further, we identify MOMTiP-4 as a likely MRO inner membrane solute transporter that structurally resembles and functionally complements the yeast SLC25A carrier, Mtm1. The GlMitoCarta resource is freely available and should serve as a valuable foundation for systematic and mechanistic studies of MRO biology and Giardia pathogenesis.