Mitochondrial proteome of Acanthamoeba delineates aerobic and anaerobic pathways dynamically regulated by oxygen

  • Cell. 2026 Oct 1;189(20):6246-6266.e19. doi: 10.1016/j.cell.2026.08.056.
Jonathan A Stefely  1 ,  Felicia G Deng  2 ,  Michael Z Chen  2 ,  Khanh Nguyen  3 ,  Bridget E Luce  4 ,  Michelle Y Fry  4 ,  Xiaoyan A Guo  5 ,  Konstantinos Maliaris  2 ,  Keith D Rivera  3 ,  Malvina Papanastasiou  3 ,  Tiana M Issa  2 ,  Bharath Kanakapura Sundararaj  6 ,  Sophia L Y Root  2 ,  D R Mani  3 ,  MitoCarta Tree of Life Consortium ,  Luke H Chao  4 ,  Sarah E Calvo  2 ,  Namrata D Udeshi  7 ,  John Samuelson  8 ,  Vamsi K Mootha  9
Affiliations
  • 1. Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, MA 02118, USA.
  • 2. Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114, USA.
  • 3. Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • 4. Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Medical School, Boston, MA 02115, USA.
  • 5. Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114, USA.
  • 6. Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, MA 02118, USA.
  • 7. Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address: [email protected].
  • 8. Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, MA 02118, USA. Electronic address: [email protected].
  • 9. Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114, USA. Electronic address: [email protected].
PMID: 42822424 DOI: 10.1016/j.cell.2026.08.056
Abstract

Acanthamoeba castellanii causes infectious blindness and resides in a key evolutionary outgroup to humans and fungi. Its divergent mitochondria are of outstanding interest due to their predicted aerobic and anaerobic functions with potential for drug targeting. However, a detailed delineation of its bioenergetic machinery, its activities, and their regulation remains lacking. Here, we integrate mitochondrial immunoprecipitation, density gradient purification, mass spectrometry, protein correlation profiling, and microscopy to generate a high-confidence inventory of the Acanthamoeba mitoproteome. The resulting AcMitoCarta contains 1,122 proteins, including 381 lacking readily identifiable homologs in human and yeast mitochondria. Complexome analysis highlights 20 macromolecular assemblies. Complementary proteomic and transcriptomic profiling reveals extensive rewiring of the organelle's bioenergetic machinery by oxygen, including induction of an anaerobic pyruvate:ferredoxin oxidoreductase-to-hydrogenase pathway under anoxia. We experimentally demonstrate that Acanthamoeba can produce H2 gas under anoxic conditions via a mitochondria-localized, oxygen-labile hydrogenase. AcMitoCarta establishes a framework for dissecting the interplay between aerobic and anaerobic Energy Metabolism.

Keywords
ATP synthase; Acanthamoeba; BN-PAGE-MS; HCP; HGT; HupA; HydA; HydE; HydF; HydG; MDH; NDH2; NirK; PFOR; SDHB; TFAM; complex I; hybrid cluster protein; mitochondria; mitochondrial ribosome.
Products