Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, United States
Xiaowen Wang
Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, United States
Jiyao Song
Institute of Biochemistry and Molecular Biology, Faculty of Medicine, University of Freiburg, Freiburg, Germany; Institute of Biochemistry and Molecular Biology, Faculty of Medicine, University of Bonn, Bonn, Germany
Ebbing de Jong
Proteomics and Mass Spectrometry Core Facility, State University of New York Upstate Medical University, Syracuse, United States
Karin Schneider
Department of Microbiology and Immunology, State University of New York Upstate Medical University, Syracuse, United States
Paul T Massa
Department of Microbiology and Immunology, State University of New York Upstate Medical University, Syracuse, United States; Department of Neurology, State University of New York Upstate Medical University, Syracuse, United States
Frank A Middleton
Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, United States; Department of Neuroscience and Physiology, State University of New York Upstate Medical University, Syracuse, United States
Thomas Becker
Institute of Biochemistry and Molecular Biology, Faculty of Medicine, University of Bonn, Bonn, Germany
Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, United States; Department of Neuroscience and Physiology, State University of New York Upstate Medical University, Syracuse, United States
Mitochondrial biogenesis requires the import of >1,000 mitochondrial preproteins from the cytosol. Most studies on mitochondrial protein import are focused on the core import machinery. Whether and how the biophysical properties of substrate preproteins affect overall import efficiency is underexplored. Here, we show that protein traffic into mitochondria can be disrupted by amino acid substitutions in a single substrate preprotein. Pathogenic missense mutations in ADP/ATP translocase 1 (ANT1), and its yeast homolog ADP/ATP carrier 2 (Aac2), cause the protein to accumulate along the protein import pathway, thereby obstructing general protein translocation into mitochondria. This impairs mitochondrial respiration, cytosolic proteostasis, and cell viability independent of ANT1’s nucleotide transport activity. The mutations act synergistically, as double mutant Aac2/ANT1 causes severe clogging primarily at the translocase of the outer membrane (TOM) complex. This confers extreme toxicity in yeast. In mice, expression of a super-clogger ANT1 variant led to neurodegeneration and an age-dependent dominant myopathy that phenocopy ANT1-induced human disease, suggesting clogging as a mechanism of disease. More broadly, this work implies the existence of uncharacterized amino acid requirements for mitochondrial carrier proteins to avoid clogging and subsequent disease.
