Mitochondrial protein import clogging as a mechanism of disease
- SUNY Upstate Medical University, United States
- University of Freiburg, Germany
- University of Bonn, Germany
Abstract
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 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 cause 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.
Data availability
The small datasets generated in the study are included in the supplemental tables. The RNA-seq data has been deposited to NCBI Gene Expression Omnibus/Sequence Read Archive with the accession number GSE227295.
-
Mitochondrial protein import clogging as a mechanism of diseaseNCBI Gene Expression Omnibus, GSE227295.
Article and author information
Author details
Funding
National Institute on Aging (R01AG063499)
- Xin Jie Chen
National Institute on Aging (R01AG061204)
- Xin Jie Chen
National Institute on Aging (F30AG060702)
- Liam P Coyne
Deutsche Forschungsgemeinschaft (project ID 269925409)
- Thomas Becker
Deutsche Forschungsgemeinschaft (BE 4679 2/2)
- Thomas Becker
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- R. Luke Wiseman, Scripps Research Institute, United States
Ethics
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (D1600318) of the State University of New York Upstate Medical University. The protocol was approved by the Committee on the Ethics of Animal Experiments of SUNY Upstate Medical University (Permit Number: #268).
Version history
- Preprint posted: September 21, 2022 (view preprint)
- Received: October 20, 2022
- Accepted: April 17, 2023
- Accepted Manuscript published: May 2, 2023 (version 1)
- Version of Record published: May 24, 2023 (version 2)
Copyright
© 2023, Coyne et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,879
- views
-
- 411
- downloads
-
- 14
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Mitochondrial protein import clogging as a mechanism of disease
eLife 12:e84330.
https://doi.org/10.7554/eLife.84330
Further reading
-
- Biochemistry and Chemical Biology
- Cell Biology
Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by the loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER-trans-Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
The mechanism underlying the preferential and cooperative binding of cofilin and the expansion of clusters toward the pointed-end side of actin filaments remains poorly understood. To address this, we conducted a principal component analysis based on available filamentous actin (F-actin) and C-actin (cofilins were excluded from cofilactin) structures and compared to monomeric G-actin. The results strongly suggest that C-actin, rather than F-ADP-actin, represented the favourable structure for binding preference of cofilin. High-speed atomic force microscopy explored that the shortened bare half helix adjacent to the cofilin clusters on the pointed end side included fewer actin protomers than normal helices. The mean axial distance (MAD) between two adjacent actin protomers along the same long-pitch strand within shortened bare half helices was longer (5.0–6.3 nm) than the MAD within typical helices (4.3–5.6 nm). The inhibition of torsional motion during helical twisting, achieved through stronger attachment to the lipid membrane, led to more pronounced inhibition of cofilin binding and cluster formation than the presence of inorganic phosphate (Pi) in solution. F-ADP-actin exhibited more naturally supertwisted half helices than F-ADP.Pi-actin, explaining how Pi inhibits cofilin binding to F-actin with variable helical twists. We propose that protomers within the shorter bare helical twists, either influenced by thermal fluctuation or induced allosterically by cofilin clusters, exhibit characteristics of C-actin-like structures with an elongated MAD, leading to preferential and cooperative binding of cofilin.
