Signal-anchored (SA) proteins are anchored into the mitochondrial outer membrane (OM) via a single transmembrane segment at their N-terminus while the bulk of the proteins is facing the cytosol. These proteins are encoded by nuclear DNA, translated on cytosolic ribosomes, and are then targeted to the organelle and inserted into its OM by import factors. Recently, research on the insertion mechanisms of these proteins into the mitochondrial OM have gained a lot of attention. In contrast, the early cytosolic steps of their biogenesis are unresolved. Using various proteins from this category and a broad set of in vivo, in organello, and in vitro assays, we reconstituted the early steps of their biogenesis. We identified a subset of molecular (co)chaperones that interact with newly synthesized SA proteins, namely, Hsp70 and Hsp90 chaperones and co-chaperones from the Hsp40 family like Ydj1 and Sis1. These interactions were mediated by the hydrophobic transmembrane segments of the SA proteins. We further demonstrate that interfering with these interactions inhibits the biogenesis of SA proteins to a various extent. Finally, we could demonstrate direct interaction of peptides corresponding to the transmembrane segments of SA proteins with the (co)chaperones and reconstitute in vitro the transfer of such peptides from the Hsp70 chaperone to the mitochondrial Tom70 receptor. Collectively, this study unravels an array of cytosolic chaperones and mitochondrial import factors that facilitates the targeting and membrane integration of mitochondrial SA proteins.
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD031610.All data generated or analyzed during this study are included in the manuscript and supporting file; Source Data files have been provided for all relevant Figures.
- Doron Rapaport
- Doron Rapaport
- Layla Drwesh
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Nikolaus Pfanner, University of Freiburg, Germany
© 2022, Drwesh 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.
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