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  1. Evaluation Summary:

    The authors dissect and reconstitute the cytosolic steps for mitochondrial signal-anchored membrane protein biogenesis focusing on post-translational precursor recognition by cytosolic chaperones and their subsequent transfer to import receptors located within the mitochondrial outer membrane. These are crucial events in order to assist proper protein biogenesis while preventing aggregation and its downstream consequences. The study is an important contribution to the understanding of cytosolic events in the biogenesis of mitochondrial proteins, and this paper will be of relevance for researchers in the fields of chaperone and mitochondrial biology as well as for cell biologists studying the biogenesis of membrane proteins.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    Protein targeting of precursor proteins to the mitochondrial surface is poorly understood. Drwesh and colleagues characterized cytosolic factors that target mitochondrial signal-anchored proteins. Using yeast translational extracts, they identified Hsp70 and Hsp90 chaperones and their co-chaperones such as Ydj1 and Sis1 as important binding partners. The transmembrane domain of the signal anchored protein is critical for the binding of the chaperones. The authors used a combination of in vitro binding assays and import experiments into mitochondria to characterize the interaction of the chaperones to the precursor proteins and their subsequent transfer to the TOM receptors. Overall, the reported findings are highly interesting and an important contribution to the field. The study provides important insights into the cytosolic network that controls protein targeting to mitochondria. The presented data are of high quality and convincing.

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  3. Reviewer #2 (Public Review):

    Drwesh and colleagues used an array of in vitro, in organello and in vivo approaches to study the early steps of mitochondrial signal-anchored (SA) membrane protein biogenesis after translation and before membrane insertion. The authors chose four distinct yeast mitochondrial outer membrane SA proteins (Tom20, Tom70, Msp1 and the outer membrane form of Mcr1, Mcr1mom) and analyzed their recognition by cytosolic chaperones and cochaperones from the Hsp70 and Hsp40 families that bind translated precursors, preventing their aggregation and keeping precursors in an import-competent state. Furthermore, they analyzed precursor protein interactions with outer mitochondrial membrane receptors, therefore covering the two major cytosolic steps required for the biogenesis of mitochondrial SA proteins.

    The authors could clearly show that SA protein recognition via co-/chaperones occurs via interactions with their transmembrane segments. Using co-immunoprecipitation of newly-translated HA-tagged precursors in yeast extract the authors found all analyzed SA proteins to interact with chaperones from the Hsp70 (Ssa1/2) and co-chaperones from the Hsp40 (Ydj1, Sis1, Djp1) families. Further mass spectrometry analysis of the Mcr1 and Msp1 eluates showed their interaction with a broader array of chaperones from the Hsp70 family.

    Downregulation of Ydj1 and Sis1 co-chaperones from the Hsp40 family showed an effect on steady-state levels and import defects for SA proteins. Interestingly, depletion of both co-chaperones showed the increase in SA precursor binding to Hsp104 and Hsp26 chaperones that prevent protein aggregation, suggesting that co-chaperones from the Hsp40 family, even if they do not directly impact steady-state levels or import of all tested SA candidates, they do prevent their aggregation.
    In vitro fluorescence anisotropy experiments demonstrated the interaction of the transmembrane segment of some SA candidates for both Ssa1 and Sis1, with a higher affinity towards the chaperone as for the co-chaperone. Interestingly, using the same approach and sequential addition of co-/chaperones and membrane receptor domains, they showed that precursors can be transferred in vitro from the co-chaperone, to the chaperone and to the receptors, therefore reconstituting the required steps in early biogenesis of SA proteins. Affinity measurements go in accordance with this sequential precursor transfer.

    Finally, the authors show that even if the studied membrane receptors are cleaved or blocked, SA proteins can still be imported into mitochondria in organello to different extents, suggesting that the cytosolic chaperone system is even more important SA anchored protein biogenesis compared to the import receptors.

    Overall, the manuscript is the first in-depth characterization of the complex transfer of mitochondrial signal-anchored proteins by cytosolic co-/chaperones to the mitochondrial membrane receptors, suggesting that cells possess redundant chaperone and receptor machineries to fulfill this task. This set of distinct approaches characterizes the cytosolic interactions required to efficiently transfer signal-anchored membrane proteins from co-/chaperones to the membrane receptors.

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  4. Reviewer #3 (Public Review):

    The authors aimed to investigate the network of interactions in the cytosol between a number of chaperones and SA targeted proteins in the mitochondrial OM. They also wanted to clarify the mechanism of insertion at the OM and the role of the main receptors in this process. They have managed to provide a wealth of interesting data on a mechanism that is very elaborate as it involves a lot of redundancy between the different chaperones. One strong point of their work is that they go beyond the typical analysis of interactions by biochemical approaches and provide some quantitative data that support a model for a cascade of interactions. This was missing in previous studies and is a notion that will likely be taken up in future studies as well. The impact of the work is substantial and represents an important step forward in our understanding of the mitochondrial protein targeting processes.

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