MitoStores: chaperone‐controlled protein granules store mitochondrial precursors in the cytosol

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Hundreds of nucleus‐encoded mitochondrial precursor proteins are synthesized in the cytosol and imported into mitochondria in a post‐translational manner. However, the early processes associated with mitochondrial protein targeting remain poorly understood. Here, we show that in Saccharomyces cerevisiae , the cytosol has the capacity to transiently store mitochondrial matrix‐destined precursors in dedicated deposits that we termed MitoStores. Competitive inhibition of mitochondrial protein import via clogging of import sites greatly enhances the formation of MitoStores, but they also form during physiological cell growth on nonfermentable carbon sources. MitoStores are enriched for a specific subset of nucleus‐encoded mitochondrial proteins, in particular those containing N‐terminal mitochondrial targeting sequences. Our results suggest that MitoStore formation suppresses the toxic potential of aberrantly accumulating mitochondrial precursor proteins and is controlled by the heat shock proteins Hsp42 and Hsp104. Thus, the cytosolic protein quality control system plays an active role during the early stages of mitochondrial protein targeting through the coordinated and localized sequestration of mitochondrial precursor proteins.

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  1. Review coordinated via ASAPbio’s crowd preprint review

    This review reflects comments and contributions by Ruchika Bajaj, Gary McDowell, Sree Rama Chaitanya Sridhara. Review synthesized by Iratxe Puebla.

    The preprint studies the process for mitochondrial targeting of mitochondrial precursor proteins. Using a yeast model, experiments show that the cytosol transiently stores matrix-destined precursors in dedicated granules which the authors name MitoStores. The formation of MitoStores is controlled by the heat shock proteins Hsp42 and Hsp104, and suppresses the toxicity arising from non-imported accumulated mitochondrial precursor proteins.

    The manuscript is clear and well-written. The reviewers raised a few comments and suggestions as outlined below:

    The introduction was extremely clear and provides a good summary of the protein homeostasis dimension of the problem in question. However, there could be a clearer discussion of the processes of import, in particular with respect to the results discussing “clogging”. It is suggested to add a penultimate transitional paragraph in the introduction that facilitates this transition e.g. this could be expansion of the first paragraph in the Results section, moved into the introduction to provide more context about the cloggers, PACE, and the Rpn4-mediated proteasomal regulation.

    Figure 2E and Figure S2 - Can some further explanation be provided about what data belongs to delta-rpn otr WT, or whether the associated fold change is reported - delta-rpn/WT.

    Results ‘while the levels of most chaperones were unaffected or even reduced in Δrpn4 cells, the disaggregase Hsp104 and the small heat shock protein Hsp42 were considerably upregulated (Fig. 2F, G)’ - Suggest adding some further clarification as to why Hsp104 and Hsp42 are selected despite perturbations in other protein partners. Are there other proteins than proteosomes and chaperones which are significantly up- or down-regulated? STRING or cytoscape tools may help with the interactome analysis.

    Figure 3

    • Figure 3A - It seems Δrpn4 cells are bigger in size than control cells, suggest commenting on this point.
    • Figure 3B ‘Hsp104-GFP was purified on nanotrap sepharose’ - Please clarify on which tag the purification was based.
    • grown at the indicated temperatures’ - Please clarify the rationale for using 30 or 40C.
    • SN, supernatant representing the non-bound fraction’ - Please report what is total, wash and elute etc.

    Results ‘protein accumulated at similar levels as Hsp104-GFP in the yeast cytosol (Fig. S4B)’ - Please clarify whether the image reports qualitative or quantitative data, and how the levels of DHFR-GFP and Hsp104-GFP are compared based on S4B.

    ‘Owing to the striking acquisition of nuclear encoded mitochondrial proteins in these structures, we termed them MitoStores’- Suggest providing some discussion about the fraction of Hsp104 that is part of the MitoStores? Does a major portion of Hsp104 in the absence of Rpn4 form MitoStore structures?