MitoStores: a place for precursors to ride out the storm

Plain language summary

The fate of unimported mitochondrial precursors has been increasingly studied in recent years, mostly focusing on protein degradation. In this issue of the EMBO journal, Krämer et al discovered MitoStores, a new protective mechanism that temporarily stores mitochondrial proteins in cytosolic deposits.

Recent work identifies MitStores as temporary cytosolic storage granules for un‐imported mitochondrial precursors.

The life cycle of organellar proteins starts in the cytosol. Many of these precursor proteins will be imported and translocated through organelle membranes only once their translation is complete. Inefficient protein import not only disrupts the organelle function, which depends on a constant supply of newly synthesized proteins, but also can lead to a toxic accumulation of proteins in other areas of the cell (Song et al, 2020). The damage from protein import defects is particularly profound in the case of mitochondria, as the large mitochondrial proteome is almost exclusively translocated posttranslationally. Sensing and responding to the accumulation of mitochondrial proteins outside the organelle is imperative, especially in highly metabolic tissues, such as muscle, which requires a relatively high mitochondrial mass.

Aberrant accumulation of mitochondrial precursors triggers mitoprotein‐induced stress responses to maintain proteostasis in the cytosol (Song et al, 2020). These responses attenuate protein translation and increase the capacity of cytosolic protein quality control pathways. Recent work in budding yeast identified a central role for the transcription factor Rpn4 in repairing damage caused by defective mitochondrial protein import. Rpn4, a master regulator of the proteasome, mediates a surge in protein degradation as a protective mechanism that helps clear unimported mitochondrial precursors from the cytosol and other compartments (Boos et al, 2019). However, massive degradation of newly synthesized mitochondrial proteins can be wasteful, especially if the inhibition of their import is temporary and can be resolved relatively quickly. Whether other pathways exist that protect the cytosol from the toxicity of unimported proteins, but at the same time preserve them to allow mitochondrial translocation once the defect is resolved, was unknown.

Krämer et al (2023) reveal a protein quality control pathway that acts in parallel to proteasomal degradation. An indication for the existence of such a pathway was initially observed in RPN4 null yeast. Unexpectedly, this mutant exhibited a higher tolerance to the cytosolic accumulation of mitochondrial precursors than wild‐type cells as demonstrated by an increased proliferation rate. The difference was particularly apparent in cells with a severe protein import defect, induced by the expression of a mitochondrial import channel clogger. Proteomic analyses revealed that, unlike other heat‐shock proteins, the cytosolic chaperones Hsp104 and Hsp42, were significantly upregulated in RPN4‐deleted cells. To investigate whether they have a central role in alleviating cytosolic damage caused by mistargeted mitochondrial proteins, the authors constitutively expressed Hsp104 and Hsp42 in wild‐type cells. These conditions, which mimic the high levels of the chaperones in RPN4 null cells, were indeed sufficient to improve proliferation upon clogging of mitochondrial channels.

Most of the recent progress in our understanding of damage caused by impaired mitochondrial protein import was derived from experiments using drugs, genetic disruption of the import machinery, and overexpression of a clogger or other slowly imported substrates (Wang & Chen, 2015; Wrobel et al, 2015; Weidberg & Amon, 2018; Boos et al, 2019; Mårtensson et al, 2019). Krämer et al (2023) used respiration obligatory conditions (a nonfermentable carbon source) to induce mitochondrial biogenesis and overwhelm the import machinery. Under these conditions, cells lacking Hsp104, Hsp42, and Rpn4 were inviable, and constitutive expression of Hsp104 and Hsp42 increased cell proliferation. Importantly, these results demonstrated a protective role for Hsp104 and Hsp42 under physiological conditions that require highly functional mitochondria.

Previous studies indicated that Hsp104 and Hsp42 are involved in mitoprotein stress. These chaperones, known to associate with protein aggregates (Mogk & Bukau, 2017), are upregulated when mitochondrial protein import is impaired (Nowicka et al, 2021). Moreover, aggregation‐prone mitochondrial precursors were shown to reside in Hsp42‐containing inclusion bodies (Nowicka et al, 2021; Xiao et al, 2021). However, the impact of Hsp104 and Hsp42 on cellular proteostasis during mitoprotein stress and the mechanism by which they act were yet to be determined. Krämer et al (2023) demonstrated that Hsp104 localizes to cytosolic foci that increase in diameter upon accumulation of unimported mitochondrial precursors. Unlike other types of Hsp42/Hsp104‐containing inclusion bodies (Mogk & Bukau, 2017), the mitochondrial stress‐related foci appeared in close proximity to mitochondria. How do these cytosolic deposits aid in tolerating unimported mitochondrial proteins? Isolation and proteomic analysis of Hsp104‐bound foci revealed that they are enriched in mitochondrial precursors, most of which contained a mitochondrial targeting signal commonly found in matrix proteins.

Identifying the content of the mitoprotein‐induced foci, MitoStores, enabled the investigation of the requirements for their formation. The authors first demonstrated that the deletion of HSP42 prevents the sequestration of mitochondrial precursors in foci. The localization of precursors to foci was then shown to be dependent on the presence of a mitochondrial targeting signal (Krämer et al, 2023). While the formation of mitochondrial precursor‐containing foci in both the cytosol and the nucleus was demonstrated in previous studies, these foci were considered to be the final stage in the proteins' life cycle, prior to degradation (Nowicka et al, 2021; Shakya et al, 2021). Krämer et al (2023), however, found that MitoStores are reversible by demonstrating that sequestered precursors can be imported into mitochondria once the import capacity is recovered (Fig 1).

Figure 1. Quality control of unimported mitochondrial proteins.

Posttranslationally translocated mitochondrial proteins are synthesized in the cytosol. Different pathways have evolved to respond to defects in the import of these precursors into mitochondria and to prevent cellular proteotoxicity. Certain mitochondrial proteins are promptly degraded by the proteosome. Two mechanisms regulate precursors containing a mitochondrial targeting signal (MTS): (i) mitoNUC—precursors are sequestered in nuclear inclusion bodies and subsequently degraded by the proteosome, (ii) MitoStores—Hsp104 and Hsp42 control the formation of mitochondria‐associated cytosolic inclusion bodies where precursors are temporarily stored. MitoStores‐sequestered precursors are translocated into mitochondria upon restoration of protein import capacity. Other quality control pathways for mitochondrial precursors such as carrier, outer membrane (OM), and intermembrane space (IMS) proteins are yet to be discovered. It is unclear whether these precursors are sequestered in different types of aggregates or regulated by other mechanisms.

Along with other groups, data from Krämer et al (2023) are consistent with the finding that different unimported mitochondrial proteins are regulated by different protective pathways: Some are primarily degraded, while others accumulate in different cellular compartments (Song et al, 2020; Nowicka et al, 2021; Shakya et al, 2021) (Fig 1). What determines the fate of individual precursors is still unclear. The presence of a matrix mitochondrial targeting signal might be crucial for recognition by aggregation machineries in the cytosol and nucleus, but whether other features, such as internal targeting signals, play a role is yet to be discovered.

In summary, Krämer et al (2023) reveal that Hsp104/Hsp42‐mediated formation of MitoStores helps cells cope with stress caused by mitochondrial protein import defects by temporarily isolating toxic mitochondrial precursors. Whether MitoStores contribute to mitochondrial health in addition to their cytosolic protective function is a key open question. It is tempting to hypothesize that these deposits contribute to the recovery from mitoprotein‐induced stress by allowing rapid reconstitution of the mitochondrial proteome. MitoStores might also promote translocation of precursors prior to resolving the defect, by cycling them in and out of the deposits and providing repeated attempts for their import. This could provide an additional explanation for MitoStore formation being superior to RPN4‐mediated proteasomal degradation in its beneficial impact on cell proliferation during stress. Thus, MitoStores could potentially have a dual role in repairing two major aspects of damage caused by mitochondrial protein import defects: depletion of the mitochondrial proteome and cytosolic proteotoxicity.

The EMBO Journal (2023) 42: e113576