Metabolic regulation of misfolded protein import into mitochondria

Reviewer #1 (Public Review):

"MAGIC" was introduced by the Rong Li lab in a Nature letters article in 2017. This manuscript is an extension of this original work and uses a genome wide screen the Baker's yeast to decipher which cellular pathways influence MAGIC. Overall, this manuscript is a logical extension of the 2017 study, however the manuscript is challenging to follow, complicated by the data often being discussed out of sequence. Although the manuscripts makes claims of a mechanism being pinpointed, there are many gaps and the true mechanisms of how the factors identified in the screen influence MAGIC is not clear. A key issue is that there are many assumptions drawn on previous literature, but central aspects of the mechanisms being proposed are not adequately shown.

Key comments:

[1] Reasoning and pipelines presented in the first two sections of the results are disordered and do not follow figure order. In some instances, the background to experimental analyses such as detailing the generation of spGFP constructs in the YKO mutant library, or validation of Snf1 activation are mentioned after respective results are discussed. This needs to be fixed.
[2] In general there is a lack of data to support microscopy data and supporting quantification analysis. The validity of this data could be significantly strengthened with accompanying western blots showing accumulation of a given constructs in mitochondrial sub compartments (as was the case in the labs original paper in 2017).
[3] Much of the mechanisms proposed relies on the Snf1 activation. This is however not shown, but assumed to be taking place. Given that this activation is central to the mechanism proposed this should be explicitly shown here - for example survey the phosophorylation status of the protein.

Reviewer #2 (Public Review):

Work of Rong Li´s lab, published in Nature 2017 (Ruan et al, 2017), led the authors to suggest that the mitochondrial protein import machinery removes misfolded/aggregated proteins from the cytosol and transports them to the mitochondrial matrix, where they are degraded by Pim1, the yeast Lon protease. The process was named mitochondria as guardian in cytosol (MAGIC).

The mechanism by which MAGIC selects proteins lacking mitochondrial targeting information, and the mechanism which allows misfolded proteins to cross the mitochondrial membranes remained, however, enigmatic. Up to my knowledge, additional support of MAGIC has not been published. Due to that, MAGIC is briefly mentioned in relevant reviews (it is a very interesting possibility!), however, the process is mentioned as a "proposal" (Andreasson et al, 2019) or is referred to require "further investigation to define its relevance for cellular protein homeostasis (proteostasis)" (Pfanner et al, 2019).

Rong Li´s lab now presents a follow-up story. As in the original Nature paper, the major findings are based on in vivo localization studies in yeast. The authors employ an aggregation prone, artificial luciferase construct (FlucSM), in a classical split-GFP assay: GFP1-10 is targeted to the matrix of mitochondria by fusion with the mitochondrial protein Grx5, while GFP11 is fused to FlucSM, lacking mitochondrial targeting information. In addition the authors perform a genetic screen, based on a similar assay, however, using the cytosolic misfolding-prone protein Lsg1 as a read-out.

My major concern about the manuscript is that it does not provide additional information which helps to understand how specifically aggregated cytosolic proteins, lacking a mitochondrial targeting signal could be imported into mitochondria. As it stands, I am not convinced that the observed FlucSM-/Lsg1-GFP signals presented in this study originate from FlucSM-/Lsg1-GFP localized inside of the mitochondrial matrix. The conclusions drawn by the authors in the current manuscript, however, rely on this single approach.

In the 2017 paper the authors state: "... we speculate that protein aggregates engaged with mitochondria via interaction with import receptors such as Tom70, leading to import of aggregate proteins followed by degradation by mitochondrial proteases such as Pim1." Based on the new data shown in this manuscript the authors now conclude "that MP (misfolded protein) import does not use Tom70/Tom71 as obligatory receptors." The new data presented do not provide a conclusive alternative. More experiments are required to draw a conclusion.
In my view: to confirm that MAGIC does indeed result in import of aggregated cytosolic proteins into the mitochondrial matrix, a second, independent approach is needed. My suggestion is to isolate mitochondria from a strain expressing FlucSM-GFP and perform protease protection assays, which are well established to demonstrate matrix localization of mitochondrial proteins. In case the authors are not equipped to do these experiments I feel that a collaboration with one of the excellent mitochondrial labs in the US might help the MAGIC pathway to become established.

Reviewer #3 (Public Review):

In this study, Wang et al extend on their previous finding of a novel quality control pathway, the MAGIC pathway. This pathway allows misfolded cytosolic proteins to become imported into mitochondria and there they are degraded by the LON protease. Using a screen, they identify Snf1 as a player that regulates MAGIC. Snf1 inhibits mitochondrial protein import via the transcription factor Hap4 via an unknown pathway. This allows cells to adapt to metabolic changes, upon high glucose levels, misfolded proteins an become imported and degraded, while during low glucose growth conditions, import of these proteins is prevented, and instead import of mitochondrial proteins is preferred.

This is a nice and well-structured manuscript reporting on important findings about a regulatory mechanism of a quality control pathway. The findings are obtained by a combination of mostly fluorescent protein-based assays. Findings from these assays support the claims well.

While this study convincingly describes the mechanisms of a mitochondria-associated import pathway using mainly model substrates, my major concern is that the physiological relevance of this pathway remains unclear: what are endogenous substrates of the pathway, to which extend are they imported and degraded, i.e. how much does MAGIC contribute to overall misfolded protein removal (none of the experiments reports quantitative "flux" information). Lastly, it remains unclear by which mechanism Snf1 impacts on MAGIC or whether it is "only" about being outcompeted by mitochondrial precursors.