Metabolic regulation of misfolded protein import into mitochondria

  1. Yuhao Wang
  2. Linhao Ruan
  3. Jin Zhu
  4. Xi Zhang
  5. Alexander Chih-Chieh Chang
  6. Alexis Tomaszewski
  7. Rong Li

  • Curated by eLife

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    This study provides an important connection between cellular metabolism and proteostasis through MAGIC, a previously proposed protein quality control pathway for clearing cytosolic misfolded and aggregated proteins by import into mitochondria. Using a split-GFP reporter system, the authors reveal the role of Snf1, a yeast AMPK, in preventing the import of misfolded proteins to mitochondria for MAGIC, controlled by transcription factor Hap4 as a function of cellular metabolic status. The experimental evidence provided by the authors is still inadequate for explaining the regulatory mechanism of MAGIC by Snf1 or HAP4 and incomplete for explaining the cellular mechanism of substrate selection for MAGIC.

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Abstract

Mitochondria are the cellular energy hub and central target of metabolic regulation. Mitochondria also facilitate proteostasis through pathways such as the ‘mitochondria as guardian in cytosol’ (MAGIC) whereby cytosolic misfolded proteins (MPs) are imported into and degraded inside mitochondria. In this study, a genome-wide screen in yeast uncovered that Snf1, the yeast AMP-activated protein kinase (AMPK), inhibits the import of MPs into mitochondria while promoting mitochondrial biogenesis under glucose starvation. We show that this inhibition requires a downstream transcription factor regulating mitochondrial gene expression and is likely to be conferred through substrate competition and mitochondrial import channel selectivity. We further show that Snf1/AMPK activation protects mitochondrial fitness in yeast and human cells under stress induced by MPs such as those associated with neurodegenerative diseases.

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  1. Version 2 published on bioRxiv
  2. eLife

    eLife assessment

    This study provides an important connection between cellular metabolism and proteostasis through MAGIC, a previously proposed protein quality control pathway for clearing cytosolic misfolded and aggregated proteins by import into mitochondria. Using a split-GFP reporter system, the authors reveal the role of Snf1, a yeast AMPK, in preventing the import of misfolded proteins to mitochondria for MAGIC, controlled by transcription factor Hap4 as a function of cellular metabolic status. The experimental evidence provided by the authors is still inadequate for explaining the regulatory mechanism of MAGIC by Snf1 or HAP4 and incomplete for explaining the cellular mechanism of substrate selection for MAGIC.

  3. eLife

    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.

  4. eLife

    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.

  5. eLife

    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.

  6. Life Science Editors Foundation

    Review coordinated by Life Science Editors Foundation

    Reviewed by: Dr. Angela Andersen, Life Science Editors Foundation

    Potential Conflicts of Interest: None

    Punch line: Activation of the yeast AMP-activated protein kinase (AMPK) negatively regulates MAGIC, inhibits the import of misfolded proteins into mitochondria & promotes mitochondrial biogenesis and fitness.

    Why is this interesting? Maybe all those healthy things like caloric restriction, intermittent fasting, exercise etc that activate AMPK & extend lifespan do so by inhibiting MAGIC & preventing mitochondrial damage from misfolded proteins.

    Background: Metabolic imbalance & loss of proteostasis are interconnected hallmarks of aging and age-related diseases. A mitochondria-mediated proteostasis mechanism called MAGIC (mitochondria as guardian in cytosol) concentrates cytosolic misfolded protein at the surface of mitochondria, where they are disaggregated by molecular chaperones, and then imported for degradation by mitochondrial proteases. Inhibition of this pathway prolongs protein aggregation in cytosol after proteotoxic stress, but excessive misfolded proteins in mitochondria can lead to mitochondrial damage.

    Results: • Genetic screen for MAGIC regulators uncovered 145 genes. Loss of Snf1 (AMPK homolog) led to increased mitochondrial import even without proteotoxic stress. In contrast indirect, constitutive activation of Snf1 (e.g. low glucose) prevented the import of misfolded proteins in mitochondria.

    • The data suggest that the reduced accumulation of misfolded proteins in mitochondria of Snf1-active cells is not due to enhanced intramitochondrial degradation nor to reduced levels of the misfolded protein, but rather due to blocked mitochondrial import.

    • Deletion of HAP4 counteracted Snf1 activation and overexpression of Hap4 alone recapitulated Snf1 activation. The Hap2/3/4/5 complex activates the expression of nuclear encoded mitochondrial proteins. Their data suggest that high expression of mitochondrial preproteins due to an elevated Snf1-Hap4 axis compete with misfolded proteins for mitochondrial import.

    • Proteotoxic stress led to a reduced growth rate & reduced mitochondrial fitness in high glucose medium but not under glucose limitation. The data suggest that low glucose, activation of Snf1 & prevention of misfolded protein import into mitochondria prevent the growth defect.

    • Many neurodegenerative disease-associated aggregation-prone proteins (α-synuclein, FUSP525L, TDP-43, amyloid beta, C9ORF72-associated poly(GR) dipeptide) are detected in mitochondria of human patients or disease models and impair mitochondrial functions. Their data suggest that the import of α-synuclein & associated reduction in mitochondrial fitness can be counteracted by indirect AMPK/Snf1 activation (i.e. glucose limitation).

    • Show data in yeast & human cells.

    Discussion: This paper revealed an unexpected link between cellular metabolism and proteostasis through MAGIC/mitochondria.

    • Snf1/AMPK is a key regulator of MAGIC & of misfolded protein import into mitochondria.

    • Snf1/AMPK balances the mitochondrial metabolic and proteostatic functions in response to glucose availability and protects mitochondrial fitness under proteotoxic stress.

    • The authors speculate that in high glucose, cells rely on glycolysis for ATP production and mitochondria ‘moonlighting’ in cellular proteostasis through MAGIC, but when glucose is limited and cells rely on oxidative phosphorylation for ATP generation, AMPK is activated and shuts down MAGIC, prioritizing the import of essential mitochondrial preproteins to ensure mitochondrial fitness and energy production.

    • Acknowledge limitations: Snf1/Hap4 activation elevates the expression of hundreds of mitochondrial preproteins, not clear whether specific preproteins or cytosolic factors directly involved in inhibiting mitochondrial import, & that more details on mechanisms will be of interest.

    • Caloric restriction & AMPK activation might contribute to lifespan extension by inhibiting MAGIC. In human, AMPK activity is elevated during health-benefitting activities such as exercise. Their data suggest that elevating AMPK activity may be beneficial in alleviating proteotoxicity associated with degenerative diseases - but hyperactivated AMPK has also been reported in several neurodegenerative diseases with proteostasis decline (Ang wonders- maybe AMPK is overwhelmed?).

    THIS IS A GORGEOUS PAPER!

    Future work - I can't wait to see the characterization of the ribosome biogenesis genes that they also pulled out as MAGIC regulators. Anticipating a translation, misfolded protein, mitochondria, aging axis :)

  7. Version 1 published on bioRxiv