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The chaperone-binding activity of the mitochondrial surface receptor Tom70 protects the cytosol against mitoprotein-induced stress.

ABSTRACT: Most mitochondrial proteins are synthesized as precursors in the cytosol and post-translationally transported into mitochondria. The mitochondrial surface protein Tom70 acts at the interface of the cytosol and mitochondria. In vitro import experiments identified Tom70 as targeting receptor, particularly for hydrophobic carriers. Using in vivo methods and high-content screens, we revisit the question of Tom70 function and considerably expand the set of Tom70-dependent mitochondrial proteins. We demonstrate that the crucial activity of Tom70 is its ability to recruit cytosolic chaperones to the outer membrane. Indeed, tethering an unrelated chaperone-binding domain onto the mitochondrial surface complements most of the defects caused by Tom70 deletion. Tom70-mediated chaperone recruitment reduces the proteotoxicity of mitochondrial precursor proteins, particularly of hydrophobic inner membrane proteins. Thus, our work suggests that the predominant function of Tom70 is to tether cytosolic chaperones to the outer mitochondrial membrane, rather than to serve as a mitochondrion-specifying targeting receptor.

SUBMITTER: Backes S

PROVIDER: S-EPMC7615001 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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The chaperone-binding activity of the mitochondrial surface receptor Tom70 protects the cytosol against mitoprotein-induced stress.

Backes Sandra S Bykov Yury S YS Flohr Tamara T Räschle Markus M Zhou Jialin J Lenhard Svenja S Krämer Lena L Mühlhaus Timo T Bibi Chen C Jann Cosimo C Smith Justin D JD Steinmetz Lars M LM Rapaport Doron D Storchová Zuzana Z Schuldiner Maya M Boos Felix F Herrmann Johannes M JM

Cell reports 20210401 1

Most mitochondrial proteins are synthesized as precursors in the cytosol and post-translationally transported into mitochondria. The mitochondrial surface protein Tom70 acts at the interface of the cytosol and mitochondria. In vitro import experiments identified Tom70 as targeting receptor, particularly for hydrophobic carriers. Using in vivo methods and high-content screens, we revisit the question of Tom70 function and considerably expand the set of Tom70-dependent mitochondrial proteins. We d ...[more]

PMID: 33826901

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Project description:Chaperone-mediated autophagy (CMA) is a major pathway of lysosomal proteolysis critical for cellular homeostasis and metabolism, and whose defects have been associated with several human pathologies. While CMA has been well described in mammals, functional evidence has only recently been documented in fish, opening up new perspectives to tackle this function under a novel angle. Now we propose to explore CMA functions in the rainbow trout (RT, Oncorhynchus mykiss), a fish species recognized as a model organism of glucose intolerance and characterized by the presence of two paralogs of the CMA-limiting factor Lamp2A (lysosomal associated membrane protein 2A). To this end, we validated a fluorescent reporter (KFERQ-PA-mCherry1) previously used to track functional CMA in mammalian cells, in an RT hepatoma-derived cell line (RTH-149). We found that incubation of cells with high-glucose levels (HG, 25 mM) induced translocation of the CMA reporter to lysosomes and/or late endosomes in a KFERQ- and Lamp2A-dependent manner, as well as reduced its half-life compared to the control (5 mM), thus demonstrating increased CMA flux. Furthermore, we observed that activation of CMA upon HG exposure was mediated by generation of mitochondrial reactive oxygen species, and involving the antioxidant transcription factor Nfe2l2/Nrf2 (nfe2 like bZIP transcription factor 2). Finally, we demonstrated that CMA plays an important protective role against HG-induced stress, primarily mediated by one of the two RT Lamp2As. Together, our results provide unequivocal evidence for CMA activity existence in RT and highlight both the role and regulation of CMA during glucose-related metabolic disorders.Abbreviations: AREs: antioxidant response elements; CHC: α-cyano -4-hydroxycinnamic acid; Chr: chromosome; CMA: chaperone-mediated autophagy; CT: control; DMF: dimethyl fumarate; Emi: endosomal microautophagy; HG: high-glucose; HMOX1: heme oxygenase 1; H2O2: hydrogen peroxide; KFERQ: lysine-phenylalanine-glutamate-arginine-glutamine; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; MCC: Manders' correlation coefficient; Manders' correlation coefficient Mo: morpholino oligonucleotide; NAC: N-acetyl cysteine; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; PA-mCherry: photoactivable mCherry; PCC: Pearson's correlation coefficient; ROS: reactive oxygen species; RT: rainbow trout; siRNAs: small interfering RNAs; SOD: superoxide dismutase; Tsg101: tumor susceptibility 101; TTFA: 2-thenoyltrifluoroacetone; WGD: whole-genome duplication.

Project description:Mitochondrial Ca2+ overload is a main contributor to mitochondrial damage hence cardiomyocyte death in myocardial ischemia/reperfusion (MI/R) injury. MICU1 has been recently identified as an important regulator of mitochondrial Ca2+ homeostasis. Here we try to identify the role of MICU1 in MI/R, and to investigate whether the mitochondrial importer receptor Tom70 possesses critical roles in the mitochondrial translocation of MICU1 and MI/R. Specific small interfering RNA (20 μg) against MICU1 and Tom70, and lentivirus vectors carrying the Tom70a sequences (3.3 × 107 TU) were delivered through intramyocardial injection. Seventy-two hours after injection, mice were subjected to 30 min of MI followed by 3 h (for cell apoptosis and mitochondrial damage assessment) or 24 h (for cardiac function and infarct size determination) of reperfusion. MI/R had no significant effect on total MICU1 expression, but caused significant reduction of MICU1 in mitochondria. Knockdown of MICU1 significantly aggravated MI/R injury, as evidenced by enlarged infarct size, depressed cardiac function and increased myocardial apoptosis. Moreover, MICU1 deficiency resulted in markedly aggravated mitochondrial Ca2+ overload, consequently destructed mitochondrial morphology and suppressed mitochondrial function (evidenced by decreased ATP production). Interestingly, mitochondrial Tom70 was also decreased in MI/R. Genetic loss-function study revealed that mitochondrial MICU1 expression was depressed by Tom70 ablation. Furthermore, Tom70 deficiency significantly aggravated MI/R injury and worsened mitochondrial Ca2+ overload. However, supplementation of Tom70 significantly attenuated MI/R injury, preserved mitochondrial morphology and function, and inhibited mitochondrial Ca2+ overload, all of which were abolished by MICU1 suppression. Mitochondrial Tom70/MICU1 pathway protects against MI/R injury, in which mitochondrial localization of MICU1 is governed by Tom70, and MICU1 serves as an indispensable factor in Tom70's cardioprotection.

Dataset Information

The chaperone-binding activity of the mitochondrial surface receptor Tom70 protects the cytosol against mitoprotein-induced stress.

Publications

The chaperone-binding activity of the mitochondrial surface receptor Tom70 protects the cytosol against mitoprotein-induced stress.

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