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Import of ADP/ATP carrier into mitochondria: Two receptors act in parallel

Rockefeller University Press
Journal of Cell Biology (JCB)
Authors:
Heinrich F. Steger
Heinrich F. Steger
Heinrich F. Steger
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Thomas H Söllner at Universität Heidelberg
Thomas H Söllner
Michael A Kiebler at Ludwig-Maximilians-University of Munich
Michael A Kiebler
KA Dietmeier
KA Dietmeier
KA Dietmeier
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Abstract and Figures

We have identified the yeast homologue of Neurospora crassa MOM72, the mitochondrial import receptor for the ADP/ATP carrier (AAC), by functional studies and by cDNA sequencing. Mitochondria of a yeast mutant in which the gene for MOM72 was disrupted were impaired in specific binding and import of AAC. Unexpectedly, we found a residual, yet significant import of AAC into mitochondria lacking MOM72 that occurred via the receptor MOM19. We conclude that both MOM72 and MOM19 can direct AAC into mitochondria, albeit with different efficiency. Moreover, the precursor of MOM72 apparently does not require a positively charged sequence at the extreme amino terminus for targeting to mitochondria.
Import of various precursor proteins into mitochondria from MOM72-deficient yeast. Mitochondria (10 #g of protein)
Import of various precursor proteins into mitochondria from MOM72-deficient yeast. Mitochondria (10 #g of protein)
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N. crassa mitochondria lacking MOM72 are able to import the ADP/ATP carrier via MOM19. (A) Selective degradation of MOM72 by elastase. N. crassa mitochondria (50 #g of protein) were treated with elastase (0.15/zg/ml [Pfaller et al., 1988; S/~llner et al., 1989]) or left untreated. Mitochondrial proteins were separated by SDS-PAGE, transferred to nitrocellulose paper, and immunodecorated with antisera directed against N. crassa MOM72 and MOM19. (B and C) Antibodies against MOM19 block import of ADP/ATP carrier into MOM72-deficient mitochondria. Mitochondria (10 /zg of protein) were pretreated with elastase (0.15 /zg/ml) and incubated with IgGs as indicated (S611ner et al., 1989, 1990). Mitochondria were isolated and incubated with reticulocyte lysate containing radiolabeled precursor of AAC in the presence of 8 mM potassium ascorbate and 0.2 mM N, N, N', N'-tetramethylphenylenediamine (TMPD) for 7 min at 25°C as described in Materials and Methods. The samples were treated with proteinase K as described (Planner and Neupert, 1987; Pfaller et al., 1988). Mitochondria were reisolated and analyzed by SDS-PAGE, fluorography, and laser densitometry.
N. crassa mitochondria lacking MOM72 are able to import the ADP/ATP carrier via MOM19. (A) Selective degradation of MOM72 by elastase. N. crassa mitochondria (50 #g of protein) were treated with elastase (0.15/zg/ml [Pfaller et al., 1988; S/~llner et al., 1989]) or left untreated. Mitochondrial proteins were separated by SDS-PAGE, transferred to nitrocellulose paper, and immunodecorated with antisera directed against N. crassa MOM72 and MOM19. (B and C) Antibodies against MOM19 block import of ADP/ATP carrier into MOM72-deficient mitochondria. Mitochondria (10 /zg of protein) were pretreated with elastase (0.15 /zg/ml) and incubated with IgGs as indicated (S611ner et al., 1989, 1990). Mitochondria were isolated and incubated with reticulocyte lysate containing radiolabeled precursor of AAC in the presence of 8 mM potassium ascorbate and 0.2 mM N, N, N', N'-tetramethylphenylenediamine (TMPD) for 7 min at 25°C as described in Materials and Methods. The samples were treated with proteinase K as described (Planner and Neupert, 1987; Pfaller et al., 1988). Mitochondria were reisolated and analyzed by SDS-PAGE, fluorography, and laser densitometry.
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Role of MOM19 in import of the ADP/ATP carder into intact N. crassa mitochondria. (A) Mitochondria loaded with antibodies against MOM72 and MOM19 are completely blocked in import of AAC. The experiment was performed as described in the legend of Fig. 7, B and C, except that IgGs were prebound to untreated mitochondria. The values given are the means of five or more experiments (with a standard error of the means <3%). (B) Both MOM72 and MOM19 direct AAC to the GIP site. Mitochondria (10 #g of protein) were preincubated with IgGs (60 #g) as indicated (S611ner et al., 1990). Mitochondria were reisolated and incubated with reticulocyte lysate containing radiolabeled precursor of AAC in the presence of 1 #M valinomycin for 20 min at 25°C as described in Materials and Methods and treated with proteinase K (10 #g/ml) (Planner and Neupert, 1987; Planner et al., 1987b).
Role of MOM19 in import of the ADP/ATP carder into intact N. crassa mitochondria. (A) Mitochondria loaded with antibodies against MOM72 and MOM19 are completely blocked in import of AAC. The experiment was performed as described in the legend of Fig. 7, B and C, except that IgGs were prebound to untreated mitochondria. The values given are the means of five or more experiments (with a standard error of the means <3%). (B) Both MOM72 and MOM19 direct AAC to the GIP site. Mitochondria (10 #g of protein) were preincubated with IgGs (60 #g) as indicated (S611ner et al., 1990). Mitochondria were reisolated and incubated with reticulocyte lysate containing radiolabeled precursor of AAC in the presence of 1 #M valinomycin for 20 min at 25°C as described in Materials and Methods and treated with proteinase K (10 #g/ml) (Planner and Neupert, 1987; Planner et al., 1987b).
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Figure L Identification of MOM72 in yeast mitochondrial outer membranes. Mitochondrial outer membranes were prepared as described in Materials and Methods. Lane I shows the protein pattern of the outer membrane (30/Lg) after separation by SDS-PAGE and staining with Coomassie blue R-250. For lane 2, 30 ~g of outer membrane proteins were separated by SDS-PAGE, electrotransferred to nitrocellulose paper, and immunodecorated with antibodies against yeast MOM72.
Figure L Identification of MOM72 in yeast mitochondrial outer membranes. Mitochondrial outer membranes were prepared as described in Materials and Methods. Lane I shows the protein pattern of the outer membrane (30/Lg) after separation by SDS-PAGE and staining with Coomassie blue R-250. For lane 2, 30 ~g of outer membrane proteins were separated by SDS-PAGE, electrotransferred to nitrocellulose paper, and immunodecorated with antibodies against yeast MOM72.
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Supplementary resource (1)

N. crassa MOM72 mRNA for 72K mitochondrial outer membrane protein
Nucleotide Sequence
July 1990
Michael A Kiebler
... The effect of Δtom70Δtom71 on MP import was consistent, albeit less pronounced, with Tom70 cd overexpression ( Figure 3D and E; Figure 3-figure supplement 1B and C). One potential explanation for the modest effect in double mutant is that given to the functional redundancy between Tom20 and Tom70 (Steger et al., 1990;Young et al., 2003), Tom20 receptors in Δtom70Δtom71 cells could instead mediate preprotein import, whereas cytosolic Tom70 cd may have a dominant inhibitory effect on preprotein import by reducing association between preproteins and mitochondrial outer membrane or TOM complexes (Brix et al., 1997;Brix et al., 2000;Schmidt et al., 2011). Together, these data suggest that increased expression and receptor-dependent import of certain mitochondrial preproteins under Snf1 activation might indirectly restrict the import of MPs. ...
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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 Saccharomyces cerevisiae 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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... Upon formation, MDCs selectively sequester Tom70, a receptor of the mitochondrial outer membrane translocase (TOM) complex Steger et al., 1990), as well as other OMM proteins, diminishing the levels of these proteins in mitochondria (Hughes et al., 2016;Schuler et al., 2021). In contrast, mitochondrial intermembrane space (IMS) proteins, matrix proteins, and mitochondrial inner membrane (IMM) proteins are excluded from MDCs (Hughes et al., 2016;Wilson et al., 2023a, Preprint). ...
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Cells utilize multiple mechanisms to maintain mitochondrial homeostasis. We recently characterized a pathway that remodels mitochondria in response to metabolic alterations and protein overload stress. This remodeling occurs via the formation of large membranous structures from the mitochondrial outer membrane called mitochondrial-derived compartments (MDCs), which are eventually released from mitochondria and degraded. Here, we conducted a microscopy-based screen in budding yeast to identify factors that regulate MDC formation. We found that two phospholipids, cardiolipin (CL) and phosphatidylethanolamine (PE), differentially regulate MDC biogenesis. CL depletion impairs MDC biogenesis, whereas blocking mitochondrial PE production leads to constitutive MDC formation. Additionally, in response to metabolic MDC activators, cellular and mitochondrial PE declines, and overexpressing mitochondrial PE synthesis enzymes suppress MDC biogenesis. Altogether, our data indicate a requirement for CL in MDC biogenesis and suggest that PE depletion may stimulate MDC formation downstream of MDC-inducing metabolic stress.
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... To this end, we purified mitochondria from yeast strains that lacked Tom70 or contained either yeast Tom70 or HsTom70 and used these in in vitro import experiments. Therefore, we radiolabeled the ATP/ADP carrier of Neurospora crassa (Aac), a well-characterized model protein for Tom70 binding (Steger et al., 1990). Upon incubation with WT mitochondria, this carrier reached a protease-resistant location indicating its translocation across the outer membrane ( Fig. 3 C). ...
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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses high amounts of the protein Orf9b to target the mitochondrial outer membrane protein Tom70. Tom70 serves as an import receptor for mitochondrial precursors and, independently of this function, is critical for the cellular antiviral response. Previous studies suggested that Orf9b interferes with Tom70-mediated antiviral signaling, but its implication for mitochondrial biogenesis is unknown. In this study, we expressed Orf9b in human HEK293 cells and observed an Orf9b-mediated depletion of mitochondrial proteins, particularly in respiring cells. To exclude that the observed depletion was caused by the antiviral response, we generated a yeast system in which the function of human Tom70 could be recapitulated. Upon expression of Orf9b in these cells, we again observed a specific decline of a subset of mitochondrial proteins and a general reduction of mitochondrial volume. Thus, the SARS-CoV-2 virus is able to modulate the mitochondrial proteome by a direct effect of Orf9b on mitochondrial Tom70-dependent protein import.
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View
Metabolic regulation of misfolded protein import into mitochondria
Preprint
Full-text available
  • Jun 2023
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 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 misfolded proteins 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 misfolded proteins such as those associated with neurodegenerative diseases.
View
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View
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