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, 30 (11), 2101-14

Mitochondria Regulate Autophagy by Conserved Signalling Pathways

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Mitochondria Regulate Autophagy by Conserved Signalling Pathways

Martin Graef et al. EMBO J.

Abstract

Autophagy is a conserved degradative process that is crucial for cellular homeostasis and cellular quality control via the selective removal of subcellular structures such as mitochondria. We demonstrate that a regulatory link exists between mitochondrial function and autophagy in Saccharomyces cerevisiae. During amino-acid starvation, the autophagic response consists of two independent regulatory arms-autophagy gene induction and autophagic flux-and our analysis indicates that mitochondrial respiratory deficiency severely compromises both. We show that the evolutionarily conserved protein kinases Atg1, target of rapamycin kinase complex I, and protein kinase A (PKA) regulate autophagic flux, whereas autophagy gene induction depends solely on PKA. Within this regulatory network, mitochondrial respiratory deficiency suppresses autophagic flux, autophagy gene induction, and recruitment of the Atg1-Atg13 kinase complex to the pre-autophagosomal structure by stimulating PKA activity. Our findings indicate an interrelation of two common risk factors-mitochondrial dysfunction and autophagy inhibition-for ageing, cancerogenesis, and neurodegeneration.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Mitochondrial respiratory deficiency impairs ATG8 induction and autophagic flux. (A) Wild-type and rho0 cells harbouring prATG8-GFP-ATG8 (upper panels) or prATG8-GFP (lower panels) were exposed to amino-acid or nitrogen starvation (-N) medium supplemented with indicated carbon sources. Cells were analysed at indicated time points by whole cell extraction and western blot analysis using α-GFP and α-Cdc11 antibodies. Quantification of ATG8 induction is shown in the lower left panel. Total GFP signals (GFP-Atg8 and free GFP) were quantified and normalized to Cdc11 signals. Normalized values at 0 h were set as one and relative changes are shown after 6 h starvation. Quantification of autophagic flux is shown in the lower right panel as ratio of free GFP to total GFP signals (GFP-Atg8 and free GFP) after 6 h starvation. The means and s.d. of four (n=4) independent experiments are indicated. (B) Fluorescence microscopical analysis. Wild-type, rho0, and Δatg7 cells expressing prATG8-GFP-ATG8 were grown as described in (A) and exposed to amino-acid (left panel, galactose) or nitrogen starvation (right panel, glucose (-N)) for 6 h. Vacuoles were visualized by over night FM4-64 (1 μM) staining (red). Arrowhead indicates a punctate GFP-Atg8 structure. Transmission and fluorescence light microscopy images were superimposed to visualize cellular boundaries. Cellular localization of GFP signal was analysed in at least 150 cells (n⩾150) for each strain and condition. Scale bars represent 1.5 μm.
Figure 2
Figure 2
Autophagic response in cells with compromised respiratory chain complex III, IV, or V activity during amino-acid starvation. Wild-type, rho0, Δatg7 cells and mutants that are selectively inhibited in the biogenesis of respiratory chain complex III (CIII: Δcbs1), complex IV (CIV: Δmss51, Δpet111, Δpet122), or the F1Fo-ATP synthase (complex V; CV: Δatp10) were exposed to amino-acid starvation medium supplemented with acetate (A) or galactose (B). When indicated, wild-type cells were exposed to antimycin A (AA) or oligomycin (O) during the amino-acid starvation period. All strains expressed prATG8-GFP-ATG8 (upper panels) or prATG8-GFP (lower panels). Samples were analysed as described in Figure 1A. The means and s.d. of five (n=5) independent experiments are indicated.
Figure 3
Figure 3
ATP level and mitochondrial membrane potential during amino-acid starvation. (A, B) Wild-type, rho0, and Δatg7 cells were exposed to amino-acid starvation medium supplemented with acetate (A) or galactose (B). When indicated, wild-type cells were exposed to antimycin A (AA) or oligomycin (O) during the amino-acid starvation period. ATP and protein from total cells (upper panels) or isolated mitochondria (lower panels) were determined at indicated time points. (C) Wild-type and Δatg7 cells were exposed to amino-acid starvation medium supplemented with acetate or galactose for 3 h. Wild-type cells were exposed to antimycin A (AA), oligomycin (O), or CCCP (50 μM) during the amino-acid starvation period when indicated. Cells were treated with the mitochondrial membrane potential-dependent dye DiOC6(3) and examined by fluorescence microscopy. Average pixel intensities for at least 10 mitochondrial tubules (n=10) in 5 representative cells were determined for each condition. Values for wild-type mitochondria in the presence of acetate were defined as 100% and relative values were calculated accordingly. The means and s.d. are shown.
Figure 4
Figure 4
Role of TORC1 in autophagy regulation under amino-acid starvation. (A) Wild-type, rho0, Δnpr2, and Δnpr2 rho0 cells expressing prATG8-GFP-ATG8 (upper panel) or prATG8-GFP (lower panel) were exposed to amino-acid starvation medium supplemented with galactose in the absence or presence of rapamycin. Samples were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated. (B) Wild-type, rho0, Δnpr2, and Δatg7 cells expressing prNPR1-NPR1-HA were exposed to amino-acid starvation medium supplemented with galactose in the absence (upper panels) or presence (lower panels) of rapamycin. The hyperphosphorylated (Npr1-P) and dephosphorylated (Npr1) forms of Npr1 are indicated. Cells were analysed at indicated time points by whole cell extraction and western blot analysis using α-HA and α-Cdc11 antibodies. (C) Wild-type, rho0, Δnpr2, and Δatg7 cells were exposed to amino-acid starvation medium supplemented with galactose. Additionally, wild-type cells were treated with rapamycin during starvation (left panel). Phosphorylated (Atg13-P) and dephosphorylated (Atg13) Atg13 was monitored at indicated time points by whole cell extraction and western blot analysis using α-Atg13 and α-Cdc11 antibodies.
Figure 5
Figure 5
Mitochondrial function controls autophagy by modulating PKA activity. (A) PKA-dependent regulation of the autophagic response under amino-acid starvation. Wild-type, rho0, pka, and ras2G19V-expressing cells harbouring prATG8-GFP-ATG8 (upper panels) or prATG8-GFP (lower panels) were exposed to amino-acid starvation medium supplemented with galactose. PKA activity in pka was inhibited by addition of 1NM-PP1 (PP1; 1 μg/ml). Samples were analysed as described in Figure 1A; autophagic flux was determined after 3 and 6 h. (B) In vivo activity of PKA. Wild-type, rho0, pka, and ras2G19V-expressing cells harbouring 6xMYC-cki12200(S125/130A) (Cki1) were grown in galactose medium. When indicated, wild-type cells were grown in galactose medium in the presence of antimycin A (AA) or oligomycin (O) for 6 h. PKA-dependent phosphorylation of Cki1 was analysed by whole cell extraction and western blot analysis using a α-Myc antibody (upper panels). Ratio of phosphorylated (Cki1-P) and non-phosphorylated (Cki1) forms of Cki1 relative to wild-type cells (wt=1) (lower panels). (C) PKA inhibition restores autophagic flux, but not ATG8 induction in the presence of mitochondrial dysfunction. Wild-type, rho0, pka, and pka rho0 cells expressing prATG8-GFP-ATG8 were treated as described in (A). Samples were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated in (AC).
Figure 6
Figure 6
Mitochondrial respiratory deficiency impairs PAS recruitment of the Atg1–Atg13 complex under amino-acid starvation. (A) ATG8 induction is independent of the Atg1–Atg13 complex and autophagic flux. Wild-type, rho0, Δatg1, Δatg7, Δatg9, and Δatg11 cells harbouring prATG8-GFP-ATG8 were analysed as described in Figure 1A. The means and s.d. of four (n=4) independent experiments are indicated. (B) Atg1 and Atg13 recruitment to the PAS depends on mitochondrial function. Wild-type and rho0 cells harbouring prATG8-GFP-ATG8 and prATG1-ATG1-mCherry (upper panels) or prATG13-ATG13-mCherry (lower panels) were exposed to amino-acid starvation medium supplemented with galactose for 3 h. Wild-type cells were treated with antimycin A (AA 30′) after 2.5 h of starvation for 30 min or with oligomycin (O) for 3 h of starvation. Arrowheads indicate the position of GFP-Atg8 puncta. Transmission and fluorescence light microscopy images were superimposed to visualize cellular boundaries. Scale bar represents 1.5 μm. (C) Steady-state levels of Atg1- and Atg13-mCherry during amino-acid starvation. Wild-type, rho0, and Δatg7 cells expressing prATG1-ATG1-mCherry (upper panels) or prATG13-ATG13-mCherry (lower panels) were exposed and treated as described in (B) and analysed by whole cell extraction and western blot analysis using α-dsRed and α-Cdc11 antibodies.
Figure 7
Figure 7
Model for the role of mitochondrial function in autophagy regulation under amino-acid starvation. Amino-acid starvation induces the two regulatory arms of the autophagic response, ATG8 induction and autophagic flux. Autophagic flux is regulated in an Atg1, PKA, and TORC1-dependent manner, while ATG8 induction is regulated by PKA. Mitochondrial respiratory deficiency induces PKA activity and, thereby, suppresses both arms of the autophagic response.

Comment in

  • Mitochondria Breathe for Autophagy
    K Okamoto. EMBO J 30 (11), 2095-6. PMID 21629271.
    Proper mitochondrial structure and function rely on fusion/fission cycles and on autophagy-mediated mitochondrial degradation. A paper in this issue of The EMBO Journa

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