The mitochondrial Hsp70 controls the assembly of the F1FO-ATP synthase

Abstract

The mitochondrial F₁F_O-ATP synthase produces the bulk of cellular ATP. The soluble F₁ domain contains the catalytic head that is linked via the central stalk and the peripheral stalk to the membrane embedded rotor of the F_O domain. The assembly of the F₁ domain and its linkage to the peripheral stalk is poorly understood. Here we show a dual function of the mitochondrial Hsp70 (mtHsp70) in the formation of the ATP synthase. First, it cooperates with the assembly factors Atp11 and Atp12 to form the F₁ domain of the ATP synthase. Second, the chaperone transfers Atp5 into the assembly line to link the catalytic head with the peripheral stalk. Inactivation of mtHsp70 leads to integration of assembly-defective Atp5 variants into the mature complex, reflecting a quality control function of the chaperone. Thus, mtHsp70 acts as an assembly and quality control factor in the biogenesis of the F₁F_O-ATP synthase.

Fig. 1. MtHsp70 binds to subunits of the ATP synthase.

a Wild-type (WT) and ρ⁰ mitochondria were analysed by SILAC labelling and mass spectrometry. Depicted are the mean log₂ value of the fold changes of protein intensities in ρ⁰ versus WT mitochondria of four replicates, plotted against their statistical significance (−log₁₀ of p values). b mtHsp70_His and mtHsp70_His ρ⁰ mitochondria from SILAC-labelled cells were subjected to affinity purification via Ni-NTA agarose in the absence of ATP followed by mass spectrometry. Depicted are the mean log₂ values of fold changes of mtHsp70_His -bound proteins in ρ⁰ versus WT background mitochondria of four replicates, plotted against their statistical significance (−log₁₀ of p values). The signals for Lys2 and Ccp1 had unusually high values, indicating an unspecific interaction, and were removed from the plot. c Differentially SILAC-labelled WT and ρ⁰ mitochondrial lysates were subjected to affinity purification via Mge1_His-coated Ni-NTA agarose in the absence of ATP followed by mass spectrometry. Depicted are the mean log₂ values of fold changes of Mge1_His-bound proteins in ρ⁰ versus WT mitochondria of four replicates, plotted against their statistical significance (-log₁₀ of p-values). d MtHsp70_His and mtHsp70_His ρ⁰ mitochondria were subjected to affinity purification via Ni-NTA agarose in the absence of ATP followed by SDS-PAGE and immunodetection. Load: (1% right panel, 2.5% left panel), elution 100%. e Left panel, WT and ρ⁰ mitochondria were subjected to affinity purification via Mge1_His in the absence of ATP followed by SDS-PAGE and immunodetection. Load, 1% (Atp1, Atp2, Atp5, mtHsp70, Mdh1) and 2.5% (other proteins), elution 100%. Right panel, Quantification of the co-purified Atp1, Atp2 and Atp5 with Mge1_His. Depicted are mean values ± SEM of 5 independent experiments. The fractions of Atp1, Atp2 or Atp5 co-eluted with Mge1_His in WT mitochondria were set to 100% (control). Subsequently, the amount of Atp1, Atp2 or Atp5 co-eluted with Mge1_His in ρ⁰ was determined and the co-purification efficiency was correlated to WT. Source data are provided as a Source Data file. f WT and Atp1_His mitochondria were subjected to affinity purification via Ni-NTA agarose followed by immunoblotting. Load: 1%, elution: 100%.

Fig. 2. MtHsp70 interacts with unassembled subunits of the ATP synthase.

a Wild-type (WT) and atp3∆ mitochondria were analysed by blue native electrophoresis and immunodetection with the indicated antisera. V_D, dimer of the ATP synthase, V_M, monomer of the ATP synthase, F₁, F₁ domain. b Upper panel, WT and atp3∆ mitochondria were subjected to affinity purification via Mge1_His coated Ni-NTA agarose. Proteins were analysed by SDS-PAGE and immunodetection with the indicated antisera. Load: 1%, elution: 100%. Lower panels, Quantification of the co-purified Atp1, Atp2 and Atp5 with Mge1_His. Depicted are mean values ± SEM of 4 independent experiments. The amount of Atp1, Atp2 or Atp5 co-eluted with mtHsp70 in WT mitochondria were set to 100% (control). Source data are provided as a Source Data file. c Upper panel, WT and atp4∆, atp5∆, atp7∆ and atp14∆ mitochondria were subjected to affinity purification via Mge1_His-coated Ni-NTA agarose. Proteins were analysed by SDS-PAGE and immunodetection with the indicated antisera. Load: 1%, elution: 100%. Lower panel, Quantification of the co-purified Atp1, Atp2 and Atp5 with Mge1_His. Depicted are mean values ± SEM of 4 independent experiments. The amount of Atp1, Atp2 or Atp5 co-eluted with mtHsp70 in WT mitochondria were set to 100% (control). Source data are provided as a Source Data file. d WT and atp4∆, atp5∆, atp7∆ and atp14∆ mitochondria were analysed by blue native electrophoresis and immunodetection with the indicated antisera. V_D, dimer of the ATP synthase, V_M, monomer of the ATP synthase, F₁, F₁ domain.

Fig. 3. mtHsp70 facilitates the assembly of the F₁ domain.

a Upper panels, [³⁵S]Atp1 or [³⁵S]Atp2 were imported into WT and ssc1-62 mitochondria from non-stressed cells for the indicated time points. In control reactions, the membrane potential (Δψ) was depleted. Samples were analysed via SDS-PAGE and autoradiography. p, precursor, m, mature band. Lower panels: Quantification of the imported Atp1 or Atp2. Depicted are mean values ± SEM of 3 independent experiments for Atp1 and mean values ± SEM of 4 independent experiments for Atp2. The amounts of mature Atp1 or Atp2 in WT mitochondria in the longest import time point were set to 100% (control). Source data are provided as a Source Data file. b Upper panels, [³⁵S]Atp1 or [³⁵S]Atp2 were imported into WT and ssc1-62 mitochondria from non-stressed cells for the indicated time points. In control reactions, the membrane potential (Δψ) was depleted. Samples were analysed via blue native electrophoresis and autoradiography. V_D, dimer of the ATP synthase, V_M, monomer of the ATP synthase, F₁, F₁ domain. Lower panels: Quantification of Atp1 or Atp2 assembled into the F₁ domain. Depicted are mean values ± SEM of 3 independent experiments. The amounts of assembled Atp1 or Atp2 in WT mitochondria at the longest import time point were set to 100% (control). Source data are provided as a Source Data file. c, d Wild-type (WT) and ssc1-62 mitochondria were isolated from cells that were shifted to non-permissive growth conditions. Mitochondrial proteins were analysed by blue native electrophoresis followed by either immunodetection with the indicated antisera (c) or by in-gel activity stain (d). V_D, dimer of the ATP synthase, V_M, monomer of the ATP synthase, F₁, F₁ domain. c Right panel, The amounts of the dimer, monomer and F₁ domain in WT and ssc1-62 was determined with the indicated antisera. The formation of the forms in WT was set to 100% (control). Depicted are mean values ± SEM of 3 independent experiments. Source data are provided as a Source Data file.

Fig. 4. mtHsp70 cooperates with Atp11 and Atp12 in the assembly of the F₁ domain.

a Wild-type (WT) and ssc1-62 mitochondria from non-stressed cells were subjected to affinity purification via Mge1_His-coated Ni-NTA agarose. Proteins were analysed by SDS-PAGE and immunodetection with the indicated antisera. The blots from the left and right panels were from two independent experiments. Load: 1% (left panel) or 3% (right panel), elution: 100%. b WT, Atp11_His, Atp11_His ssc1-62, Atp12_His, and Atp12_His ssc1-62 were shifted to non-permissive growth conditions. Isolated mitochondria were subjected to affinity purification via Ni-NTA agarose. Proteins were analysed by SDS-PAGE and immunodetection with the indicated antisera. Load: 1%, elution: 100%. c Lysed WT, Atp11_His and Atp12_His mitochondria were pre-incubated with or without ATP and subsequently subjected to affinity purification via Ni-NTA agarose. Proteins were analysed by SDS-PAGE and immunodetection with the indicated antisera. Load: 0.2%, elution: 100%. d Serial dilutions of the indicated yeast strains were grown on full medium containing either glycerol or glucose as carbon source grown at 23 °C. e WT, fmc1∆, ssc1-62, and fmc1∆ ssc1-62 mitochondria from non-stressed cells were analysed by blue native electrophoresis and immunodetection with the indicated antisera.

Fig. 5. mtHsp70 cooperates with INAC to link the F₁ domain to the peripheral stalk.

a Left panel, Wild-type (WT), ina17∆ and ina22∆ mitochondria were subjected to affinity purification via Mge1_His coated Ni-NTA agarose. Proteins were analysed by SDS-PAGE and immunodetection with the indicated antisera. Load: 1%, elution: 100%. Right panel, Quantification of Atp1, Atp2 and Atp5 co-purified with Mge1_His. Depicted are mean values ± SEM of 5 independent experiments. The amount of Atp1, Atp2 or Atp5 co-eluted with mtHsp70 in WT mitochondria was set to 100% (control). Source data are provided as a Source Data file. b Lysed WT, Ina17_His and Ina22_His mitochondria were pre-incubated with or without ATP and subsequently subjected to affinity purification via Ni-NTA agarose. Proteins were analysed by SDS-PAGE and immunodetection with the indicated antisera. Load: 0.5%, elution: 100%. c WT, Ina17_His and Ina22_His mitochondria were subjected to affinity purification via Ni-NTA agarose. Where indicated, bound proteins were eluted by incubation with ATP. Proteins were analysed by SDS-PAGE and immunodetection with the indicated antisera. Load: 1%, elution: 100%. d Serial dilutions of the indicated yeast strains were grown on full medium containing either glycerol or glucose as carbon source grown at 23 °C. e WT, ina17∆, ssc1-62, and ina17∆ ssc1-62 mitochondria from non-stressed cells were analysed by blue native electrophoresis and immunodetection with the indicated antisera. Asterisk marks unknown Atp1 and Atp2-containing protein complex in ina17∆ mitochondria.

Fig. 6. mtHsp70 controls the assembly of Atp5.

a [³⁵S]Atp4, [³⁵S]Atp5, or [³⁵S]Atp14 were imported into wild-type (WT) and ssc1-62 mitochondria from non-stressed cells for the indicated time period. Non-imported precursor proteins were removed by proteinase K (Prot. K). In control reactions, the membrane potential (Δψ) was depleted. Samples were analysed via SDS-PAGE and autoradiography. p, precursor, m, mature band. b Upper panels, [³⁵S]Atp5, [³⁵S]Atp4 or [³⁵S]Atp14 were imported into WT and ssc1-62 mitochondria from non-stressed cells for the indicated time periods. In control reactions, the membrane potential (Δψ) was depleted. Samples were analysed via blue native electrophoresis and autoradiography. V_D, dimer of the ATP synthase, V_M, monomer of the ATP synthase. Lower panels: Quantification of Atp5, Atp4 or Atp14 assembled into the dimer of the ATP synthase. Depicted are mean values ± SEM of 3 independent experiments for Atp4 and Atp14, and mean values ± SEM of 4 independent experiments for Atp5. The amounts of Atp5, Atp4 or Atp14 assembled into the ATP synthase dimer in WT mitochondria at the longest import time point were set to 100% (control). Source data are provided as a Source Data file. c, d [³⁵S]Atp5 (c) or [³⁵S]Atp5^G183A (d) were imported into WT and ssc1-62 mitochondria isolated from non-stressed cells for the indicated time periods. In control reactions, the membrane potential (Δψ) was depleted. Samples were analysed via blue native electrophoresis and autoradiography. V_D, dimer of the ATP synthase, V_M, monomer of the ATP synthase. d Right panel: quantification of Atp5^G183A assembled into the monomer and dimer of the ATP synthase. Depicted are mean values ± SEM of 4 independent experiments. The amounts of assembled Atp5^G183A in WT mitochondria at the longest import time point were set to 100% (control). Source data are provided as a Source Data file.

Fig. 7. mtHsp70 and Pim1 promotes degradation of unassembled Atp5.

a Wild-type (WT) and ssc1-62 cells with or without overexpression of either Atp5 or Atp5^G183A were grown under permissive conditions and subjected to affinity purification via Mge1_His coated Ni-NTA agarose. Proteins were analysed by SDS-PAGE and immunodetection with the indicated antisera. Load: 1%, elution: 100%. b Serial dilutions of the indicated yeast strains were grown on selective medium containing either glucose or galactose as carbon source grown at 33 °C. The overexpression of Atp5 and Atp5^G183A is induced upon growth on galactose. c Mitochondria isolated from non-stressed WT and ssc1-62 cells expressing HA-tagged Atp5 or Atp5^G183A were lysed under denaturing conditions and proteins were analysed with the indicated antisera. d Total cell extracts from WT, pim1∆, or yta12∆ strains expressing HA-tagged Atp5 or Atp5^G183A were lysed under denaturing conditions and proteins were analysed with the indicated antisera. e Hypothetical model of the role of mtHsp70 in the formation of the ATP synthase. MtHsp70 promotes import of ATP synthase subunits into the mitochondrial matrix. The chaperone cooperates with Atp11 and Atp12 to assemble Atp2 and Atp1 subunits into the F₁ domain. MtHsp70 delivers Atp5 to the assembly line to link the peripheral stalk and the F₁ domain. Unassembled Atp5 can be degraded by Pim1.