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.2018 Aug 16;71(4):567-580.e4.
doi: 10.1016/j.molcel.2018.06.039.

ACP Acylation Is an Acetyl-CoA-Dependent Modification Required for Electron Transport Chain Assembly

Affiliations

ACP Acylation Is an Acetyl-CoA-Dependent Modification Required for Electron Transport Chain Assembly

Jonathan G Van Vranken et al. Mol Cell..

Abstract

The electron transport chain (ETC) is an important participant in cellular energy conversion, but its biogenesis presents the cell with numerous challenges. To address these complexities, the cell utilizes ETC assembly factors, which include the LYR protein family. Each member of this family interacts with the mitochondrial acyl carrier protein (ACP), the scaffold protein upon which the mitochondrial fatty acid synthesis (mtFAS) pathway builds fatty acyl chains from acetyl-CoA. We demonstrate that the acylated form of ACP is an acetyl-CoA-dependent allosteric activator of the LYR protein family used to stimulate ETC biogenesis. By tuning ETC assembly to the abundance of acetyl-CoA, which is the major fuel of the TCA cycle and ETC, this system could provide an elegant mechanism for coordinating the assembly of ETC complexes with one another and with substrate availability.

Keywords: acetyl-CoA; assembly factors; electron transport chain; mitochondria; mitochondrial fatty acid synthesis; respiration.

Copyright © 2018 Elsevier Inc. All rights reserved.

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Conflict of interest statement

Declaration of interests

Nothing to declare.

Figures

Figure 1
Figure 1. LYR proteins require ACP to execute their functions as complex-specific ETC assembly factors
A. A structural model depicting the interaction between ACP (blue) and ISD11 (LYR; green). The 4′-PP and a bound acyl chain are colored by element. PDB ID: 5USR (Cory et al., 2017). B. Mitochondrial lysates generated from the indicated strains harboring the indicated plasmids were separated by BN-PAGE and immunoblotted with the indicated antibodies (EV – empty vector). C-F. Acp1HA/FLAG or Acp1S82A-HA/FLAG were immunoprecipitated from cells expressing Sdh7V5 (C,D) or Mzm1V5 (E,F). The inputs and eluates were immunoblotted with the indicated antibodies. G,H. Acp1HA/FLAG was immunprecipitated from WT,mct1Δ, andoar1Δ cells expressing Mzm1V5 (G) or Sdh7V5 (H). The inputs and eluates were immunoblotted with the indicated antibodies. Inputs represent 5% of the total lysate.
Figure 2
Figure 2. Yeast mitochondria accumulate an acylated form of ACP
A. Depiction of various ACP species. B. Mitochondrial lysates expressing Acp1HA/FLAG were separated by SDS-PAGE in the presence of 10mM NEM and immunoblotted for HA. * indicates non-specific bands. C. The fraction of acylated Acp1HA/FLAG from (B) was quantified using densitometry. N≥3. Error bars represent standard deviation. ****p<0.0001. D. WT oroar1Δ mitochondrial lysates expressing Acp1HA/FLAG or Acp1S82A-HA/FLAG were separated by SDS-PAGE in the presence of 10mM NEM or 1% βME and immunoblotted for HA. E. Acp1HA/FLAG or Sdh6V5 (expressed from theGPD1 promoter) were immunoprecipitated from mitochondrial lysates expressing the indicated proteins, separated by SDS-PAGE in the presence of 10 mM NEM, and immunoblotted for HA (Acp1HA/FLAG) and V5 (Sdh6V5). Inputs represent 5% of the total lysate.
Figure 3
Figure 3. ACP acylation is required for ETC biogenesis
A. Ten-fold serial dilutions of the indicated strains harboring the indicated plasmids were plated on synthetic medium with either glucose or glycerol and grown for 2-3 days. B. Mitochondrial lysates generated from the indicated strains harboring the indicated plasmids were separated by BN-PAGE (upper panel) or SDS-PAGE (lower panel) and immunoblotted with the indicated antibodies. C. Mitochondrial lysates expressing Acp1HA/FLAG were separated by SDS-PAGE in the presence of 10mM NEM or 1% βME and immunoblotted for HA. * indicates non-specific bands.
Figure 4
Figure 4. ACP acylation is sensitive to perturbation in mitochondrial acetyl-CoA
A. Mitochondrial lysates of the indicated strains were separated by BN-PAGE (upper panel) or SDS-PAGE (lower panel) and immunoblotted with the indicated antibodies. B,D. Mitochondrial lysates generated frommpc1Δ (B) orlip5Δ (D) strains expressing Acp1HA/FLAG were separated by SDS-PAGE in the presence of 10mM NEM and immunoblotted for HA. * indicates non-specific bands. C,E. The fraction of acylated Acp1HA/FLAG from B and D was quantified using densitometry formpc1Δ (C) andlip5Δ (E) cells. N≥3. Error bars represent standard deviation. ****p<0.0001.
Figure 5
Figure 5. Expression of mito-FAA2 restores ACP acylation, OXPHOS assembly and growth inmpc1Δ cells
A. Ten-fold serial dilutions of the indicated strains harboring the indicated plasmids were plated on synthetic medium with either glucose or glycerol and grown for 2-3 days. B. Mitochondrial lysates generated from the indicated strains harboring the indicated plasmids were separated by BN-PAGE (upper panel) or SDS-PAGE (lower panel) and immunoblotted with the indicated antibodies. C. Mitochondrial lysates expressing Acp1HA/FLAG were separated by SDS-PAGE in the presence of 10mM NEM or 1% βME and immunoblotted for HA. * indicates non-specific bands.
Figure 6
Figure 6. Assessing the phenotypic differences betweenmct1Δ andmpc1Δ cells
A. Proteomics experimental overview. B. Heat map and dendrogram plot demonstrating that all biological replicates cluster as expected. C,D. Volcano plots highlighting significantly (p≤0.01) up-(fold change ≥ 1.25) and down-(fold change ≤ 0.75) regulated proteins comparing WT andmct1Δ (C) and WT andmpc1Δ (D). Proteins involved in oxidative phosphorylation are emphasized in red. E. Heat map showing the fold change (log2) of the indicated proteins involved in acetyl-CoA generation.
Figure 7
Figure 7. Acetyl-CoA limitation prevents LYR-mediated ETC assembly and activation
A,B. Heat map showing the fold change (log2) of the indicated proteins involved in mitochondrial respiration (A) and mitochondrial protein synthesis (B) inmct1Δ andmpc1Δ cells compared to WT. ^LYR target proteins. Proteins in red are encoded by the mitochondrial genome. C. Relative abundance of the indicated proteins in WT,mct1Δ, andmpc1Δ cells (N=3). Error bars represent the standard deviation. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.00005. D,E. Mitochondrial lysates isolated from cells expressing either Qcr7HA (D) or Qcr2HA (E) were separated by BN-PAGE (upper panel) or SDS-PAGE (lower panel) and immunoblotted with the indicated antibodies.
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