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. 2014 May 8;7(3):907-17.
doi: 10.1016/j.celrep.2014.03.065. Epub 2014 Apr 24.

A mitochondrial RNAi screen defines cellular bioenergetic determinants and identifies an adenylate kinase as a key regulator of ATP levels

Nathan J Lanning  1 , Brendan D Looyenga  2 , Audra L Kauffman  1 , Natalie M Niemi  1 , Jessica Sudderth  3 , Ralph J DeBerardinis  3 , Jeffrey P MacKeigan  4
Affiliations

A mitochondrial RNAi screen defines cellular bioenergetic determinants and identifies an adenylate kinase as a key regulator of ATP levels

Nathan J Lanning et al. Cell Rep. .

Abstract

Altered cellular bioenergetics and mitochondrial function are major features of several diseases, including cancer, diabetes, and neurodegenerative disorders. Given this important link to human health, we sought to define proteins within mitochondria that are critical for maintaining homeostatic ATP levels. We screened an RNAi library targeting >1,000 nuclear-encoded genes whose protein products localize to the mitochondria in multiple metabolic conditions in order to examine their effects on cellular ATP levels. We identified a mechanism by which electron transport chain (ETC) perturbation under glycolytic conditions increased ATP production through enhanced glycolytic flux, thereby highlighting the cellular potential for metabolic plasticity. Additionally, we identified a mitochondrial adenylate kinase (AK4) that regulates cellular ATP levels and AMPK signaling and whose expression significantly correlates with glioma patient survival. This study maps the bioenergetic landscape of >1,000 mitochondrial proteins in the context of varied metabolic substrates and begins to link key metabolic genes with clinical outcome.

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Figures

Figure 1
Figure 1. A Sensitized RNAi Screen to Identify Regulators Glycolytic and OXHPOS Bioenergetics
(A) Depiction of the nutrient source strategy utilized in this study. (B) Relative ATP/cell measurements from cells after a four hour treatment with DMSO, iodoacetic acid (IAA, 10 μM), or rotenone (500 nM). Error bars represent SD values. (C) Compliment of metabolic pathways and functions targeted in the RNAi screen. (D) RNAi screen results. Labeled data points indicate siRNAs that increased or decreased ATP levels 25% or more compared to control siRNAs in all four conditions. See also Figure S1.
Figure 2
Figure 2. Mitochondrial Functions Impacting Cellular Bioenergetics
Cytoscape (see Methods section) was used to define mitochondrial functions affecting ATP levels in response to each metabolic condition. Central colored nodes represent individual nutrient sources. Gray nodes radiating from the central node represent all mitochondrial functions associated with siRNAs altering ATP by ≥25%. The distance between each functional node and the central node is proportional to the number of genes associated with that function that increased or decreased ATP by ≥ 25% in that condition. Functions with five or more associated genes altering ATP by ≥ 25% are labeled and include the associated genes, represented as nodes with colored edges. Solid colored edges represent increased ATP levels; dotted edges represent decreased ATP levels. The distance between each gene node and the associated functional node is proportional to the magnitude of ATP level change. See also Table S3 and Figure S2.
Figure 3
Figure 3. Suppression of OXPHOS in Glycolytic Conditions Increases Energy Production
(A) Electron transport chain schematic. Each subunit is colored according to the results of the RNAi screen in ATP/cell standard deviations from control siRNA. (B) Results of the RNAi screen presented as percent change in ATP/cell for each subunit. (C) Percent change in relative ATP/cell was determined compared to DMSO treatment. Measurements were obtained after 4 hours of treatment with ETC complex inhibitors (n=5 for each dose). Error bars represent SD values. *p < 0.05. (D) Relative ATP/cell was measured in cells transfected with control or selected RC I subunit siRNAs and in cells treated for 500 nM rotenone for 4 hours (n=3 for each condition). Error bars represent SD values. **p < 0.01; ***p < 0.001. (E) Glucose-to-lactate conversion in control and selected RC I subunit siRNA transfected HeLa cells. Cells were cultured with medium containing 1,6 13C-labeled glucose, and enrichment of 13C-lactate (m+3) in the extracellular medium was measured after 6 hours (n=3 for each condition). Error bars represent SD values. **p < 0.01; ***p < 0.001.
Figure 4
Figure 4. Mitochondrial Kinome and Phosphatome Impact on ATP Levels
(A) Volcano plot depicting the results of a second RNAi screen targeting the mitochondrial kinome and phosphatome in glucose and pyruvate conditions. (B) Relative ATP/cell was measured in cells transfected with control, AK4, or PPTC7 siRNAs. Error bars represent SD values (n=4 for each siRNA). **p < 0.01. (C) Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse Biosciences XFe96 Extracellular Flux Analyzer. Values were normalized to cell number. Cells were cultured in 10mM glucose, 1mM pyruvate, 1mM glutamine. Error bars represent SD values (n=10 for each siRNA).
Figure 5
Figure 5. AK4 Controls ATP Levels, Proliferation, and Expression Levels Correlate with Glioma Patient Survival
(A), (B) Relative ATP/cell was measured in cells transfected with control or AK4 siRNAs (n=3 for each siRNA). Error bars represent SD values. **p < 0.01. Western blot depicting level of AK4 knockdown. (C) Cell proliferation was measured by xCELLigence RTCA. Cell density measurements were taken every hour for 96 hours (n=6 for each siRNA). Error bars represent SD values. (D) The Rembrandt database was used to perform a Kaplan-Meier survival analysis of glioma patients with differential AK4 expression and copy number.
Figure 6
Figure 6. AK4 Regulates Cellular Energy Charge and AMPK Signaling
(A), ATP levels, ADP/ATP ratio, and phospho-AMPK were measured after 72 hours in the indicated medias (n=6 for each condition). Error bars represent SD values. (B) ADP/ATP ratio was measured in cells transfected with control or AK4 siRNAs (n=3 for each condition). Error bars represent SD values. (C,D) Western blot analyses of AMPK signaling pathway from cells transfected with control or AK4 siRNAs. Blots are representative of three independent experiments. See also Figure S3.
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