Defining the ATPome reveals cross-optimization of metabolic pathways.

Disrupted energy metabolism drives cell dysfunction and disease, but approaches to increase or preserve ATP are lacking. To generate a comprehensive metabolic map of genes and pathways that regulate cellular ATP-the ATPome-we conducted a genome-wide CRISPR interference/activation screen integrated with an ATP biosensor. We show that ATP level is modulated by distinct mechanisms that promote energy production or inhibit consumption. In our system HK2 is the greatest ATP consumer, indicating energy failure may not be a general deficiency in producing ATP, but rather failure to recoup the ATP cost of glycolysis and diversion of glucose metabolites to the pentose phosphate pathway. We identify systems-level reciprocal inhibition between the HIF1 pathway and mitochondria; glycolysis-promoting enzymes inhibit respiration even when there is no glycolytic ATP production, and vice versa. Consequently, suppressing alternative metabolism modes paradoxically increases energy levels under substrate restriction. This work reveals mechanisms of metabolic control, and identifies therapeutic targets to correct energy failure.

A high-throughput screen of real-time ATP levels in individual cells reveals mechanisms of energy failure.

Insufficient or dysregulated energy metabolism may underlie diverse inherited and degenerative diseases, cancer, and even aging itself. ATP is the central energy carrier in cells, but critical pathways for regulating ATP levels are not systematically understood. We combined a pooled clustered regularly interspaced short palindromic repeats interference (CRISPRi) library enriched for mitochondrial genes, a fluorescent biosensor, and fluorescence-activated cell sorting (FACS) in a high-throughput genetic screen to assay ATP concentrations in live human cells. We identified genes not known to be involved in energy metabolism. Most mitochondrial ribosomal proteins are essential in maintaining ATP levels under respiratory conditions, and impaired respiration predicts poor growth. We also identified genes for which coenzyme Q10 (CoQ10) supplementation rescued ATP deficits caused by knockdown. These included CoQ10 biosynthetic genes associated with human disease and a subset of genes not linked to CoQ10 biosynthesis, indicating that increasing CoQ10 can preserve ATP in specific genetic contexts. This screening paradigm reveals mechanisms of metabolic control and genetic defects responsive to energy-based therapies.

Endoplasmic Reticulum Transport of Glutathione by Sec61 Is Regulated by Ero1 and Bip.

In the endoplasmic reticulum (ER), Ero1 catalyzes disulfide bond formation and promotes glutathione (GSH) oxidation to GSSG. Since GSSG cannot be reduced in the ER, maintenance of the ER glutathione redox state and levels likely depends on ER glutathione import and GSSG export. We used quantitative GSH and GSSG biosensors to monitor glutathione import into the ER of yeast cells. We found that glutathione enters the ER by facilitated diffusion through the Sec61 protein-conducting channel, while oxidized Bip (Kar2) inhibits transport. Increased ER glutathione import triggers H2O2-dependent Bip oxidation through Ero1 reductive activation, which inhibits glutathione import in a negative regulatory loop. During ER stress, transport is activated by UPR-dependent Ero1 induction, and cytosolic glutathione levels increase. Thus, the ER redox poise is tuned by reciprocal control of glutathione import and Ero1 activation. The ER protein-conducting channel is permeable to small molecules, provided the driving force of a concentration gradient.