Genetically encoded manipulation of ATP/ADP ratio in human cells uncovers proteomic and physiological signatures of energy stress

The ability of cells to power energy-demanding processes depends on maintaining the ATP hydrolysis reaction a billion-fold away from equilibrium. Cells respond to alterations in the energy state by sensing changes in the ratio of ATP, ADP, AMP, and inorganic phosphate levels. A key barrier to understanding how this happens is a lack of tools for direct manipulation of the energy state in living cells. Here, we introduce ATPGobble-a genetically encoded tool that hydrolyzes ATP in vivo. ATPGobble increases the metabolic rate, decreases [ATP]/[ADP] and [ATP]/[AMP] ratios, and activates AMPK in human cells. We performed a systematic analysis of proteome and phosphoproteome changes caused by ATPGobble, and found that it remodels cytoskeleton, cell cycle, and translation machinery. Our results establish ATPGobble as a powerful new tool for dissecting the regulatory roles of energy state in living cells.