Increased mitochondrial protein import and cardiolipin remodelling upon early mtUPR

Author summary Mitochondria are essential organelles and involved in numerous important functions like ATP production, biosynthesis of metabolites and co-factors or regulation of programmed cell death. To fulfill this plethora of different tasks, mitochondria require an extensive proteome, which is build by import of nuclear-encoded precursor proteins from the cytosol. Mitochondrial defects can cause a variety of severe human disorders that often affect tissues with high energy demand e.g. heart, muscle or brain. However, protective mechanisms exist that are triggered upon mitochondrial dysfunction: Imbalances in mitochondrial proteostasis are sensed by the cell and elicit a nuclear transcriptional response, termed mitochondrial unfolded protein response (mtUPR). Transcription of mitochondrial chaperones and proteases is increased to counteract mitochondrial dysfunctions. In this study, we investigated if mtUPR progresses in different temporal stages and how protein import is affected upon mtUPR. We discover that mtUPR is subdivided into an early phase, in which protein import increases and a late phase, in which it declines. Stimulation of protein import is accompanied by an increase and remodelling of the mitochondrial signature lipid cardiolipin. Our work establishes a novel model how cells respond to dysfunctional mitochondria, in which cardiolipin and protein import are modulated as first protective measures. Mitochondrial defects can cause a variety of human diseases and protective mechanisms exist to maintain mitochondrial functionality. Imbalances in mitochondrial proteostasis trigger a transcriptional program, termed mitochondrial unfolded protein response (mtUPR). However, the temporal sequence of events in mtUPR is unclear and the consequences on mitochondrial protein import are controversial. Here, we have quantitatively analyzed all main import pathways into mitochondria after different time spans of mtUPR induction. Kinetic analyses reveal that protein import into all mitochondrial subcompartments strongly increases early upon mtUPR and that this is accompanied by rapid remodelling of the mitochondrial signature lipid cardiolipin. Genetic inactivation of cardiolipin synthesis precluded stimulation of protein import and compromised cellular fitness. At late stages of mtUPR upon sustained stress, mitochondrial protein import efficiency declined. Our work clarifies the enigma of protein import upon mtUPR and identifies sequential mtUPR stages, i