Kathayat RS Cao Y Elvira PD Sandoz PA Zaballa ME Springer MZ Drake LE Macleod KF van der Goot FG Dickinson BC 2018 The reversible modification of cysteine residues by thioester formation with palmitate (S-palmitoylation) is an abundant lipid post-translational modification (PTM) in mammalian systems. S-palmitoylation has been observed on mitochondrial proteins, providing an intriguing potential connection between metabolic lipids and mitochondrial regulation. However, it is unknown whether and/or how mitochondrial S-palmitoylation is regulated. Here we report the development of mitoDPPs, targeted fluorescent probes that measure the activity levels of "erasers" of S-palmitoylation, acyl-protein thioesterases (APTs), within mitochondria of live cells. Using mitoDPPs, we discover active S-depalmitoylation in mitochondria, in part mediated by APT1, an S-depalmitoylase previously thought to reside in the cytosol and on the Golgi apparatus. We also find that perturbation of long-chain acyl-CoA cytoplasm and mitochondrial regulatory proteins, respectively, results in selective responses from cytosolic and mitochondrial S-depalmitoylases. Altogether, this work reveals that mitochondrial S-palmitoylation is actively regulated by "eraser" enzymes that respond to alterations in mitochondrial lipid homeostasis. application/pdf https://sonar.ch/global/documents/69510 eng info:eu-repo/semantics/altIdentifier/doi/10.1038/s41467-017-02655-1 info:eu-repo/semantics/altIdentifier/pmid/29362370 info:eu-repo/semantics/openAccess Nature communications. - 2018 A549 Cells Acyl Coenzyme A Fluorescent Dyes HEK293 Cells HeLa Cells Humans Kinetics Lipoylation MCF-7 Cells Microscopy, Confocal Mitochondria Mitochondrial Dynamics RNA Interference Thiolester Hydrolases Active and dynamic mitochondrial S-depalmitoylation revealed by targeted fluorescent probes. http://purl.org/coar/resource_type/c_6501