Identification of palmitoyl protein thioesterase 1 substrates defines roles for synaptic depalmitoylation

SUMMARYWe report here the identification of substrates of the depalmitoylating enzyme PPT1 by quantitative mass spectrometry. We first used a stringent Acyl Resin-Assisted Capture (Acyl RAC) screen in which PPT1 knockout (KO) mouse brain proteins showing increased in vivo palmitoylation are identified as putative PPT1 substrates. We then validated hits by directly depalmitoylating with PPT1 to confirm bona fide substrates. This novel two-step screen identified >100 substrates not previously known to be depalmitoylated by PPT1, including a wide range of channels/transporters, G-proteins, endo/exocytic components, synaptic adhesion molecules, and mitochondrial proteins. Interestingly, many sites of depalmitoylation on transmembrane proteins were located in extracellular domains facing the synaptic cleft. For this group of substrates, depalmitoylation appears to facilitate disulfide bond formation. Collectively, these diverse substrates may explain the many facets of CLN1 disease caused by the loss of PPT1 function.Highlights10% of palmitoylated proteins are palmitoyl protein thioesterase 1 (PPT1) substratesUnbiased proteomic approaches identify 9 broad classes of substrates, including synaptic adhesion molecules and endocytic proteinsPPT1 depalmitoylates several transmembrane proteins in their extracellular domainsDepalmitoylation allows for disulfide bond formation in some PPT1 substratesProtein degradation does not require depalmitoylation by PPT1Other palmitoylated Neuronal Ceroid Lipofuscinosis proteins are impacted by deficiency of PPT1, indicating a common disease pathway