Identification of palmitoyl protein thioesterase 1 substrates defines roles for synaptic depalmitoylation Erica L. Gorenberg 1,2 , Helen R. Zhao 1 , Jason Bishai 3 , Vicky Chou 1 , Gregory S. Wirak 1 , TuKiet T. Lam 4,5 , Sreeganga S. Chandra 1, * 1 Departments of Neurology; Neuroscience, Yale University, New Haven, CT 06536 2 Interdepartmental Neuroscience Program, Yale University, New Haven, CT 06510 3 Department of Microbial Pathogenesis, Yale University, New Haven, CT 06510 4 Departments of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520 5 Keck MS & Proteomics Resource, WM Keck Biotechnology Resource Laboratory, New Haven, CT 06510 * To whom correspondence should be addressed to: sreeganga.chandra@yale.edu; 203-785-6172 Author contributions: ELG, HRZ, VC, and GSW conducted experiments. ELG and SSC designed experiments. ELG and JB performed data analysis. TTL conducted mass spectrometry. ELG and SSC wrote paper. All authors read and edited text. Highlights • 10% of palmitoylated proteins are palmitoyl protein thioesterase 1 (PPT1) substrates • Unbiased proteomic approaches identify 9 broad classes of substrates, including synaptic adhesion molecules and endocytic proteins • PPT1 depalmitoylates several transmembrane proteins in their extracellular domains • Depalmitoylation allows for disulfide bond formation in some PPT1 substrates • Protein degradation does not require depalmitoylation by PPT1 • Other palmitoylated Neuronal Ceroid Lipofuscinosis proteins are impacted by deficiency of PPT1, indicating a common disease pathway Keywords palmitoylation, synapse, Palmitoyl protein thioesterase 1, neuronal ceroid lipofuscinoisis, neurodegeneration, synaptic adhesion molecules, synaptic vesicle endocytosis, AMPA receptor, synaptic cleft was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which this version posted May 3, 2020. ; https://doi.org/10.1101/2020.05.02.074302 doi: bioRxiv preprint