Fig 3. Direct depalmitoylation with PPT1 validates putative synaptic substrates.

(A) (i) Schematic of PPT1-mediated depalmitoylation assay, in which solubilized synaptosomes are treated with recombinant mPPT1 or GFP generated from HEK293T cells during Acyl RAC (Fig 1Bii). (ii) Venn diagram of PPT1-mediated depalmitoylation assay (n = 3 biological replicates, n = 3 technical replicates) shows 975 proteins common to replicates. (B) Comparison of PPT1-mediated depalmitoylation proteins to putative substrates results in 138 validated PPT1 substrates. (C) The 138 combined high- (red) and medium- (green) confidence validated proteins fall into curated UniProt functional and subcellular location groupings (UniProt gene names are indicated). (D) Western blots of subcellular fractionation samples prepared from whole brains of WT and PPT1 KO mice. (E) Xenopus laevis oocytes were injected with WT GluA1 (GluA1.WT) or C323A mutant GluA1 cRNA (100 pg) alone or with auxiliary factor TARPγ8 (100 pg). Glutamate (5 μm)-evoked current with cyclothiazide (50 μM) was measured by 2-electrode voltage-clamp recording (n = 4; *** p < 0.001). GluA1.WT (2 ng) or GluA1.C323 (2 ng) were injected at higher concentrations to test spontaneous channel formation in the absence of auxiliary factors and glutamate (1 mM)-evoked current with cyclothiazide (50 μm) was recorded (n = 6; *** p < 0.001). Data are presented as mean ± SEM (S1 Data). (F) IPA displays enrichment of high- and medium-confidence substrates for synaptic long-term depression, synaptogenesis signaling, GABA receptor signaling, endocannabinoid neural synapses, and CREB signaling in neurons. Acyl RAC, Acyl Resin-Assisted Capture; IPA, Ingenuity Pathway Analysis; KO, knockout; PPT1, palmitoyl protein thioesterase 1; WT, wild-type.