Fig 2: Furin and PC7 are both palmitoylated, primarily by ZDHHC5. (A) Diagrams of Furin and PC7 with the transmembrane and cytosolic cysteines shown. Abbreviations: EC, extracellular; PP, propeptide; SP, signal peptide; TM, transmembrane; with “N-” and “-C” referring to the termini. The main palmitoylated cysteines are in purple (C771 for Furin, FurinCS; and C699/C704 for PC7, PC7CCSS), while the rest are shown in pink. (B) Representative blot of Acyl-RAC performed on RPE-1 cells transfected with V5-tagged Furin or PC7 WT or mutants. Hydroxylamine (NH2OH) was used to cleave off acyl chains before capturing their resultant free cysteines on beads. (C) RPE-1 cells were transfected as in B, then labeled for 2 h with 3H-palmitic acid, after which cells were lysed and immunoprecipitated. Statistics: ratio paired two-tailed t test on the original data. (D) Representative blot of Acyl-RAC performed as in B on WT and ΔZDHHC5 HAP1 cells. Endogenous Furin and PC7 are shown. (E) RPE-1 cells were silenced for Control or ZDHHC5 and then transfected with V5-tagged Furin or PC7. They were treated with 3H-palmitic acid as in C. Statistics: ratio paired two-tailed t test on the original data. (F) RPE-1 cells were transfected with V5-tagged WT and mutant Furin and PC7 and then lysed and immunoprecipitated (IP) with anti-ZDHHC5 antibody. Both ZDHHC5 (direct) and V5 Co-IP blots are shown. Statistics: ratio paired two-tailed t test on the original data. *P < 0.05, **P < 0.01.

Fig 3: While ZDHHC5 does not affect processing of other Furin/PC7 substrates, it does affect cleavage in endosomes and lipid rafts. (A) HA-tagged E-cadherin was transfected into RPE-1 cells that were silenced for Control, Furin (F here), PC7, F/PC7, or ZDHHC5. Western blots using HA antibody show both uncleaved (u) and cleaved (c) versions of E-cadherin. Statistics: unpaired two-tailed t test. (B) Representative blot of IGF-1Rβ levels in cells silenced for F/PC7 or ZDHHC5, with or without transfection of Furin-V5. Both uncleaved (u) and cleaved (c) IGF-1Rβ are labeled. (C) The FLAG-tagged late endosome (LE-FLAG) sensor was transfected into RPE-1 cells and the PC-specific inhibitor chloromethyl ketone (CMK) was added for 6 h at 50 μM, after which cells were lysed and subjected to SDS/PAGE, transferred, and probed with an anti-FLAG antibody. (D) PC FLAG-tagged sensors targeting different subcellular compartments (PM for plasma membrane, TGN for trans-Golgi network, and GPI for glycosylphosphatidylinositol-anchored proteins primarily found in lipid rafts) were transfected in cells either silenced for Control or ZDHHC5. Cells were lysed and the levels of the cleaved sensors were measured using Western blots with the anti-FLAG antibody. Statistics: unpaired two-tailed t test. *P < 0.05, **P < 0.01.