Phosphatidylinositol 4-Kinase IIα Is Palmitoylated by Golgi-localized Palmitoyltransferases in Cholesterol-dependent Manner

Phosphatidylinositol 4-kinase II alpha (PI4KII alpha) is predominantly Golgi-localized, and it generates >50% of the phosphatidylinositol 4-phosphate in the Golgi. The lipid kinase activity, Golgi localization, and integral membrane binding of PI4KII alpha and its association with low buoyant density raft domains are critically dependent on palmitoylation of its cysteine-rich (CCPCC177)-C-173 motif and are also highly cholesterol-dependent. Here, we identified the palmitoyl acyltransferases (Asp-His-His-Cys (DHHC) PATs) that palmitoylate PI4KII alpha and show for the first time that palmitoylation is cholesterol-dependent. DHHC3 and DHHC7 PATs, which robustly palmitoylated PI4KII alpha and were colocalized with PI4KII alpha in the trans-Golgi network (TGN), were characterized in detail. Overexpression of DHHC3 or DHHC7 increased PI4KII alpha palmitoylation by >3-fold, whereas overexpression of the dominant-negative PATs or PAT silencing by RNA interference decreased PI4KII alpha palmitoylation, integral membrane association, and Golgi localization. Wild-type and dominant-negative DHHC3 and DHHC7 co-immunoprecipitated with PI4KII alpha, whereas non-candidate DHHC18 and DHHC23 did not. The PI4KII alpha (CCPCC177)-C-173 palmitoylation motif is required for interaction because the palmitoylation-defective SSPSS mutant did not co-immunoprecipitate with DHHC3. Cholesterol depletion and repletion with methyl-beta-cyclodextrin reversibly altered PI4KII alpha association with these DHHCs as well as PI4KII alpha localization at the TGN and integral membrane association. Significantly, the Golgi phosphatidylinositol 4-phosphate level was altered in parallel with changes in PI4KII alpha behavior. Our study uncovered a novel mechanism for the preferential recruitment and activation of PI4KII alpha to the TGN by interaction with Golgi- and raft-localized DHHCs in a cholesterol-dependent manner.