Phosphatidylinositol 4-kinase IIα is palmitoylated by golgi-localized palmitoyltransferases in cholesterol-dependent manner

Phosphatidylinositol 4-kinase IIα (PI4KIIα) 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 PI4KIIαand its association with low buoyant density "raft" domains are critically dependent on palmitoylation of its cysteine-rich ¹⁷³CCPCC¹⁷⁷ motif and are also highly cholesterol-dependent. Here, we identified the palmitoyl acyltransferases (Asp-His- His-Cys (DHHC) PATs) that palmitoylate PI4KIIα and show for the first time that palmitoylation is cholesterol-dependent. DHHC3 and DHHC7 PATs, which robustly palmitoylated PI4KIIα and were colocalized with PI4KIIα in the trans-Golgi network (TGN), were characterized in detail. Overexpression of DHHC3 or DHHC7 increased PI4KIIα palmitoylation by >3-fold, whereas overexpression of the dominant-negative PATs or PAT silencing by RNA interference decreased PI4KIIαpalmitoylation, "integral" membrane association, and Golgi localization. Wild-type and dominant-negative DHHC3 and DHHC7 co-immunoprecipitated with PI4KIIα, whereas noncandidate DHHC18 and DHHC23 did not. The PI4KIIα ¹⁷³CCPCC¹⁷⁷ palmitoylation motif is required for interaction because the palmitoylation-defective SSPSS mutant did not coimmunoprecipitate with DHHC3. Cholesterol depletion and repletion with methyl-β-cyclodextrin reversibly altered PI4KIIα association with these DHHCs as well as PI4KIIα localization at theTGNand "integral" membrane association. Significantly, the Golgi phosphatidylinositol 4-phosphate level was altered in parallel with changes in PI4KIIα behavior. Our study uncovered a novel mechanism for the preferential recruitment and activation of PI4KIIα to theTGNby interaction with Golgiand raft-localized DHHCs in a cholesterol-dependent manner.