Palmitoylation at Cys 1145 in the Carboxyl Terminus of Human Type 6 Adenylyl Cyclase is Not Required for Targeting to Lipid Rafts and Caveolae

Abstract

Palmitoylation is important for targeting certain membrane-associated and integral membrane proteins to lipid rafts and caveolae. Previous data have shown that adenylyl cyclase type 6 (AC6) is enriched in lipid rafts or caveolae while other isoforms of AC, such as AC2 and AC4, are excluded from these domains. We hypothesized that palmitoylation on a cysteine residue in the carboxyl terminus (C-terminus) of AC6 or other elements encoded in this region are required for AC6 expression in lipid rafts and caveolae. Thus, we expressed in Cos-7 cells epitope-tagged full length human AC6 and three different C-terminally truncated AC6 proteins, one (AC6 1-1148) retaining Cys 1145, a putative palmitoylation site, and two others (AC6 1-1144 and AC6 1-1127) lacking this residue. We used several approaches for assessing the subcellular localization of these expressed proteins: non-detergent biochemical isolation of lipid rafts and immunoblotting, immunoprecipitation of caveolin-1, Triton-X-100 insolubility, and immunoisolation of caveolae followed by adenylyl cyclase activity assays. We found that AC6 1-1144, AC6 1-1127 and AC6 1–1148 truncation proteins were each localized similarly to full-length AC6. We conclude that neither the putative palmitoylation site Cys 1145, nor other elements in the distal portion of the carboxyl terminus of AC6, are important for targeting of this effector enzyme to lipid rafts and caveolae. Supported by AHA grant 0555291B.