Artificial Palmitoylation of Proteins Controls the Lipid Domain-Selective Anchoring on Biomembranes and the Raft-Dependent Cellular Internalization

Protein palmitoylation, a post-translational modification, is universally observed in eukaryotic cells. The localization of palmitoylated proteins to highly dynamic, sphingolipid- and cholesterol-rich microdomains (called lipid rafts) on the plasma membrane has been shown to play an important role in signal transduction in cells. However, this complex biological system is not yet completely understood. Here, we used a combined approach where an artificial lipidated protein was applied to biomimetic model membranes and plasma membranes in cells to illuminate chemical and physiological properties of the rafts. Using cell-sized giant unilamellar vesicles, we demonstrated the selective partitioning of enhanced green fluorescent protein modified with a C-terminal palmitoyl moiety (EGFP-Pal) into the liquid-ordered phase consisting of saturated phospholipids and cholesterol. Using Jurkat T cells treated with an immunostimulant (concanavalin A), we observed the vesicular transport of EGFP-Pal. Further cellular studies with the treatment of methyl beta-cyclodextrin revealed the cholesterol-dependent internalization of EGFP-Pal, which can be explained by a raft-dependent, caveolae-mediated endocytic pathway. The present synthetic approach using artificial and natural membrane systems can be further extended to explore the potential utility of artificially lipidated proteins at biological and artificial interfaces.

Automated summary

Protein palmitoylation plays an important role in signal transduction by localizing proteins to lipid rafts on the plasma membrane. This study used an artificial lipidated protein to investigate the properties of lipid rafts and demonstrated their selective partitioning and vesicular transport. It was also found that the internalization of the protein is cholesterol-dependent and may occur through a raft-dependent, caveolae-mediated endocytic pathway.