N-terminal protein acylation confers localization to cholesterol, sphingolipid-enriched membranes but not to lipid rafts/caveolae

• Published in: Molecular biology of the cell Vol. 12; no. 11; pp. 3601 - 3617
• Main Authors: McCabe, J B, Berthiaume, L G
• Format: Journal Article
• Language: English
• Published: United States The American Society for Cell Biology 01.11.2001
• Subjects: Acylation; Animals; Biomarkers; Caveolae - metabolism; Caveolin 1; Caveolins - metabolism; Cell Fractionation; Cell Membrane - metabolism; Cercopithecus aethiops; Cholesterol - metabolism; COS Cells; Detergents; Filipin - metabolism; G(M1) Ganglioside - metabolism; Green Fluorescent Proteins; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Membrane Microdomains - metabolism; Membrane Proteins - metabolism; Octoxynol; Organelles; Proteins - metabolism; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-yes; Solubility; Sphingolipids - metabolism; src-Family Kinases; Transferrin - metabolism; Vesicular Transport Proteins; Xanthenes
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.12.11.3601

When variably fatty acylated N-terminal amino acid sequences were appended to a green fluorescent reporter protein (GFP), chimeric GFPs were localized to different membranes in a fatty acylation-dependent manner. To explore the mechanism of localization, the properties of acceptor membranes and their interaction with acylated chimeric GFPs were analyzed in COS-7 cells. Myristoylated GFPs containing a palmitoylated or polybasic region colocalized with cholesterol and ganglioside GM(1), but not with caveolin, at the plasma membrane and endosomes. A dipalmitoylated GFP chimera colocalized with cholesterol and GM(1) at the plasma membrane and with caveolin in the Golgi region. Acylated GFP chimeras did not cofractionate with low-density caveolin-rich lipid rafts prepared with Triton X-100 or detergent-free methods. All GFP chimeras, but not full-length p62(c-yes) and caveolin, were readily solubilized from membranes with various detergents. These data suggest that, although N-terminal acylation can bring GFP to cholesterol and sphingolipid-enriched membranes, protein-protein interactions are required to localize a given protein to detergent-resistant membranes or caveolin-rich membranes. In addition to restricting acceptor membrane localization, N-terminal fatty acylation could represent an efficient means to enrich the concentration of signaling proteins in the vicinity of detergent-resistant membranes and facilitate protein-protein interactions mediating transfer to a detergent-resistant lipid raft core.