title: N-terminal protein acylation confers localization to cholesterol, sphingolipid-enriched membranes but not to lipid rafts/caveolae.
abstract: 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.
authors.affiliation: M.D./Ph.D. Program, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: J B
authors.initials: JB
authors.lastname: McCabe
authors.forename: L G
authors.initials: LG
authors.lastname: Berthiaume
mesh_terms: D000215:Acylation; D000818:Animals; D015415:Biomarkers; D019556:COS Cells; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D002784:Cholesterol / Q000378:metabolism*; D003902:Detergents; D005372:Filipin / Q000378:metabolism; D005677:G(M1) Ganglioside / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D017830:Octoxynol; D015388:Organelles; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051575:Proto-Oncogene Proteins c-yes; D012995:Solubility; D013107:Sphingolipids / Q000378:metabolism*; D014168:Transferrin / Q000378:metabolism; D033921:Vesicular Transport Proteins; D014966:Xanthenes; D019061:src-Family Kinases
chemical_list: D015415:Biomarkers; D051242:Caveolin 1; D022461:Caveolins; D003902:Detergents; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D013107:Sphingolipids; D014168:Transferrin; D033921:Vesicular Transport Proteins; D014966:Xanthenes; C093846:early endosome antigen 1; D049452:Green Fluorescent Proteins; D005677:G(M1) Ganglioside; C034657:Texas red; D005372:Filipin; D017830:Octoxynol; D002784:Cholesterol; D051575:Proto-Oncogene Proteins c-yes; D019061:src-Family Kinases