title: Glycolipid Crosslinking Is Required for Cholera Toxin to Partition Into and Stabilize Ordered Domains.
abstract: Current models of lipid rafts propose that lipid domains exist as nanoscale compositional fluctuations and these fluctuations can potentially be stabilized into larger domains, consequently better compartmentalizing cellular functions. However, the mechanisms governing stabilized raft assembly and function remain unclear. Here, we test the role of glycolipid crosslinking as a raft targeting and ordering mechanism using the well-studied raft marker cholera toxin B pentamer (CTxB) that binds up to five GM1 glycosphingolipids to enter host cells. We show that when applied to cell-derived giant plasma membrane vesicles, a variant of CTxB containing only a single functional GM1 binding site exhibits significantly reduced partitioning to the ordered phase compared to wild-type CTxB with five binding sites. Moreover, monovalent CTxB does not stabilize membrane domains, unlike wild-type CTxB. These results support the long-held hypothesis that CTxB stabilizes raft domains via a lipid crosslinking mechanism and establish a role for crosslinking in the partitioning of CTxB to ordered domains.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee.
authors.forename: Krishnan
authors.initials: K
authors.lastname: Raghunathan
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee.
authors.forename: Tiffany H
authors.initials: TH
authors.lastname: Wong
authors.affiliation: Harvard Medical School and the Harvard Digestive Diseases Center, Boston, Massachusetts.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Chinnapen
authors.affiliation: Harvard Medical School and the Harvard Digestive Diseases Center, Boston, Massachusetts.
authors.forename: Wayne I
authors.initials: WI
authors.lastname: Lencer
authors.affiliation: Department of Immunology and Microbiology, University of Colorado, Aurora, Colorado.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Jobling
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee; Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee. Electronic address: anne.kenworthy@vanderbilt.edu.
authors.forename: Anne K
authors.initials: AK
authors.lastname: Kenworthy
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002772:Cholera Toxin / Q000378:metabolism*; D006028:Glycosphingolipids / Q000737:chemistry* / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D021381:Protein Transport
chemical_list: D006028:Glycosphingolipids; D002772:Cholera Toxin