title: Potential of Proapoptotic Peptides to Induce the Formation of Giant Plasma Membrane Vesicles with Lipid Domains.
abstract: We have established a method of preparing giant plasma membrane vesicles (GPMVs) by using cysteine mutants of the proapoptotic peptide (PAP) Ac-R7-GG-KLAKLAKKLAKLAK. A cysteine scan revealed that cytotoxicity and GPMV formation were dependent on the cysteine position within the PAP sequence. In comparison to GPMVs prepared by extensive treatment with paraformaldehyde (PFA) and dithiothreitol (DTT), our GPMVs were produced from HeLa cells at much lower concentrations of the blebbing agent. We found that only GPMVs derived from cysteine-containing PAP showed lipid phase separation. This membrane model was applied to investigate the phase partitioning of two relevant membrane proteins: influenza virus hemagglutinin (HA) and tetherin, which clamps budding HIV to infected cells. For tetherin, we show for the first time exclusion from cholesterol-rich domains in a GPMV model, thus documenting the potential of our approach for membrane-partitioning studies.
authors.affiliation: Department of Biology, Molecular Biophysics, Humboldt-Universität zu Berlin, Invalidenstrasse 42, 10115 Berlin (Germany).
authors.forename: Daniel
authors.initials: D
authors.lastname: Lauster
authors.affiliation: Department of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin (Germany).
authors.forename: Olalla
authors.initials: O
authors.lastname: Vazquez
authors.affiliation: Department of Biology, Molecular Biophysics, Humboldt-Universität zu Berlin, Invalidenstrasse 42, 10115 Berlin (Germany).
authors.forename: Roland
authors.initials: R
authors.lastname: Schwarzer
authors.affiliation: Department of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin (Germany).
authors.forename: Oliver
authors.initials: O
authors.lastname: Seitz
authors.affiliation: Department of Biology, Molecular Biophysics, Humboldt-Universität zu Berlin, Invalidenstrasse 42, 10115 Berlin (Germany). andreas.herrmann@rz.hu-berlin.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Herrmann
mesh_terms: D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D058851:GPI-Linked Proteins / Q000378:metabolism; D006367:HeLa Cells; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D008969:Molecular Sequence Data; D009975:Orthomyxoviridae / Q000502:physiology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D044367:Phase Transition; D000074010:Bone Marrow Stromal Antigen 2
chemical_list: D015703:Antigens, CD; D002784:Cholesterol; D003545:Cysteine; D058851:GPI-Linked Proteins; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D010455:Peptides; C094572:BST2 protein, human; D000074010:Bone Marrow Stromal Antigen 2
keywords: cysteine; giant vesicles; hemagglutinin; influenza virus; phase separation; tetherin