Determination of the lipid composition of the GPI anchor
| Autor: | Ana Maria Perez-Linero, Sofia Rodriguez-Gallardo, Susana Sabido-Bozo, Sergio López, Manuel Muñiz, Alejandro Cortes-Gomez, Auxiliadora Aguilera-Romero, Isabelle Riezman, Howard Riezman |
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| Přispěvatelé: | Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), European Commission, Universidad de Sevilla, Swiss National Science Foundation, Ministerio de Educación, Cultura y Deporte (España), Universidad de Sevilla. Departamento de Biología Celular, Ministerio de Ciencia, Innovación y Universidades (MICINN). España, European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER), Swiss National Science Foundation (SNFS), [Aguilera-Romero,A, Sabido-Bozo,S, Lopez,S, Cortes-Gomez,A, Rodriguez-Gallardo,S, Perez-Linero,AM, Muñiz,M] Departamento de Biología Celular, Facultad de Biología, Universidad de Sevilla e Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain. [Riezman,I, Riezman,H] Department of Biochemistry, NCCR Chemical Biology, University of Geneva, Geneva, Switzerland., This research was funded by the FEDER/Ministerio de Ciencia, Innovación y Universidades—Agencia Estatal de Investigación/BFU2017-89700-P to Manuel Muñiz, 'VI Own Research Plan' of the University of Seville VIPPIT-2020-I.5 to Manuel Muñiz, by the Incentivo al Grupo de Investigación BIO-271 (2019/BIO-271) and the NCCR Chemical Biology and the Swiss National Science Foundation (51NF40-185898 and 310030_184949) to Howard Riezman. ‘V Own Research Plan’ of the University of Seville (VPPIUS) contract (cofounded by the European Social Fund) to Sergio Lopez, University of Seville fellowships to Sofia Rodriguez-Gallardo and Ana Maria Perez-Linero, Ministry of Education, Culture and Sport (MECD) fellowship to Susana Sabido Bozo and contract from the University of Seville by the Youth Employment Initiative to Alejandro Cortes-Gomez. |
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Chemicals and Drugs::Amino Acids
Peptides and Proteins::Proteins::Glycoproteins::Membrane Glycoproteins::GPI-Linked Proteins [Medical Subject Headings] Glycosylphosphatidylinositols Chemicals and Drugs::Amino Acids Peptides and Proteins::Proteins::Fungal Proteins::Saccharomyces cerevisiae Proteins [Medical Subject Headings] Cell Membranes Yeast and Fungal Models Protein lipidation Tandem mass spectrometry Endoplasmic Reticulum Biochemistry Mass Spectrometry Chemicals and Drugs::Organic Chemicals::Amides::Ceramides [Medical Subject Headings] Lab Protocol Genes Reporter Macromolecular Structure Analysis Chemicals and Drugs::Amino Acids Peptides and Proteins::Proteins::Glycoproteins::Membrane Glycoproteins [Medical Subject Headings] Staining Multidisciplinary Membrane Glycoproteins Secretory Pathway Lipid Analysis biology Chemistry Eukaryota Genes reporter Lipids Blot Membrane Staining Chemicals and Drugs::Carbohydrates::Glycoconjugates::Glycolipids::Glycosylphosphatidylinositols [Medical Subject Headings] Experimental Organism Systems Cell Processes Lípidos Medicine lipids (amino acids peptides and proteins) Cellular Structures and Organelles Saccharomyces cerevisiae Proteins Immunoprecipitation Science Saccharomyces cerevisiae Green Fluorescent Proteins Phenomena and Processes::Genetic Phenomena::Genetic Structures::Genome::Genome Components::Genes::Genes Reporter [Medical Subject Headings] Ceramides GPI-Linked Proteins Research and Analysis Methods Saccharomyces Glycolipid Model Organisms Organisms::Eukaryota::Fungi::Ascomycota::Saccharomycetales::Saccharomyces [Medical Subject Headings] Integral Membrane Proteins Molecular Biology Anatomy::Cells::Cellular Structures::Cell Membrane [Medical Subject Headings] Membranes Glicosilfosfatidilinositoles Cell Membrane Genes informadores Organisms Fungi Biology and Life Sciences Membrane Proteins Cell Biology biology.organism_classification Yeast Membranas Ceramidas carbohydrates (lipids) Membrane protein Chemicals and Drugs::Amino Acids Peptides and Proteins::Proteins::Luminescent Proteins::Green Fluorescent Proteins [Medical Subject Headings] Specimen Preparation and Treatment Animal Studies Analytical Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Chemistry Techniques Analytical::Mass Spectrometry [Medical Subject Headings] |
| Zdroj: | PLoS ONE idUS. Depósito de Investigación de la Universidad de Sevilla instname PLoS ONE, Vol 16, Iss 8, p e0256184 (2021) Digital.CSIC. Repositorio Institucional del CSIC |
| Popis: | In eukaryotic cells, a subset of cell surface proteins is attached by the glycolipid glycosylphosphatidylinositol (GPI) to the external leaflet of the plasma membrane where they play important roles as enzymes, receptors, or adhesion molecules. Here we present a protocol for purification and mass spectrometry analysis of the lipid moiety of individual GPI-anchored proteins (GPI-APs) in yeast. The method involves the expression of a specific GPI-AP tagged with GFP, solubilization, immunoprecipitation, separation by electrophoresis, blotting onto PVDF, release and extraction of the GPI-lipid moiety and analysis by mass spectrometry. By using this protocol, we could determine the precise GPI-lipid structure of the GPI-AP Gas1-GFP in a modified yeast strain. This protocol can be used to identify the lipid composition of the GPI anchor of distinct GPI-APs from yeast to mammals and can be adapted to determine other types of protein lipidation. This research was funded by the FEDER/Ministerio de Ciencia, Innovación y Universidades—Agencia Estatal de Investigación/BFU2017-89700-P to Manuel Muñiz, "VI Own Research Plan" of the University of Seville VIPPIT-2020-I.5 to Manuel Muñiz, by the Incentivo al Grupo de Investigación BIO-271 (2019/BIO-271) and the NCCR Chemical Biology and the Swiss National Science Foundation (51NF40-185898 and 310030_184949) to Howard Riezman. ‘V Own Research Plan’ of the University of Seville (VPPI-US) contract (cofounded by the European Social Fund) to Sergio Lopez, University of Seville fellowships to Sofia Rodriguez-Gallardo and Ana Maria Perez-Linero, Ministry of Education, Culture and Sport (MECD) fellowship to Susana Sabido-Bozo and contract from the University of Seville by the Youth Employment Initiative to Alejandro Cortes-Gomez. |
| Databáze: | OpenAIRE |
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