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1. Determination of the lipid composition of the GPI anchor

Author

Ana Maria Perez-Linero, Sofia Rodriguez-Gallardo, Susana Sabido-Bozo, Sergio López, Manuel Muñiz, Alejandro Cortes-Gomez, Auxiliadora Aguilera-Romero, Isabelle Riezman, Howard Riezman, 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., and 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.

Subjects
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]
Abstract

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.

Published
2021

2. Ceramide chain length-dependent protein sorting into selective endoplasmic reticulum exit sites

Author

Stefano Vanni, Miyako Nakano, Misako Araki, Alejandro Cortes-Gomez, Sofia Rodriguez-Gallardo, Auxiliadora Aguilera-Romero, Valeria Zoni, Atsuko Ikeda, Sergio López, Susana Sabido-Bozo, Howard Riezman, Kazuo Kurokawa, Manuel Muñiz, Miho Waga, Kouichi Funato, Akihiko Nakano, Ana Maria Perez-Linero, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Universidad de Sevilla, Japan Society for the Promotion of Science, Swiss National Science Foundation, Swiss National Supercomputing Centre, National Centres of Competence in Research (Switzerland), Ministerio de Educación, Cultura y Deporte (España), [Rodriguez-Gallardo,S, Sabido-Bozo,S, Cortes-Gomez,A, Aguilera-Romero,A, Perez-Linero,AM, Lopez,S, Muñiz,M] Department of Cell Biology, Faculty of Biology, University of Seville and Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain. [Kurokawa,K, Waga,M, Nakano,A] Live Cell Super-Resolution Imaging Research Team, RIKEN Center for Advanced Photonics, Saitama, Japan. [Zoni,V, Vanni, S] Department of Biology, University of Fribourg, Fribourg, Switzerland. [Ikeda,A, Araki,M, Nakano,M, Funato,K] Graduate School of Integrated Sciences for Life, Hiroshima University, Hiroshima, Japan. [Riezman,H] NCCR Chemical Biology, Department of Biochemistry, University of Geneva, Geneva, Switzerland., This research was funded by the Spanish Ministry of Economy and Competitiveness (MINECO, grant number BFU2017-89700-P to M.M.), 'VI Own Research Plan' of the University of Seville (VIPPIT-2020-I.5 to M.M.), and Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science (JP25221103, JP17H06420, and JP18H05275 to A.N. and K. K., and JP19H02922 to M.N. and K.F.). S.V. and V.Z. acknowledge support from the Swiss National Science Foundation (grant no. 163966) and from the Swiss National Supercomputing Centre (CSCS) under project IDs s726 and s842. H.R. is supported by the Swiss National Science Foundation and the NCCR Chemical Biology (grants 184949 and 185898). This research was funded by the 'VI Own Research Plan 'of the University of Seville (VIPPIT-2020-I.5), 'V Own Research Plan' of the University of Seville (VPPI-US) contract (cofounded by the European Social Fund) to S.L., University of Seville fellowship to S.R.-G., Ministry of Education, Culture, and Sport (MECD) fellowship to S.S.-B., and contract from the University of Seville by the Youth Employment Initiative to A.C.-G., Universidad de Sevilla. Departamento de Biología Celular, Ministerio de Economía y Competitividad (MINECO). España, Swiss National Science Foundation (SNFS), and Swiss National Supercomputing Centre (CSCS)

Subjects
Vías secretorias, Endoplasmic Reticulum, medicine.disease_cause, Biochemistry, Chemicals and Drugs::Organic Chemicals::Amides::Ceramides [Medical Subject Headings], chemistry.chemical_compound, 0302 clinical medicine, Anatomy::Cells::Cellular Structures::Intracellular Space::Cytoplasm::Cytoplasmic Structures::Organelles::Endoplasmic Reticulum [Medical Subject Headings], Protein targeting, Research Articles, 0303 health sciences, Secretory Pathway, Multidisciplinary, Phenomena and Processes::Metabolic Phenomena::Metabolism::Biological Transport::Protein Transport [Medical Subject Headings], Chemistry, SciAdv r-articles, Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins [Medical Subject Headings], Compartmentalization (psychology), Lipids, Anatomy::Tissues::Membranes [Medical Subject Headings], Transmembrane protein, Cell biology, Protein Transport, Lípidos, Proteínas, Reticulum, Research Article, Anatomy::Animal Structures::Stomach, Ruminant::Reticulum [Medical Subject Headings], Ceramide, Cell signaling, Chemicals and Drugs::Lipids [Medical Subject Headings], Ceramides, Retículo endoplásmico, Chemicals and Drugs::Lipids::Glycolipids::Glycosylphosphatidylinositols [Medical Subject Headings], 03 medical and health sciences, Live cell imaging, medicine, Secretory pathway, 030304 developmental biology, Membranes, Endoplasmic reticulum, Glicosilfosfatidilinositoles, Proteins, Membrane Proteins, Membranas, Ceramidas, Glycosylphosphatidylinositol, Phenomena and Processes::Metabolic Phenomena::Metabolism::Biological Transport::Secretory Pathway [Medical Subject Headings], Phenomena and Processes::Physiological Phenomena::Physiological Processes::Homeostasis [Medical Subject Headings], 030217 neurology & neurosurgery
Abstract

Protein sorting in the secretory pathway is crucial to maintain cellular compartmentalization and homeostasis. In addition to coat-mediated sorting, the role of lipids in driving protein sorting during secretory transport is a longstanding fundamental question that still remains unanswered. Here, we conduct 3D simultaneous multicolor high-resolution live imaging to demonstrate in vivo that newly synthesized glycosylphosphatidylinositol-anchored proteins having a very long chain ceramide lipid moiety are clustered and sorted into specialized endoplasmic reticulum exit sites that are distinct from those used by transmembrane proteins. Furthermore, we show that the chain length of ceramide in the endoplasmic reticulum membrane is critical for this sorting selectivity. Our study provides the first direct in vivo evidence for lipid chain length-based protein cargo sorting into selective export sites of the secretory thway., This research was funded by the Spanish Ministry of Economy and Competitiveness (MINECO; grant number BFU2017-89700-P to M.M.), “VI Own Research Plan” of the University of Seville (VIPPIT-2020-I.5 to M.M.), and Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science (JP25221103, JP17H06420, and JP18H05275 to A.N. and K. K.; and JP19H02922 to M.N. and K.F.). S.V. and V.Z. acknowledge support from the Swiss National Science Foundation (grant no. 163966) and from the Swiss National Supercomputing Centre (CSCS) under project IDs s726 and s842. H.R. is supported by the Swiss National Science Foundation and the NCCR Chemical Biology (grants 184949 and 185898). This research was funded by the “VI Own Research Plan “of the University of Seville (VIPPIT-2020-I.5); “V Own Research Plan” of the University of Seville (VPPI-US) contract (cofounded by the European Social Fund) to S.L.; University of Seville fellowship to S.R.-G.; Ministry of Education, Culture, and Sport (MECD) fellowship to S.S.-B.; and contract from the University of Seville by the Youth Employment Initiative to A.C.-G.

Published
2020

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