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5 results on '"[Rodriguez-Gallardo,S"'

1. Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM)

Author

Miho Waga, Akihiko Nakano, Auxiliadora Aguilera-Romero, Alejandro Cortes-Gomez, Sergio López, Sofia Rodriguez-Gallardo, Manuel Muñiz, Kazuo Kurokawa, Susana Sabido-Bozo, Ana Maria Perez-Linero, [Rodriguez-Gallardo,S, Sabido-Bozo,S, Cortes-Gomez,A, Perez-Linero,AM, Aguilera-Romero,A, Lopez,S, Muñiz,M] Department of Cell 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., MM, BFU2017-89700-P, FEDER/Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investigación, https://www.ciencia.gob.es/portal/site/MICINN/aei AN and KK, JP25221103, JP17H06420 and JP18H05275, Japan Society for the Promotion of Science (JSPS), https://www.jsps.go.jp/english/index.html, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), European Commission, Japan Society for the Promotion of Science, and Universidad de Sevilla. Departamento de Biología Celular

Subjects
Fluorescence-lifetime imaging microscopy, Cell Membranes, Golgi Apparatus, Endoplasmic Reticulum, Biochemistry, Anatomy::Cells::Cellular Structures::Cell Membrane::Intracellular Membranes [Medical Subject Headings], Ceramide, Fluorescence Microscopy, Lab Protocol, Cargo Proteins, Microscopía, Anatomy::Cells::Cellular Structures::Intracellular Space::Cytoplasm::Cytoplasmic Structures::Organelles::Endoplasmic Reticulum [Medical Subject Headings], ERES, COPII, Microscopy, Multidisciplinary, Secretory Pathway, Microscopy, Confocal, Analytical, Diagnostic and Therapeutic Techniques and Equipment::Diagnosis::Diagnostic Techniques and Procedures::Diagnostic Imaging::Imaging, Three-Dimensional [Medical Subject Headings], Chemistry, Phenomena and Processes::Metabolic Phenomena::Metabolism::Biological Transport::Protein Transport [Medical Subject Headings], Membrane, Eukaryota, Light Microscopy, ER retention, Lipids, Cell biology, Protein Transport, Cell Processes, symbols, Medicine, Phenomena and Processes::Metabolic Phenomena::Metabolism::Biological Transport [Medical Subject Headings], Cellular Structures and Organelles, Analytical, Diagnostic and Therapeutic Techniques and Equipment::Diagnosis::Diagnostic Techniques and Procedures::Diagnostic Imaging::Microscopy::Microscopy, Confocal [Medical Subject Headings], Anatomy::Cells::Cellular Structures::Intracellular Space::Cytoplasm::Cytoplasmic Structures::Organelles::Golgi Apparatus [Medical Subject Headings], Imaging Techniques, Science, Research and Analysis Methods, Retículo endoplásmico, symbols.namesake, Imaging, Three-Dimensional, Live cell imaging, Fluorescence Imaging, Secretory pathway, Endoplasmic reticulum, Protein, Organisms, Fungi, Biology and Life Sciences, Proteins, Membrane Proteins, Biological Transport, Cell Biology, Intracellular Membranes, Golgi apparatus, Yeast, Membranas, Ceramidas, Secretory protein, Proteína
Abstract

Understanding how in eukaryotic cells thousands of proteins are sorted from each other through the secretory pathway and delivered to their correct destinations is a central issue of cell biology. We have further investigated in yeast how two distinct types of cargo proteins are sorted into different endoplasmic reticulum (ER) exit sites (ERES) for their differential ER export to the Golgi apparatus. We used an optimized protocol that combines a live cell dual-cargo ER export system with a 3D simultaneous multi-color high-resolution live cell microscopy called Super-resolution Confocal Live Imaging Microscopy (SCLIM). Here, we describe this protocol, which is based on the reversible ER retention of two de novo co-expressed cargos by blocking COPII function upon incubation of the thermo-sensitive COPII allele sec31-1 at restrictive temperature (37°C). ER export is restored by shifting down to permissive temperature (24°C) and progressive incorporation of the two different types of cargos into the fluorescently labelled ERES can be then simultaneously captured at 3D high spatial resolution by SCLIM microscopy. By using this protocol, we have shown that newly synthesized glycosylphosphatidylinositol (GPI)-anchored proteins having a very long chain ceramide lipid moiety are clustered and sorted into specialized ERES that are distinct from those used by transmembrane secretory proteins. Furthermore, we showed that the chain length of the ceramide present in the ER membrane is critical for this sorting selectivity. Therefore, thanks to the presented method we could obtain the first direct in vivo evidence for lipid chain length-based protein cargo sorting into selective ERES., MM, BFU2017-89700-P, FEDER/Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investigación, https://www.ciencia.gob.es/portal/site/MICINN/aei AN and KK, JP25221103, JP17H06420 and JP18H05275, Japan Society for the Promotion of Science (JSPS), https://www.jsps.go.jp/english/index.html

Published
2021

2. Structural analysis of the GPI glycan

Author

Kouichi Funato, Manuel Muñiz, Miyako Nakano, Kouta Okazaki, Atsuko Ikeda, Alejandro Cortes-Gomez, Auxiliadora Aguilera-Romero, Susana Sabido-Bozo, Sofia Rodriguez-Gallardo, Sergio López, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), European Commission, Japan Society for the Promotion of Science, [Nakano,M, Okazaki,K, Ikeda,A, Funato,K] Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan. [Sabido-Bozo,S, Aguilera-Romero,A, Rodriguez-Gallardo,S, Cortes-Gomez,A, Lopez,S, Muñiz,M] Facultad de Biologia, Departamento de Biologia Celular, Hospital Universitario Virgen del Rocio/CSIC/Universidad de Sevilla, Universidad de Sevilla e Instituto de Biomedicina de Sevilla (IBiS), Sevilla, Spain., MM. BFU2017-89700-P. FEDER/Ministerio de Ciencia, Innovación y Universidades—Agencia Estatal de Investigación. http://www.aei.gob.es/. MN and KF. JP19H02922. Japan Society for the Promotion of Science. https://www.jsps.go.jp/english/. The funders had and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., Universidad de Sevilla. Departamento de Biología Celular, Ministerio de Ciencia, Innovación y Universidades (MICINN). España, and European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER)

Subjects
Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Glycoproteins::Membrane Glycoproteins::GPI-Linked Proteins [Medical Subject Headings], Cell Membranes, Cell, Electrophoretic techniques, Endoplasmic Reticulum, Biochemistry, Phenomena and Processes::Digestive System and Oral Physiological Phenomena::Digestive System Physiological Phenomena::Digestive System Processes::Digestion [Medical Subject Headings], Green fluorescent protein, Lab Protocol, Tandem Mass Spectrometry, Anatomy::Cells::Cellular Structures::Intracellular Space::Cytoplasm::Cytoplasmic Structures::Organelles::Endoplasmic Reticulum [Medical Subject Headings], Trypsin, Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Chemistry Techniques, Analytical::Electrophoresis [Medical Subject Headings], Organisms::Eukaryota::Fungi::Ascomycota::Saccharomycetales::Saccharomyces::Saccharomyces cerevisiae [Medical Subject Headings], Staining, Secretory Pathway, Multidisciplinary, biology, Chemistry, Phenomena and Processes::Chemical Phenomena::Biochemical Phenomena::Biochemical Processes::Protein Modification, Translational::Protein Processing, Post-Translational [Medical Subject Headings], Eukaryota, Proteases, Specimen preparation and treatment, Enzyme structure, Precipitation Techniques, Enzymes, Cell biology, medicine.anatomical_structure, Chemicals and Drugs::Carbohydrates::Glycoconjugates::Glycolipids::Glycosylphosphatidylinositols [Medical Subject Headings], Cell Processes, Digestión, Medicine, Digestion, lipids (amino acids, peptides, and proteins), Cellular Structures and Organelles, Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Chemistry Techniques, Analytical::Mass Spectrometry::Tandem Mass Spectrometry [Medical Subject Headings], Electrophoresis, Glycan, Immunoprecipitation, Science, Saccharomyces cerevisiae, Chemicals and Drugs::Enzymes and Coenzymes::Enzymes::Hydrolases::Peptide Hydrolases::Endopeptidases::Serine Endopeptidases::Trypsin [Medical Subject Headings], Research and Analysis Methods, GPI-Linked Proteins, Retículo endoplásmico, Coomassie Blue staining, Polysaccharides, medicine, Electroforesis, Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Peptides [Medical Subject Headings], Phenomena and Processes::Chemical Phenomena::Biochemical Phenomena::Biochemical Processes::Biosynthetic Pathways [Medical Subject Headings], Mass spectrometry, Endoplasmic reticulum, Glicosilfosfatidilinositoles, Organisms, Fungi, Biology and Life Sciences, Membrane Proteins, Proteins, Péptidos, Cell Biology, Gel electrophoresis, Yeast, Tripsina, carbohydrates (lipids), Glycosylphosphatidylinositol, Membrane protein, Electrophoretic staining, Enzyme Structure, Enzymology, biology.protein, Espectrometría de masas, Serine Proteases, Peptides, Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Chemistry Techniques, Analytical::Mass Spectrometry [Medical Subject Headings]
Abstract

Glycosylphosphatidylinositol (GPI) anchoring of proteins is an essential post-translational modification in all eukaryotes that occurs at the endoplasmic reticulum (ER) and serves to deliver GPI-anchored proteins (GPI-APs) to the cell surface where they play a wide variety of vital physiological roles. This paper describes a specialized method for purification and structural analysis of the GPI glycan of individual GPI-APs in yeast. The protocol involves the expression of a specific GPI-AP tagged with GFP, enzymatic release from the cellular membrane fraction, immunopurification, separation by electrophoresis and analysis of the peptides bearing GPI glycans by mass spectrometry after trypsin digestion. We used specifically this protocol to address the structural remodeling that undergoes the GPI glycan of a specific GPI-AP during its transport to the cell surface. This method can be also applied to investigate the GPI-AP biosynthetic pathway and to directly confirm predicted GPI-anchoring of individual proteins., MM. BFU2017-89700-P. FEDER/Ministerio de Ciencia, Innovación y Universidades—Agencia Estatal de Investigación. http://www.aei.gob.es/. MN and KF. JP19H02922. Japan Society for the Promotion of Science. https://www.jsps.go.jp/english/.

Published
2021

3. 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

4. The p24 Complex Contributes to Specify Arf1 for COPI Coat Selection

Author

Javier Manzano-López, Manuel Muñiz, Sergio López, Ralf Erik Wellinger, Ana Maria Perez-Linero, Alejandro Cortes-Gomez, Susana Sabido-Bozo, Sofia Rodriguez-Gallardo, Auxiliadora Aguilera-Romero, Universidad de Sevilla. Departamento de Biología Celular, [Sabido-Bozo,S, Perez-Linero,AM, Manzano-Lopez,J, Rodriguez-Gallardo,S, Aguilera-Romero,A, Cortes-Gomez,A, Lopez,S, Muñiz,M] Department of Cell Biology, Faculty of Biology, University of Seville, Seville, Spain. [Sabido-Bozo,S, Muñiz,M] Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain. [Manzano-Lopez,J, Wellinger,RE] Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, Sevilla, Spain. [Wellinger,RE] Department of Genetics, University of Seville, Seville, Spain., and This research was funded by the FEDER/Ministerio de Ciencia, Innovación y Universi dades—Agencia Estatal de Investigación/BFU2017-89700-P to Manuel Muñiz and to and BFU2015-69183-P (MINECO/FEDER, UE) to Ralf Erik Wellinger, and by the 'VI Own Research Plan' of the University of Seville VIPPIT-2020-I.5 to Manuel Muñiz. University of Seville fellowships to Sofia Rodriguez-Gallardo, Ana Maria Perez-Linero and Javier Manzano-Lopez. Ministry of Education, Cul ture and Sport (MECD) fellowship to Susana Sabido-Bozo. ‘V Own Research Plan’ of the University of Seville (VPPI-US) contract (cofounded by the European Social Fund) to Sergio Lopez.

Subjects
0301 basic medicine, Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Fungal Proteins::Saccharomyces cerevisiae Proteins [Medical Subject Headings], Golgi Apparatus, Chemicals and Drugs::Enzymes and Coenzymes::Enzymes::Hydrolases::Acid Anhydride Hydrolases::GTP Phosphohydrolases::GTP-Binding Proteins::Monomeric GTP-Binding Proteins::ADP-Ribosylation Factors::ADP-Ribosylation Factor 1 [Medical Subject Headings], Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::GTP-Binding Protein Regulators::Guanine Nucleotide Exchange Factors [Medical Subject Headings], Coat Protein Complex I, lcsh:Chemistry, Arf1, Golgi, Guanine Nucleotide Exchange Factors, Small GTPase, Organisms::Eukaryota::Fungi::Ascomycota::Saccharomycetales::Saccharomyces::Saccharomyces cerevisiae [Medical Subject Headings], lcsh:QH301-705.5, Spectroscopy, Proteína coat de complejo I, biology, Phenomena and Processes::Metabolic Phenomena::Metabolism::Biological Transport::Protein Transport [Medical Subject Headings], Effector, Chemistry, Vesicle, General Medicine, COPI, Computer Science Applications, Cell biology, Protein Transport, Factor 1 de ribosilacion-ADP, symbols, Guanine nucleotide exchange factor, COP-Coated Vesicles, Anatomy::Cells::Cellular Structures::Intracellular Space::Cytoplasm::Cytoplasmic Structures::Organelles::Golgi Apparatus [Medical Subject Headings], P24 complex, Saccharomyces cerevisiae Proteins, p24 complex, Saccharomyces cerevisiae, Clathrin, Catalysis, Article, Inorganic Chemistry, 03 medical and health sciences, symbols.namesake, Compartment (development), ArfGEFs, Physical and Theoretical Chemistry, Molecular Biology, Anatomy::Cells::Cellular Structures::Intracellular Space::Cytoplasm::Cytoplasmic Structures::Organelles::Cytoplasmic Vesicles::Transport Vesicles::Coated Vesicles::COP-Coated Vesicles [Medical Subject Headings], 030102 biochemistry & molecular biology, Factores de intercambio de guanina nucleótido, Organic Chemistry, Golgi apparatus, Aparato de Golgi, 030104 developmental biology, Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Membrane Proteins::Vesicular Transport Proteins::Coat Protein Complex I [Medical Subject Headings], lcsh:Biology (General), lcsh:QD1-999, biology.protein, ADP-Ribosylation Factor 1
Abstract

Golgi trafficking depends on the small GTPase Arf1 which, upon activation, drives the assembly of different coats onto budding vesicles. Two related types of guanine nucleotide exchange factors (GEFs) activate Arf1 at different Golgi sites. In yeast, Gea1 in the cis-Golgi and Gea2 in the medial-Golgi activate Arf1 to form COPI&shy, coated vesicles for retrograde cargo sorting, whereas Sec7 generates clathrin/adaptor&shy, coated vesicles at the trans-Golgi network (TGN) for forward cargo transport. A central question is how the same activated Arf1 protein manages to assemble different coats depending on the donor Golgi compartment. A previous study has postulated that the interaction between Gea1 and COPI would channel Arf1 activation for COPI vesicle budding. Here, we found that the p24 complex, a major COPI vesicle cargo, promotes the binding of Gea1 with COPI by increasing the COPI association to the membrane independently of Arf1 activation. Furthermore, the p24 complex also facilitates the interaction of Arf1 with its COPI effector. Therefore, our study supports a mechanism by which the p24 complex contributes to program Arf1 activation by Gea1 for selective COPI coat assembly at the cis-Golgi compartment.

Published
2021

5. 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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