Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM).
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 differen...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2021-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0258111 |
| _version_ | 1831583512339152896 |
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| author | Sofia Rodriguez-Gallardo Kazuo Kurokawa Susana Sabido-Bozo Alejandro Cortes-Gomez Ana Maria Perez-Linero Auxiliadora Aguilera-Romero Sergio Lopez Miho Waga Akihiko Nakano Manuel Muñiz |
| author_facet | Sofia Rodriguez-Gallardo Kazuo Kurokawa Susana Sabido-Bozo Alejandro Cortes-Gomez Ana Maria Perez-Linero Auxiliadora Aguilera-Romero Sergio Lopez Miho Waga Akihiko Nakano Manuel Muñiz |
| author_sort | Sofia Rodriguez-Gallardo |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-17T20:43:33Z |
| format | Article |
| id | doaj.art-559aca166abe4d3295be01873b9fb3a5 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-17T20:43:33Z |
| publishDate | 2021-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-559aca166abe4d3295be01873b9fb3a52022-12-21T21:33:15ZengPublic Library of Science (PLoS)PLoS ONE1932-62032021-01-011610e025811110.1371/journal.pone.0258111Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM).Sofia Rodriguez-GallardoKazuo KurokawaSusana Sabido-BozoAlejandro Cortes-GomezAna Maria Perez-LineroAuxiliadora Aguilera-RomeroSergio LopezMiho WagaAkihiko NakanoManuel MuñizUnderstanding 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.https://doi.org/10.1371/journal.pone.0258111 |
| spellingShingle | Sofia Rodriguez-Gallardo Kazuo Kurokawa Susana Sabido-Bozo Alejandro Cortes-Gomez Ana Maria Perez-Linero Auxiliadora Aguilera-Romero Sergio Lopez Miho Waga Akihiko Nakano Manuel Muñiz Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM). PLoS ONE |
| title | Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM). |
| title_full | Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM). |
| title_fullStr | Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM). |
| title_full_unstemmed | Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM). |
| title_short | Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM). |
| title_sort | assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3d super resolution confocal live imaging microscopy sclim |
| url | https://doi.org/10.1371/journal.pone.0258111 |
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