A Three-Dimensional Model of the Yeast Transmembrane Sensor Wsc1 Obtained by SMA-Based Detergent-Free Purification and Transmission Electron Microscopy
The cell wall sensor Wsc1 belongs to a small family of transmembrane proteins, which are crucial to sustain cell integrity in yeast and other fungi. Wsc1 acts as a mechanosensor of the cell wall integrity (CWI) signal transduction pathway which responds to external stresses. Here we report on the pu...
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MDPI AG
2021-02-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/7/2/118 |
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| author | Natalia Voskoboynikova Maria Karlova Rainer Kurre Armen Y. Mulkidjanian Konstantin V. Shaitan Olga S. Sokolova Heinz-Jürgen Steinhoff Jürgen J. Heinisch |
| author_facet | Natalia Voskoboynikova Maria Karlova Rainer Kurre Armen Y. Mulkidjanian Konstantin V. Shaitan Olga S. Sokolova Heinz-Jürgen Steinhoff Jürgen J. Heinisch |
| author_sort | Natalia Voskoboynikova |
| collection | DOAJ |
| description | The cell wall sensor Wsc1 belongs to a small family of transmembrane proteins, which are crucial to sustain cell integrity in yeast and other fungi. Wsc1 acts as a mechanosensor of the cell wall integrity (CWI) signal transduction pathway which responds to external stresses. Here we report on the purification of Wsc1 by its trapping in water-soluble polymer-stabilized lipid nanoparticles, obtained with an amphipathic styrene-maleic acid (SMA) copolymer. The latter was employed to transfer tagged sensors from their native yeast membranes into SMA/lipid particles (SMALPs), which allows their purification in a functional state, i.e., avoiding denaturation. The SMALPs composition was characterized by fluorescence correlation spectroscopy, followed by two-dimensional image acquisition from single particle transmission electron microscopy to build a three-dimensional model of the sensor. The latter confirms that Wsc1 consists of a large extracellular domain connected to a smaller intracellular part by a single transmembrane domain, which is embedded within the hydrophobic moiety of the lipid bilayer. The successful extraction of a sensor from the yeast plasma membrane by a detergent-free procedure into a native-like membrane environment provides new prospects for in vitro structural and functional studies of yeast plasma proteins which are likely to be applicable to other fungi, including plant and human pathogens. |
| first_indexed | 2024-03-09T05:32:43Z |
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| id | doaj.art-db6e338c1a314ca1ac9d56e2d30bd94b |
| institution | Directory Open Access Journal |
| issn | 2309-608X |
| language | English |
| last_indexed | 2024-03-09T05:32:43Z |
| publishDate | 2021-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Fungi |
| spelling | doaj.art-db6e338c1a314ca1ac9d56e2d30bd94b2023-12-03T12:31:39ZengMDPI AGJournal of Fungi2309-608X2021-02-017211810.3390/jof7020118A Three-Dimensional Model of the Yeast Transmembrane Sensor Wsc1 Obtained by SMA-Based Detergent-Free Purification and Transmission Electron MicroscopyNatalia Voskoboynikova0Maria Karlova1Rainer Kurre2Armen Y. Mulkidjanian3Konstantin V. Shaitan4Olga S. Sokolova5Heinz-Jürgen Steinhoff6Jürgen J. Heinisch7Faculty of Physics, University of Osnabrück, Barbarastrasse 7, D-49076 Osnabrück, GermanyDepartment of Bioengineering, Faculty of Biology, M. V. Lomonosov Moscow State University, 119234 Moscow, RussiaCenter of Cellular Nanoanalytics, Integrated Bioimaging Facility iBiOs, Faculty of Biology/Chemistry, University of Osnabrück, Barbarastrasse 11, D-49076 Osnabrück, GermanyFaculty of Physics, University of Osnabrück, Barbarastrasse 7, D-49076 Osnabrück, GermanyDepartment of Bioengineering, Faculty of Biology, M. V. Lomonosov Moscow State University, 119234 Moscow, RussiaDepartment of Bioengineering, Faculty of Biology, M. V. Lomonosov Moscow State University, 119234 Moscow, RussiaFaculty of Physics, University of Osnabrück, Barbarastrasse 7, D-49076 Osnabrück, GermanyDepartment of Genetics, Faculty of Biology/Chemistry, University of Osnabrück, Barbarastrasse 11, D-49076 Osnabrück, GermanyThe cell wall sensor Wsc1 belongs to a small family of transmembrane proteins, which are crucial to sustain cell integrity in yeast and other fungi. Wsc1 acts as a mechanosensor of the cell wall integrity (CWI) signal transduction pathway which responds to external stresses. Here we report on the purification of Wsc1 by its trapping in water-soluble polymer-stabilized lipid nanoparticles, obtained with an amphipathic styrene-maleic acid (SMA) copolymer. The latter was employed to transfer tagged sensors from their native yeast membranes into SMA/lipid particles (SMALPs), which allows their purification in a functional state, i.e., avoiding denaturation. The SMALPs composition was characterized by fluorescence correlation spectroscopy, followed by two-dimensional image acquisition from single particle transmission electron microscopy to build a three-dimensional model of the sensor. The latter confirms that Wsc1 consists of a large extracellular domain connected to a smaller intracellular part by a single transmembrane domain, which is embedded within the hydrophobic moiety of the lipid bilayer. The successful extraction of a sensor from the yeast plasma membrane by a detergent-free procedure into a native-like membrane environment provides new prospects for in vitro structural and functional studies of yeast plasma proteins which are likely to be applicable to other fungi, including plant and human pathogens.https://www.mdpi.com/2309-608X/7/2/118Wsc1membrane sensorSMALPdetergent-free extractionfluorescence correlation spectroscopytransmission electron microscopy |
| spellingShingle | Natalia Voskoboynikova Maria Karlova Rainer Kurre Armen Y. Mulkidjanian Konstantin V. Shaitan Olga S. Sokolova Heinz-Jürgen Steinhoff Jürgen J. Heinisch A Three-Dimensional Model of the Yeast Transmembrane Sensor Wsc1 Obtained by SMA-Based Detergent-Free Purification and Transmission Electron Microscopy Journal of Fungi Wsc1 membrane sensor SMALP detergent-free extraction fluorescence correlation spectroscopy transmission electron microscopy |
| title | A Three-Dimensional Model of the Yeast Transmembrane Sensor Wsc1 Obtained by SMA-Based Detergent-Free Purification and Transmission Electron Microscopy |
| title_full | A Three-Dimensional Model of the Yeast Transmembrane Sensor Wsc1 Obtained by SMA-Based Detergent-Free Purification and Transmission Electron Microscopy |
| title_fullStr | A Three-Dimensional Model of the Yeast Transmembrane Sensor Wsc1 Obtained by SMA-Based Detergent-Free Purification and Transmission Electron Microscopy |
| title_full_unstemmed | A Three-Dimensional Model of the Yeast Transmembrane Sensor Wsc1 Obtained by SMA-Based Detergent-Free Purification and Transmission Electron Microscopy |
| title_short | A Three-Dimensional Model of the Yeast Transmembrane Sensor Wsc1 Obtained by SMA-Based Detergent-Free Purification and Transmission Electron Microscopy |
| title_sort | three dimensional model of the yeast transmembrane sensor wsc1 obtained by sma based detergent free purification and transmission electron microscopy |
| topic | Wsc1 membrane sensor SMALP detergent-free extraction fluorescence correlation spectroscopy transmission electron microscopy |
| url | https://www.mdpi.com/2309-608X/7/2/118 |
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