Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Voskoboynikova, Natalia, Karlova, Maria, Kurre, Rainer, Mulkidjanian, Armen Y., Shaitan, Konstantin V., Sokolova, Olga S., Steinhoff, Heinz-Jürgen, Heinisch, Jürgen J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915640/
https://www.ncbi.nlm.nih.gov/pubmed/33562593
http://dx.doi.org/10.3390/jof7020118
_version_ 1783657290538156032
author Voskoboynikova, Natalia
Karlova, Maria
Kurre, Rainer
Mulkidjanian, Armen Y.
Shaitan, Konstantin V.
Sokolova, Olga S.
Steinhoff, Heinz-Jürgen
Heinisch, Jürgen J.
author_facet Voskoboynikova, Natalia
Karlova, Maria
Kurre, Rainer
Mulkidjanian, Armen Y.
Shaitan, Konstantin V.
Sokolova, Olga S.
Steinhoff, Heinz-Jürgen
Heinisch, Jürgen J.
author_sort Voskoboynikova, Natalia
collection PubMed
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.
format Online
Article
Text
id pubmed-7915640
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-79156402021-03-01 A Three-Dimensional Model of the Yeast Transmembrane Sensor Wsc1 Obtained by SMA-Based Detergent-Free Purification and Transmission Electron Microscopy Voskoboynikova, Natalia Karlova, Maria Kurre, Rainer Mulkidjanian, Armen Y. Shaitan, Konstantin V. Sokolova, Olga S. Steinhoff, Heinz-Jürgen Heinisch, Jürgen J. J Fungi (Basel) Article 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. MDPI 2021-02-05 /pmc/articles/PMC7915640/ /pubmed/33562593 http://dx.doi.org/10.3390/jof7020118 Text en © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Voskoboynikova, Natalia
Karlova, Maria
Kurre, Rainer
Mulkidjanian, Armen Y.
Shaitan, Konstantin V.
Sokolova, Olga S.
Steinhoff, Heinz-Jürgen
Heinisch, Jürgen J.
A Three-Dimensional Model of the Yeast Transmembrane Sensor Wsc1 Obtained by SMA-Based Detergent-Free Purification and 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 Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915640/
https://www.ncbi.nlm.nih.gov/pubmed/33562593
http://dx.doi.org/10.3390/jof7020118
work_keys_str_mv AT voskoboynikovanatalia athreedimensionalmodeloftheyeasttransmembranesensorwsc1obtainedbysmabaseddetergentfreepurificationandtransmissionelectronmicroscopy
AT karlovamaria athreedimensionalmodeloftheyeasttransmembranesensorwsc1obtainedbysmabaseddetergentfreepurificationandtransmissionelectronmicroscopy
AT kurrerainer athreedimensionalmodeloftheyeasttransmembranesensorwsc1obtainedbysmabaseddetergentfreepurificationandtransmissionelectronmicroscopy
AT mulkidjanianarmeny athreedimensionalmodeloftheyeasttransmembranesensorwsc1obtainedbysmabaseddetergentfreepurificationandtransmissionelectronmicroscopy
AT shaitankonstantinv athreedimensionalmodeloftheyeasttransmembranesensorwsc1obtainedbysmabaseddetergentfreepurificationandtransmissionelectronmicroscopy
AT sokolovaolgas athreedimensionalmodeloftheyeasttransmembranesensorwsc1obtainedbysmabaseddetergentfreepurificationandtransmissionelectronmicroscopy
AT steinhoffheinzjurgen athreedimensionalmodeloftheyeasttransmembranesensorwsc1obtainedbysmabaseddetergentfreepurificationandtransmissionelectronmicroscopy
AT heinischjurgenj athreedimensionalmodeloftheyeasttransmembranesensorwsc1obtainedbysmabaseddetergentfreepurificationandtransmissionelectronmicroscopy
AT voskoboynikovanatalia threedimensionalmodeloftheyeasttransmembranesensorwsc1obtainedbysmabaseddetergentfreepurificationandtransmissionelectronmicroscopy
AT karlovamaria threedimensionalmodeloftheyeasttransmembranesensorwsc1obtainedbysmabaseddetergentfreepurificationandtransmissionelectronmicroscopy
AT kurrerainer threedimensionalmodeloftheyeasttransmembranesensorwsc1obtainedbysmabaseddetergentfreepurificationandtransmissionelectronmicroscopy
AT mulkidjanianarmeny threedimensionalmodeloftheyeasttransmembranesensorwsc1obtainedbysmabaseddetergentfreepurificationandtransmissionelectronmicroscopy
AT shaitankonstantinv threedimensionalmodeloftheyeasttransmembranesensorwsc1obtainedbysmabaseddetergentfreepurificationandtransmissionelectronmicroscopy
AT sokolovaolgas threedimensionalmodeloftheyeasttransmembranesensorwsc1obtainedbysmabaseddetergentfreepurificationandtransmissionelectronmicroscopy
AT steinhoffheinzjurgen threedimensionalmodeloftheyeasttransmembranesensorwsc1obtainedbysmabaseddetergentfreepurificationandtransmissionelectronmicroscopy
AT heinischjurgenj threedimensionalmodeloftheyeasttransmembranesensorwsc1obtainedbysmabaseddetergentfreepurificationandtransmissionelectronmicroscopy