Determination of the lipid composition of the GPI anchor.

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 sp...

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Main Authors: Auxiliadora Aguilera-Romero, Susana Sabido-Bozo, Sergio Lopez, Alejandro Cortes-Gomez, Sofia Rodriguez-Gallardo, Ana Maria Perez-Linero, Isabelle Riezman, Howard Riezman, Manuel Muñiz
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2021-01-01
Series:PLoS ONE
Online Access:https://doi.org/10.1371/journal.pone.0256184
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author Auxiliadora Aguilera-Romero
Susana Sabido-Bozo
Sergio Lopez
Alejandro Cortes-Gomez
Sofia Rodriguez-Gallardo
Ana Maria Perez-Linero
Isabelle Riezman
Howard Riezman
Manuel Muñiz
author_facet Auxiliadora Aguilera-Romero
Susana Sabido-Bozo
Sergio Lopez
Alejandro Cortes-Gomez
Sofia Rodriguez-Gallardo
Ana Maria Perez-Linero
Isabelle Riezman
Howard Riezman
Manuel Muñiz
author_sort Auxiliadora Aguilera-Romero
collection DOAJ
description 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.
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spelling doaj.art-2907df15772340d8b321990edcd67aef2022-12-21T19:23:19ZengPublic Library of Science (PLoS)PLoS ONE1932-62032021-01-01168e025618410.1371/journal.pone.0256184Determination of the lipid composition of the GPI anchor.Auxiliadora Aguilera-RomeroSusana Sabido-BozoSergio LopezAlejandro Cortes-GomezSofia Rodriguez-GallardoAna Maria Perez-LineroIsabelle RiezmanHoward RiezmanManuel MuñizIn 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.https://doi.org/10.1371/journal.pone.0256184
spellingShingle Auxiliadora Aguilera-Romero
Susana Sabido-Bozo
Sergio Lopez
Alejandro Cortes-Gomez
Sofia Rodriguez-Gallardo
Ana Maria Perez-Linero
Isabelle Riezman
Howard Riezman
Manuel Muñiz
Determination of the lipid composition of the GPI anchor.
PLoS ONE
title Determination of the lipid composition of the GPI anchor.
title_full Determination of the lipid composition of the GPI anchor.
title_fullStr Determination of the lipid composition of the GPI anchor.
title_full_unstemmed Determination of the lipid composition of the GPI anchor.
title_short Determination of the lipid composition of the GPI anchor.
title_sort determination of the lipid composition of the gpi anchor
url https://doi.org/10.1371/journal.pone.0256184
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