Determination of the lipid composition of the GPI anchor

PLoS One. 2021 Aug 13;16(8):e0256184. doi: 10.1371/journal.pone.0256184. eCollection 2021.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Membrane / chemistry
  • Cell Membrane / metabolism
  • Ceramides / chemistry
  • Ceramides / isolation & purification*
  • GPI-Linked Proteins / chemistry*
  • GPI-Linked Proteins / metabolism
  • Genes, Reporter
  • Glycosylphosphatidylinositols / chemistry*
  • Glycosylphosphatidylinositols / metabolism
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Mass Spectrometry
  • Membrane Glycoproteins / chemistry*
  • Membrane Glycoproteins / metabolism
  • Saccharomyces cerevisiae / chemistry*
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / metabolism

Substances

  • Ceramides
  • GAS1 protein, S cerevisiae
  • GPI-Linked Proteins
  • Glycosylphosphatidylinositols
  • Membrane Glycoproteins
  • Saccharomyces cerevisiae Proteins
  • Green Fluorescent Proteins

Grants and funding

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. The funders had and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.