Interaction of the Spo20 membrane-sensor motif with phosphatidic acid and other anionic lipids, and influence of the membrane environment

PLoS One. 2014 Nov 26;9(11):e113484. doi: 10.1371/journal.pone.0113484. eCollection 2014.

Abstract

The yeast protein Spo20 contains a regulatory amphipathic motif that has been suggested to recognize phosphatidic acid, a lipid involved in signal transduction, lipid metabolism and membrane fusion. We have investigated the interaction of the Spo20 amphipathic motif with lipid membranes using a bioprobe strategy that consists in appending this motif to the end of a long coiled-coil, which can be coupled to a GFP reporter for visualization in cells. The resulting construct is amenable to in vitro and in vivo experiments and allows unbiased comparison between amphipathic helices of different chemistry. In vitro, the Spo20 bioprobe responded to small variations in the amount of phosphatidic acid. However, this response was not specific. The membrane binding of the probe depended on the presence of phosphatidylethanolamine and also integrated the contribution of other anionic lipids, including phosphatidylserine and phosphatidyl-inositol-(4,5)bisphosphate. Inverting the sequence of the Spo20 motif neither affected the ability of the probe to interact with anionic liposomes nor did it modify its cellular localization, making a stereo-specific mode of phosphatidic acid recognition unlikely. Nevertheless, the lipid binding properties and the cellular localization of the Spo20 alpha-helix differed markedly from that of another amphipathic motif, Amphipathic Lipid Packing Sensor (ALPS), suggesting that even in the absence of stereo specific interactions, amphipathic helices can act as subcellular membrane targeting determinants in a cellular context.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Cell Line
  • Epithelial Cells / cytology
  • Epithelial Cells / metabolism
  • Gene Expression Regulation
  • Genes, Reporter
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Ligands
  • Liposomes / chemistry
  • Liposomes / metabolism
  • Molecular Probes
  • Molecular Sequence Data
  • Phosphatidic Acids / metabolism*
  • Phosphatidylethanolamines / chemistry
  • Phosphatidylethanolamines / metabolism
  • Phosphatidylinositol Phosphates / chemistry
  • Phosphatidylinositol Phosphates / metabolism
  • Phosphatidylserines / chemistry
  • Phosphatidylserines / metabolism
  • Protein Binding
  • Qb-SNARE Proteins / chemistry
  • Qb-SNARE Proteins / genetics
  • Qb-SNARE Proteins / metabolism*
  • Qc-SNARE Proteins / chemistry
  • Qc-SNARE Proteins / genetics
  • Qc-SNARE Proteins / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Signal Transduction

Substances

  • Ligands
  • Liposomes
  • Molecular Probes
  • Phosphatidic Acids
  • Phosphatidylethanolamines
  • Phosphatidylinositol Phosphates
  • Phosphatidylserines
  • Qb-SNARE Proteins
  • Qc-SNARE Proteins
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Spo20 protein, S cerevisiae
  • Green Fluorescent Proteins
  • phosphatidylethanolamine

Grant support

This work was supported by grants from the Agence Nationale de la Recherche (ANR-08-0060-01 and “Investments for the Future” LABEX SIGNALIFE ANR-11-LABX-0028-01) and by an advanced grant from the European Research Council (ERC 268888). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.