Binding of GAPR-1 to negatively charged phospholipid membranes: Unusual binding characteristics to phosphatidylinositol

Abstract

Golgi-Associated Plant Pathogenesis-Related protein 1 (GAPR-1) is a mammalian protein that belongs to the superfamily of plant pathogenesis-related proteins group 1 (PR-1). GAPR-1 strongly associates with lipid rafts at the cytosolic leaflet of the Golgi membrane. The myristoyl moiety at the N-terminus of GAPR-1 contributes to membrane binding but is not sufficient for stable membrane anchorage. GAPR-1 is positively charged at physiological pH, which allows for additional membrane interactions with proteins or lipids. To determine the potential contribution of lipids to membrane binding of GAPR-1, we used a liposome binding assay. Here we report that non-myristoylated GAPR-1 stably binds liposomes that contain the negatively charged lipids phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, or phosphatidic acid. GAPR-1 displays the highest preference for phosphatidic acid-containing liposomes. In contrast, lysozyme, which contains a similar surface charge, did not bind to these liposomes, except for a weak membrane association with PA-containing liposomes. Interestingly, GAPR-1 binds to phosphatidylinositol with unusual characteristics. Denaturation or organic extraction of GAPR-1 does not result in dissociation of phosphatidylinositol from GAPR-1. The association of phosphatidylinositol with GAPR-1 results in a diffuse gel-shift in SDS-PAGE. Mass spectrometric analysis of gel-shifted GAPR-1 showed the association of up to 3 molecules of phosphatidylinositol with GAPR-1. These results suggest that the lipid composition contributes to the GAPR-1 binding to biological membranes.

Keywords::

• GAPR-1
• Plant pathogenesis-related proteins
• phosphatidylinositol
• membrane binding
• protein-lipid interaction

Acknowledgements

We thank Jos Brouwers for help with initial mass spectroscopic analyses of lipids bound to GAPR-1, and Coos Batenburg for critically reviewing our manuscript.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.