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Molecular Membrane Biology
ISSN: 0968-7688 (Print) 1464-5203 (Online) Journal homepage: https://www.tandfonline.com/loi/imbc20
Binding of GAPR-1 to negatively charged phospholipid membranes: Unusual binding characteristics to phosphatidylinositol
Josse Van Galen, Bas W. M. Van Balkom, Ramon L. Serrano, Dora Kaloyanova, Ruud Eerland, Ernstpeter Stüven & J. Bernd Helms
To cite this article: Josse Van Galen, Bas W. M. Van Balkom, Ramon L. Serrano, Dora Kaloyanova, Ruud Eerland, Ernstpeter Stüven & J. Bernd Helms (2010) Binding of GAPR-1 to negatively charged phospholipid membranes: Unusual binding characteristics to phosphatidylinositol, Molecular Membrane Biology, 27:2-3, 81-91, DOI:
10.3109/09687680903507080
To link to this article: https://doi.org/10.3109/09687680903507080
Published online: 25 Jan 2010.
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Molecular Membrane Biology, February –April 2010; 27(2–3): 81–91
Binding of GAPR-1 to negatively charged phospholipid membranes:
Unusual binding characteristics to phosphatidylinositol
JOSSE VAN GALEN 1 *, BAS W. M. VAN BALKOM 2‡ , RAMON L. SERRANO 1 , DORA KALOYANOVA 1 , RUUD EERLAND 1 , ERNSTPETER STÜVEN 1 , &
J. BERND HELMS 1
1 Department of Biochemistry and Cell Biology and Institute of Biomembranes, Utrecht University, Utrecht, and
2 Institute for Veterinary Research, IVW/GSAH, Utrecht; Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands
(Received 17 June 2009; and in revised form 29 October 2009)
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 lea flet of the Golgi membrane. The myristoyl moiety at the N-terminus of GAPR-1 contributes to membrane binding but is not suf ficient 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 Abbreviations: CAPproteins, cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins;
GAPR-1, Golgi Associated plant Pathogenesis Related protein 1; GPI, glycosylphosphatidylinositol; MALDI-TOF, Matrix-assisted LASER desorption/ionization time-of-flight; PA, Phosphatidic acid; PC, Phosphatidylcholine; PE, Phosphatidylethanolamine; PI, Phosphatidylinositol; PR-1, plant pathogenesis-related proteins group 1; PS, Phospha- tidylserine; SCP, Sperm-coating protein; SDS-PAGE, Sodium dodecyl sulphate polyacrylamide gelelectrophoresis;
SM, sphingomyelin; TX114, Triton X-114.
Introduction
Golgi-Associated Plant Pathogenesis-Related protein 1 (GAPR-1) is a mammalian protein, which is well conserved among vertebrates and is known under
different aliases such as GLIPR-2 and C9orf19 [1 –3]. In mice, GAPR-1 is predominantly expressed in the lung, spleen, monocytes, uterus and embryonic tissue [3]. In humans, GAPR-1 expression has thus far been described in peripheral leukocytes and
*
Current address: Department of Cell and Developmental Biology, CRG-Centre de Regulació Genòmica, C/. Dr. Aiguader, 88 08003 Barcelona, Spain
‡
Current address: Department of Nephrology and Hypertension, G 02.402, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
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