Van Galen Josse, Van Balkom Bas W M, Serrano Ramon L, Kaloyanova Dora, Eerland Ruud, Stüven Ernstpeter, Helms J Bernd
Department of Biochemistry and Cell Biology and Institute of Biomembranes, Utrecht University, Utrecht.
Mol Membr Biol. 2010 Apr;27(2-3):81-91. doi: 10.3109/09687680903507080.
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.
高尔基体相关植物病程相关蛋白1（GAPR-1）是一种哺乳动物蛋白，属于植物病程相关蛋白1组（PR-1）超家族。GAPR-1与高尔基体膜胞质小叶中的脂筏紧密结合。GAPR-1 N端的肉豆蔻酰基部分有助于膜结合，但不足以实现稳定的膜锚定。GAPR-1在生理pH值下带正电荷，这使得它能够与蛋白质或脂质进行额外的膜相互作用。为了确定脂质对GAPR-1膜结合的潜在贡献，我们使用了脂质体结合试验。在此我们报告，非肉豆蔻酰化的GAPR-1能稳定结合含有带负电荷脂质磷脂酰丝氨酸、磷脂酰甘油、磷脂酰肌醇或磷脂酸的脂质体。GAPR-1对含磷脂酸的脂质体表现出最高的偏好性。相比之下，表面电荷相似的溶菌酶除了与含PA的脂质体有微弱的膜结合外，不与这些脂质体结合。有趣的是，GAPR-1以不同寻常的方式结合磷脂酰肌醇。GAPR-1的变性或有机溶剂提取不会导致磷脂酰肌醇从GAPR-1上解离。磷脂酰肌醇与GAPR-1的结合导致SDS-PAGE中出现弥散的凝胶迁移。对凝胶迁移的GAPR-1进行质谱分析表明，GAPR-1最多可与3个磷脂酰肌醇分子结合。这些结果表明脂质组成有助于GAPR-1与生物膜的结合。
