Molecular aspects of GAPR-1 interactions with biological and model membranes

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). It is a peripheral membrane protein that strongly associates with the cytosolic leaflet of Golgi membranes and is enriched in lipid rafts. Although the majority of GAPR-1 is localized to the Golgi membranes, some recent evidence suggests that it may also localize to other membranes. The determinants of GAPR-1 membrane binding and cellular localization are unknown. It has been suggested that GAPR-1 membrane binding may be regulated by a combination of factors including a myristoyl anchor, interactions with lipids, and protein-protein interactions. By use of a liposome binding assay and a protein-lipid overlay we show that non-myristoylated GAPR-1 binds to negatively charged lipids with highest affinity for phosphoinositides. Although phosphoinositide binding of GAPR-1 shows some degree of promiscuity, the binding efficiency to different phosphoinositides did not correlate with negative charges of the lipid, implying that GAPR-1 recognizes other features of the phosphoinositide molecules. 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, which may indicate that GAPR-1 binds to phosphatidylinositol with a covalent bond. Mass spectrometric analysis showed that up to 3 molecules of phosphatidylinositol can bind to GAPR-1. We also found factors that inhibit membrane binding of GAPR-1. Phosphorylation may be involved in membrane localization as a small but distinct pool of highly phosphorylated GAPR-1 could be identified in cytosol. Furthermore, we show that phytic acid is a potent inhibitor of membrane binding of GAPR-1 to liposomes. Previously, we suggested that dimerization plays a role in the function of GAPR-1 and therefore the effect of phytic acid on the dimerization characteristics was investigated. By size-exclusion chromatography, it was found that phytic acid promotes dimer formation of GAPR-1 in solution. Elucidation of the crystal structure of GAPR-1 in the presence of phytic acid revealed that the GAPR-1 dimer differs from the GAPR-1 dimer formed in the absence of phytic acid. In the presence of phytic acid, the monomeric subunits of the dimer appear to be rotated by 28.3 relative to each other. As a consequence the dimer interface displays a different geometry with different amino acids stabilizing the dimer conformation. Mutation of alanine 68 to a lysine (A68K), an interface contact site unique to the rotated dimer structure, did not prevent the induction of dimer formation by phytic acid. This indicates that mutant GAPR-1 is still capable of binding to phytic acid. However, phytic acid did not inhibit the membrane binding of A68K GAPR-1 to liposomes anymore. The existence of different GAPR-1 dimer conformations may be an important regulatory mechanism of GAPR-1 function. The results obtained in this study suggest that multiple signals act in concert and that changes in each signal may determine membrane binding and subcellular localization of GAPR-1. As binding of GAPR-1 to membranes is multi-factorial, this allows cells to regulate GAPR-1 at multiple levels.