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

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

Performance of the commercially available SERION ELISA classic Echinococcus IgG test for the detection of cystic echinococcosis in clinical practice

Diagnosis of cystic echinococcosis (CE) is at present mainly based on imaging techniques. Serology has a complementary role, partly due to the small number of standardized and commercially available assays. Therefore we examined the clinical performance of the SERION ELISA classic Echinococcus IgG test. Using 10 U/ml as a cut-off point, and serum samples from 50 CE patients and 105 healthy controls, the sensitivity and specificity were 98.0% and 96.2%, respectively. If patients with other infectious diseases were used as negative controls, the specificity decreased to 76.9%, which causes poor positive predictive values. However, if results between 10 and 15 U/ml are classified as indecisive, the specificity of positive results (≥15 U/ml) increased to 92.5% without greatly affecting the sensitivity (92.0%). Using this approach in combination with imaging studies, the SERION ELISA classic Echinococcosis IgG test can be a useful aid in the diagnosis of CE

Phosphatidylinositol-3-Phosphate Clearance Plays a Key Role in Autophagosome Completion

SummaryBackgroundThe biogenesis of autophagosomes, the hallmark of autophagy, depends on the function of the autophagy-related (Atg) proteins and the generation of phosphatidylinositol-3-phosphate (PtdIns3P) at the phagophore assembly site (PAS), the location where autophagosomes arise. The current model is that PtdIns3P is involved primarily in the recruitment of Atg proteins to the PAS and that once an autophagosome is complete, the Atg machinery is released from its surface back into the cytoplasm and reused for the formation of new vesicles.ResultsWe have identified a PtdIns3P phosphatase, Ymr1, that is essential for the normal progression of both bulk and selective types of autophagy. This protein is recruited to the PAS at an early stage of formation of this structure through a process that requires both its GRAM domain and its catalytic activity. In the absence of Ymr1, Atg proteins fail to dissociate from the limiting membrane of autophagosomes, and these vesicles accumulate in the cytoplasm.ConclusionsOur data thus reveal a key role for PtdIns3P turnover in the regulation of the late steps of autophagosome biogenesis and indicate that the disassembly of the Atg machinery from the surface of autophagosomes is a requisite for their fusion with the vacuole