FIGURE 8.

Phospholipid binding by Age1ΔN. Phospholipid membrane strips were incubated overnight at 4 °C with proteins (0.5 μg/ml), affinity-purified under native conditions from bacterial cells, and then washed and treated with Age1-specific affinity-purified antibodies; proteins bound to the phospholipid membrane strip were detected by enhanced chemiluminescence. No signal was detected when a membrane strip was treated with proteins isolated from bacteria carrying empty vector and probed with Age1-specific antibodies (data not shown). Incubation with 2.5 μg of C-terminal fragment of SidC, a phosphatidylinositol (PI) 4-phosphate (PI(4)P)-binding protein (29), demonstrated preferential binding of the protein to phosphatidylinositol 4-phosphate and to a much lesser extent to phosphatidylinositol 3,4-diphosphate (PI(3,4)P2) and phosphatidylinositol 4,5-diphosphate (PI(4,5)P2) (data not shown). The different pattern of binding for this phosphatidylinositol 4-phosphate (PI(4)P)-binding protein versus Age1ΔN-His₆ demonstrates that phospholipid binding is protein-specific. PE, phosphatidylethanolamine; PC, phosphatidylcholine; PA, phosphatidic acid; PS, phosphatidylserine; PI, phosphatidylinositol; LPA, lysophosphatidic acid; LPC, lysophosphocholine; S1P, sphingosine-1-phosphate.