We have previously succeeded in measuring intervesicular transfer of phospholipids by time-resolved small-angle neutron scattering (TR-SANS)1 . This technique is applicable to the assay of lipid transfer activities of proteins. Sec14, a major yeast phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein, is known to regulate metabolism of PI and PC and vesicular transport from the trans-Golgi network (TGN). Sec14 transfers PC and PI in vitro, but the details of its mechanism, especially, how this activity is related to the intracellular functions, are still unknown. We carried out TR-SANS experiments with this protein.
Neutron scattering intensity change after mixing large unilamellar vesicles consisting of either 1-palmitoyl-2-oleoylphosphatidylcholine (H-LUVs) or 1-palmitoyl(d31)-2- oleoylphosphatidylcholine (D-LUVs) was monitored at 37˚C. Sec14 decayed the scattering intensity in a concentration-dependent manner, suggesting that Sec14 stimulates transfer of phospholipids between H- and D-LUVs.
It was also shown by TR-SANS that the PC transfer activity of Sec14 was increased in the presence of PI and phosphatidylserine (PS) in vesicles. On the other hand, surface plasmon resonance revealed that the membrane-binding property of Sec14 was unaffected by PI and PS. These results imply that these acidic lipids raise the lipid transfer activity not by increasing membrane-binding rate but by increasing lipid-exchanging rate of Sec14 on the membranes.