NUCLEOTIDE AND NEGATIVELY CHARGED LIPID-DEPENDENT VESICLE AGGREGATIONCAUSED BY SECA - EVIDENCE THAT SECA CONTAINS 2 LIPID-BINDING SITES

SecA which is an overall acidic protein was found to induce an increas e in the turbidity of a solution of vesicles consisting of negatively charged phospholipids. This increase was found to be due to an aggrega tion of the vesicles mediated by SecA. The SecA-mediated vesicle aggre gation was not found for zwitterionic 1,2-dioleoyl-sn-glycero-3-phosph ocholine and showed a large dependence on both temperature and ionic s trength. Furthermore it was shown that ATP and to a lesser extent ADPP(i) were able to reduce the SecA-mediated vesicle aggregation, while no effect could be seen for a non-hydrolysable ATP analog AMP-PNP. Usi ng the steady state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexa triene present in 1,2-dioleoyl-sn-glycero-3-phosphoglycerol vesicles w e could show that SecA inserts in the bilayer. Monolayer studies confi rmed that SecA is able to cause close contact between two membranes an d gave a direct insight into the different types of lipid protein inte ractions involved. From our results we propose that the SecA monomer p ossesses two lipid-binding sites which in the functional dimer conform ation are responsible for the SecA-mediated vesicle aggregation.