Nucleotide and negatively charged lipid-dependent vesicle aggregation caused by SecA: Evidence that SecA contains two lipid-binding sites

• Published in: FEBS letters 1993-09, Vol.331 (1), p.19-24
• Main Authors: Breukink, Eefjan, Keller, Rob C.A., Kruijff, Ben de
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; Bacterial Proteins - metabolism; Bacteriology; Binding Sites; Biological and medical sciences; Escherichia coli Proteins; Fluorescence Polarization; Fundamental and applied biological sciences. Psychology; Hydrolysis; Lipid Bilayers; Lipid packing; Membrane Transport Proteins; Microbiology; Model membrane; Osmolar Concentration; Permeability, membrane transport, intracellular transport; Phosphatidylcholines - metabolism; Phosphatidylglycerols - metabolism; Protein translocation; SEC Translocation Channels; SecA; Temperature; Vesicle aggregation; Viscosity
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(93)80289-7

SecA which is an overall acidic protein was found to induce an increase in the turbidity of a solution of vesicles consisting of negatively charged phospholipids. This increase was found to be due to an aggregation of the vesicles mediated by SecA. The SecA-mediated vesicle aggregation was not found for zwitterionic 1,2-dioleoyl- sn-glycero-3-phosphocholine and showed a large dependence on both temperature and ionic strength. Furthermore it was shown that ATP and to a lesser extent ADP+P i were able to reduce the SecA-mediated vesicle aggregation, while no effect could be seen for a non-hydrolysable ATP analog AMP-PNP. Using the steady state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene present in 1,2-dioleoyl- sn-glycero-3-phosphoglycerol vesicles we could show that SecA inserts in the bilayer. Monolayer studies confirmed that SecA is able to cause close contact between two membranes and gave a direct insight into the different types of lipid-protein interactions involved. From our results we propose that the SecA monomer possesses two lipid-binding sites which in the functional dimer conformation are responsible for the SecA-mediated vesicle aggregation.