Nucleotide and negatively charged lipid-dependent vesicle aggregation caused by SecA. Evidence that SecA contains two lipid-binding sites.
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+Pi 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.
Breukink
E
E
Department of Biochemistry of Membranes, University of Utrecht, The Netherlands.
Keller
R C
RC
de Kruijff
B
B
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D029968:Escherichia coli Proteins; D005454:Fluorescence Polarization; D006868:Hydrolysis; D008051:Lipid Bilayers; D026901:Membrane Transport Proteins; D009994:Osmolar Concentration; D010713:Phosphatidylcholines / Q000378:metabolism*; D010715:Phosphatidylglycerols / Q000378:metabolism*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D013696:Temperature; D014783:Viscosity
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D026901:Membrane Transport Proteins; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D000069816:SEC Translocation Channels; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins; C017251:1,2-oleoylphosphatidylcholine