Deep penetration of a portion of Escherichia coli SecA protein into model membranes is promoted by anionic phospholipids and by partial unfolding.
SecA protein, a principal component of the protein export machinery of Escherichia coli, is found both in the cytoplasm and inner membrane of cells. Previous in vitro and in vivo studies demonstrated that the interaction of SecA with the inner membrane requires the presence of physiological levels of anionic (acidic) phospholipids. In this report the degree of SecA insertion into model membranes and the conformational changes associated with this event have been examined. The extent of association of SecA with model membranes was determined by photolabeling with a hydrophobic reagent, and the depth of insertion of the protein into the phospholipid bilayer was determined by the amount of quenching of SecA fluorescence by both brominated and spin-labeled phospholipids. These methods demonstrated that SecA penetrates deep within the acyl chain region of the phospholipid bilayer. It was also found that SecA penetration into vesicles was associated with a major conformational change in the protein. This change can be induced by higher temperatures and involves a partial unfolding event as judged by differential scanning calorimetry, SecA fluorescence and increased sensitivity to proteolysis. These properties suggest the induction of a molten-globule-like conformation in a portion of the SecA polypeptide. This change was also induced at lower temperatures by the presence of membranes containing a physiological amount of the anionic phospholipid, phosphatidylglycerol. The partial unfolding and concomitant deep insertion of SecA into membranes may aid in the insertion of precursor proteins into the inner membrane and may influence possible interactions between SecA and the integral membrane export machinery components.
Ulbrandt
N D
ND
Department of Microbiology, State University of New York, Stony Brook 11794-5215.
London
E
E
Oliver
D B
DB
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000838:Anions; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005454:Fluorescence Polarization; D007425:Intracellular Membranes / Q000378:metabolism*; D026901:Membrane Transport Proteins; D010743:Phospholipids / Q000378:metabolism*; D011487:Protein Conformation; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D013696:Temperature
D000838:Anions; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D010743:Phospholipids; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins