Interaction between SecA and SecYEG in micellar solution and formation of the membrane-inserted state.
Preprotein translocation in Escherichia coli is mediated by the translocase with SecA as peripheral ATPase and SecY, SecE, and SecG as membrane domain. To facilitate large-scale purification of the SecYEG heterotrimer, SecY was fused at its amino terminus with a hexahistidine tag and co-overexpressed with SecE and SecG. The presence of the His tag allowed purification of homogeneously pure SecYEG complex by a single anion-exchange chromatographic step starting from octyl glucoside-solubilized inner membranes. Endogenous levels of SecD and SecF copurified with the SecYEG protein. Purified SecYEG complex retained a nativelike, alpha-helical conformation in octyl glucoside and in micellar solution binds SecA with high affinity. In the presence of the nonhydrolyzable nucleotide analogue adenosine 5'-(beta, gamma-imidotriphosphate), octyl glucoside-solubilized SecYEG is nearly as effective as the reconstituted enzyme in inducing the formation of a proteinase K-protected 30 kDa fragment of 125I-labeled SecA, while SecYEG is proteolyzed to fragments smaller than 6 kDa. These data demonstrate that the 30-kDa SecA fragment is not protected by the lipid phase nor by SecYEG but rather indicate that it represents a SecYEG- and nucleotide-induced stable conformational state of a SecA domain.
van der Does
C
C
Department of Microbiology, Groningen Biomolecularp5 Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
Manting
E H
EH
Kaufmann
A
A
Lutz
M
M
Driessen
A J
AJ
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000502:physiology; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001692:Biological Transport; D003902:Detergents; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005960:Glucosides; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008823:Micelles; D008969:Molecular Sequence Data; D009876:Operon; D011485:Protein Binding; D017433:Protein Structure, Secondary; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D012995:Solubility
D001426:Bacterial Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D005960:Glucosides; D046911:Macromolecular Substances; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008823:Micelles; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; C088616:SecG protein, E coli; C057841:SecY protein, E coli; C018619:octyl-beta-D-glucoside; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins