Interaction between SecA and SecYEG in micellar solution and formation of the membrane-inserted state

Biochemistry. 1998 Jan 6;37(1):201-10. doi: 10.1021/bi972105t.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / metabolism*
  • Adenosine Triphosphate / physiology
  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism*
  • Biological Transport
  • Detergents
  • Enzyme Stability
  • Escherichia coli / enzymology*
  • Escherichia coli Proteins*
  • Glucosides
  • Macromolecular Substances
  • Membrane Proteins / chemistry
  • Membrane Proteins / isolation & purification
  • Membrane Proteins / metabolism*
  • Membrane Transport Proteins*
  • Micelles
  • Molecular Sequence Data
  • Operon
  • Protein Binding
  • Protein Structure, Secondary
  • SEC Translocation Channels
  • SecA Proteins
  • Solubility


  • Bacterial Proteins
  • Detergents
  • Escherichia coli Proteins
  • Glucosides
  • Macromolecular Substances
  • Membrane Proteins
  • Membrane Transport Proteins
  • Micelles
  • SEC Translocation Channels
  • SecE protein, E coli
  • SecG protein, E coli
  • SecY protein, E coli
  • octyl-beta-D-glucoside
  • Adenosine Triphosphate
  • Adenosine Triphosphatases
  • SecA Proteins