Evaluating the oligomeric state of SecYEG in preprotein translocase.
SecA insertion and deinsertion through SecYEG drive preprotein translocation at the Escherichia coli inner membrane. We present three assessments of the theory that oligomers of SecYEG might form functional translocation sites. (i) Formaldehyde cross- linking of translocase reveals cross-links between SecY, SecE and SecG, but not higher order oligomers. (ii) Cross-linking of membranes containing unmodified SecE and hemagglutinin-tagged SecE (SecE(HA)) reveals cross-links between SecY and SecE and between SecY and SecE(HA). However, anti-HA immunoprecipitates contain neither untagged SecE nor SecY cross-linked to SecE. (iii) Membranes containing similar amounts of SecE and SecE(HA) were saturated with translocation intermediate (I(29)) and detergent solubilized. Anti-HA immunoprecipitation of I(29) required SecYE(HA)G and SecA, yet untagged SecE was not present in this translocation complex. Likewise, anti-HA immunoprecipitates of membranes containing equal amounts of SecY and SecY(HA) were found to contain SecY(HA) but not SecY. Both immunoprecipitates contain more moles of I(29) than of the untagged subunit, again suggesting that translocation intermediates are not engaged with multiple copies of SecYEG. These studies suggest that the active form of preprotein translocase is monomeric SecYEG.
Yahr
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Department of Biochemistry, Dartmouth Medical School, 7200 Vail Building, Hanover, NH 03755-3844, USA.
Wickner
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D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D018988:Bacterial Translocation; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D029968:Escherichia coli Proteins; D005557:Formaldehyde / Q000494:pharmacology; D006388:Hemagglutinins / Q000378:metabolism; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008954:Models, Biological; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D006388:Hemagglutinins; D008024:Ligands; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; C088616:SecG protein, E coli; C057841:SecY protein, E coli; D005557:Formaldehyde; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins