Requirements for the translocation of elongation-arrested, ribosome-associated OmpA across the plasma membrane of Escherichia coli.
An oligodeoxynucleotide-dependent method to generate nascent polypeptide chains was adopted for use in a cell-free translation system prepared from Escherichia coli. In this way, NH2-terminal pOmpA fragments of distinct sizes were synthesized. Because most of these pOmpA fragments could be covalently linked to puromycin, precipitated with cetyltrimethylammonium bromide, and were enriched by sedimentation, they represent a population of elongation-arrested, ribosome-associated nascent chains. Translocation of these nascent pOmpA chains into inside-out membrane vesicles of E. coli required SecA and (depending on size) SecB. Whereas their translocation was strictly dependent on the H+-motive force of the vesicles, no indication for the involvement of the bacterial signal recognition particle was obtained. SecA and SecB, although required for translocation, did not mediate binding of the ribosome-associated pOmpA to membrane vesicles. However, SecA and SecB cotranslationally associated with nascent pOmpA, since they could be co-isolated with the ribosome-associated nascent chains and as such catalyzed translocation subsequent to the release of the ribosome. These results indicate that in E. coli, SecA also functionally interacts with preproteins before they are targeted to the translocase of the plasma membrane.
Behrmann
M
M
Adolf Butenandt Institut für Physikalische Biochemie, Ludwig-Maximilians-Universität München, D-80336 München, Germany.
Koch
H G
HG
Hengelage
T
T
Wieseler
B
B
Hoffschulte
H K
HK
Müller
M
M
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D014176:Protein Biosynthesis; D012270:Ribosomes / Q000378:metabolism*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D014908:Triticum / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; C057842:SecB protein, Bacteria; C078167:OMPA outer membrane proteins; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins