title: The SecA and SecY subunits of translocase are the nearest neighbors of a translocating preprotein, shielding it from phospholipids.
abstract: To study the environment of a preprotein as it crosses the plasma membrane of Escherichia coli, unique cysteinyl residues were introduced into proOmpA and the genes for these mutant preproteins were fused to the gene of dihydrofolate reductase (Dhfr). A photoactivable, radiolabeled and reducible cross-linker was then attached to the unique cysteinyl residue of each purified protein. Partially translocated polypeptides were generated and arrested in their membrane transit by the folded structure of the dihydrofolate reductase domain. After photolysis to label their nearest neighbors and reduction of the disulfide bond between proOmpA-Dhfr and the cross-linker, radiolabeled cross-linker was selectively recovered with the SecA and SecY subunits of preprotein translocase. Strikingly, neither the SecE nor Band 1 subunits were cross-linked to any of the constructs and the membrane phospholipids were almost entirely shielded from cross-linking. The fact that SecY and SecA are the only membrane proteins cross-linked to the translocating chains suggests that they may form an entirely proteinaceous pathway through which secreted proteins pass during membrane transit.
authors.affiliation: Molecular Biology Institute, University of California, Los Angeles 90024-1570.
authors.forename: J C
authors.initials: JC
authors.lastname: Joly
authors.forename: W
authors.initials: W
authors.lastname: Wickner
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001436:Bacteriorhodopsins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D008563:Membrane Lipids / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008961:Models, Structural; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D010743:Phospholipids / Q000378:metabolism*; D011487:Protein Conformation; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D008563:Membrane Lipids; D026901:Membrane Transport Proteins; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D010743:Phospholipids; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D000069816:SEC Translocation Channels; C057841:SecY protein, E coli; C057184:outer membrane protein A precursor (E coli); D001436:Bacteriorhodopsins; C017827:bacterio-opsin; D013762:Tetrahydrofolate Dehydrogenase; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins