A mutant hunt for defects in membrane protein assembly yields mutations affecting the bacterial signal recognition particle and Sec machinery.
We describe an Escherichia coli genetic screen that yields mutations affecting two different cellular processes: disulfide bond formation and membrane protein assembly. The mutants defective in disulfide bond formation include additional classes of dsbA and dsbB mutations. The membrane protein assembly defective mutants contain a mutation in the secA operon and three mutations in the ffs gene, which encodes 4.5S RNA. These latter mutations are the only ones to be isolated in a gene encoding a component of the bacterial signal recognition particle by screening in vivo for defects in membrane protein insertion. A sensitive method for examining membrane protein localization shows that the ffs and secA locus mutations affect membrane assembly of the polytopic membrane protein, MalF. The ffs mutations also affect the membrane insertion of the FtsQ and the AcrB proteins. Although both the ffs and the secA locus mutations interfere with membrane protein assembly, only the latter also reduces export of a protein containing a cleavable signal sequence.
Tian
H
H
Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.
Boyd
D
D
Beckwith
J
J
D000251:Adenosine Triphosphatases / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D009154:Mutation; D009876:Operon; D010957:Plasmids; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D018271:Signal Recognition Particle / Q000235:genetics*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; C079503:DsbB protein, Bacteria; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D000069816:SEC Translocation Channels; D018271:Signal Recognition Particle; C078167:OMPA outer membrane proteins; D000251:Adenosine Triphosphatases; D019704:Protein Disulfide-Isomerases; D000081416:SecA Proteins