Membrane insertion of the mannitol permease of Escherichia coli occurs under conditions of impaired SecA function.
The membrane insertion of the mannitol permease (MtlA protein) of Escherichia coli, a polytopic cytoplasmic membrane protein possessing an uncleaved amphiphilic signal sequence, was studied using a cell-free protein synthesis system. The MtlA protein synthesized in the presence of inside-out cytoplasmic membrane vesicles was shown to insert into the membranes based on the following criteria: (a) co-sedimentation of the majority of the MtlA protein with the vesicles; (b) selective extraction of the membrane-associated MtlA by doxycholate but not by urea treatment; and (c) protease resistance of a defined MtlA fragment observed exclusively in the presence of membranes. Post-translational addition of membrane vesicles allowed membrane association of MtlA but did not allow efficient integration. In cell-free systems having reduced levels of the export factors SecA and SecB and exhibiting defective translocation of preOmpA and preLamB, insertion of the in vitro synthesized MtlA apparently occurred normally. In contrast, when membranes from the secY24ts mutant or trypsin-treated membranes were used, insertion of MtlA was reduced. In vivo experiments monitoring the permease activity of MtlA in the secA and secY mutants supported the conclusions of the in vitro results. Thus, the insertion of MtlA is essentially SecA- and SecB-independent but may be dependent on SecY and/or an as yet unidentified membrane protein. The requirements for the insertion of the mannitol permease are therefore clearly different from those for the translocation of most proteins having a cleavable hydrophobic signal sequence.
Werner
P K
PK
Biochemical Institute, University of Freiburg, Federal Republic of Germany.
Saier
M H
MH
Müller
M
M
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D007700:Kinetics; D008353:Mannitol / Q000378:metabolism*; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D013401:Sugar Alcohol Dehydrogenases / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011498:Protein Precursors; D000069816:SEC Translocation Channels; C057842:SecB protein, Bacteria; C057841:SecY protein, E coli; D008353:Mannitol; D013401:Sugar Alcohol Dehydrogenases; C021544:mannitol-1-phosphate dehydrogenase; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins