Synergistic insertion of two hydrophobic regions drives Sec-independent membrane protein assembly.
We have studied the membrane insertion of two proteins from the inner membrane of Escherichia coli, both with two transmembrane segments connected by a short periplasmic loop: the M13 procoat protein and a mutant "inverted" leader peptidase. Neither molecule depends on the Sec machinery for insertion. We show that the introduction of a charged residue in the second transmembrane segment completely blocks insertion of both proteins. In contrast, a Sec-dependent procoat mutant, where the periplasmic region has been lengthened, inserts into the membrane even in the presence of a charged residue in the second hydrophobic domain. In addition, a large deletion within the second transmembrane domain of the leader peptidase mutant allows membrane translocation, but only under conditions where the SecA protein is functional. Furthermore, we show that the first hydrophobic domain is required for insertion of the short periplasmic loop of the "inverted" leader peptidase. These results suggest that Sec-independent insertion occurs by a synergistic entry of the two neighboring hydrophobic domains into the lipid bilayer.
Cao
G
G
Department of Chemistry, Ohio State University, Columbus 43210.
Cheng
S
S
Whitley
P
P
von Heijne
G
G
Kuhn
A
A
Dalbey
R E
RE
D001426:Bacterial Proteins / Q000378:metabolism; D002213:Capsid / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins; D002462:Cell Membrane / Q000378:metabolism*; D016764:Cell Polarity; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008961:Models, Structural; D009154:Mutation; D011487:Protein Conformation; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism
D001426:Bacterial Proteins; D036022:Capsid Proteins; D008565:Membrane Proteins; D011498:Protein Precursors; C025668:coat protein, Bacteriophage M13; D010450:Endopeptidases; D012697:Serine Endopeptidases; D014357:Trypsin; C026515:type I signal peptidase