Requirements for translocation of periplasmic domains in polytopic membrane proteins.
Periplasmic domains of cytoplasmic membrane proteins require export signals for proper translocation. These signals were studied by using a MalF-alkaline phosphatase fusion in a genetic selection that allowed the isolation of mislocalization mutants. In the original construct, alkaline phosphatase is fused to the second periplasmic domain of the membrane protein, and its activity is thus confined exclusively to the periplasm. Mutants that no longer translocated alkaline phosphatase were selected by complementation of a serB mutation. A total of 11 deletions in the amino terminus were isolated, all of which spanned at least the third transmembrane segment. This domain immediately precedes the periplasmic domain to which alkaline phosphatase was fused. Our results obtained in vivo support the model that amino-terminal membrane-spanning segments are required for translocation of large periplasmic domains. In addition, we found that the inability to export the alkaline phosphatase domain could be suppressed by a mutation, prlA4, in the secretion apparatus.
Uhland
K
K
Fakultät für Biologie, Universität Konstanz, Germany.
Ehrle
R
R
Zander
T
T
Ehrmann
M
M
D018528:ATP-Binding Cassette Transporters; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002451:Cell Compartmentation / Q000235:genetics*; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000378:metabolism*; D008958:Models, Molecular; D008961:Models, Structural; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D000069816:SEC Translocation Channels; D012641:Selection, Genetic; D013329:Structure-Activity Relationship; D013879:Thioredoxins / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; C057841:SecY protein, E coli; C461599:maltose transport system, E coli; D013879:Thioredoxins; D000469:Alkaline Phosphatase; D010744:Phosphoric Monoester Hydrolases; C022394:phosphoserine phosphatase