Disruption of the gene encoding p12 (SecG) reveals the direct involvement and important function of SecG in the protein translocation of Escherichia coli at low temperature.
The Escherichia coli cytoplasmic membrane protein, p12, stimulates the protein translocation activity reconstituted with SecY, SecE and SecA. The gene encoding p12, which is located at 69 min on the E. coli chromosome, was deleted to examine the role of p12 in protein translocation in vivo. The deletion strain exhibited cold-sensitive growth. Pulse-chase experiments revealed that precursors of outer membrane protein A, maltose binding protein and beta-lactamase accumulated at 20 degrees C but not at 37 degrees C. The deletion strain harboring a plasmid which carries the gene encoding p12 under the control of the araBAD promoter was able to grow in the cold when p12 was expressed with the addition of arabinose. Furthermore, the accumulated precursors were rapidly processed to the mature forms upon the expression of p12. Immunoblot analysis revealed the steady-state accumulation of precursor proteins at 20 degrees C, whereas the accumulation was only marginal at 37 degrees C, indicating that the function of p12 is more critical at 20 degrees C than at 37 degrees C. Finally, proteoliposomes were reconstituted with or without p12 to demonstrate that the stimulation of the activity by p12 increases with a decrease in temperature. From these results, we concluded that p12 is directly involved in protein translocation in E. coli and plays a critical role in the cold. We propose the more systematic name, SecG, for p12.
Nishiyama
K
K
Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
Hanada
M
M
Tokuda
H
H
D018528:ATP-Binding Cassette Transporters; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism; D003080:Cold Temperature; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000235:genetics*; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D011498:Protein Precursors / Q000378:metabolism; D011510:Proteolipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D000069816:SEC Translocation Channels; D017384:Sequence Deletion; D001618:beta-Lactamases / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D011498:Protein Precursors; D011510:Proteolipids; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; C088616:SecG protein, E coli; C461599:maltose transport system, E coli; C020485:proteoliposomes; D001618:beta-Lactamases