The early interaction of the outer membrane protein phoe with the periplasmic chaperone Skp occurs at the cytoplasmic membrane.
Spheroplasts were used to study the early interactions of newly synthesized outer membrane protein PhoE with periplasmic proteins employing a protein cross-linking approach. Newly translocated PhoE protein could be cross-linked to the periplasmic chaperone Skp at the periplasmic side of the inner membrane. To study the timing of this interaction, a PhoE-dihydrofolate reductase hybrid protein was constructed that formed translocation intermediates, which had the PhoE moiety present in the periplasm and the dihydrofolate reductase moiety tightly folded in the cytoplasm. The hybrid protein was found to cross-link to Skp, indicating that PhoE closely interacts with the chaperone when the protein is still in a transmembrane orientation in the translocase. Removal of N-terminal parts of PhoE protein affected Skp binding in a cumulative manner, consistent with the presence of two Skp-binding sites in that region. In contrast, deletion of C-terminal parts resulted in variable interactions with Skp, suggesting that interaction of Skp with the N-terminal region is influenced by parts of the C terminus of PhoE protein. Both the soluble as well as the membrane-associated Skp protein were found to interact with PhoE. The latter form is proposed to be involved in the initial interaction with the N-terminal regions of the outer membrane protein.
Department of Molecular Microbiology, Institute of Molecular Biological Sciences, Biocentrum Amsterdam, De Boelelaan 1087, Amsterdam 1081 HV, The Netherlands.
N
N
Harms
G
G
Koningstein
W
W
Dontje
M
M
Muller
B
B
Oudega
J
J
Luirink
H
H
de Cock
D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D018272:Porins / Q000737:chemistry* / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D018272:Porins; D011993:Recombinant Fusion Proteins; C072721:Skp protein, E coli; C082922:PhoE protein, E coli; D013762:Tetrahydrofolate Dehydrogenase