The protein sequence responsible for lipoprotein membrane localization in Escherichia coli exhibits remarkable specificity.
Structural information defining an N-terminal sequence required for the membrane sorting of bacterial lipoproteins has been previously garnered through the study of a hybrid outer membrane (OM) lipo-beta-lactamase (LL) (Ghrayeb and Inouye (1984) J. Biol. Chem. 259, 463-467). Introduction of an aspartate as the second residue of mature LL (D2 mutant) causes an inner membrane (IM) localization of this protein (Yamaguchi, K., Yu, F., and Inouye, M. (1988) Cell 53, 423-432). Introduction of as aspartate at the third residue of mature LL (D3) causes a weaker IM sorting signal and when present as the fourth residue (D4), normal OM sorting occurs. A positively charged residue at the second position (K2) has no effect on OM localization. Remarkably, glutamate substitution at either the second (E2) or third (E3) position does not interfere with OM sorting. Sorting of the mutant D2 LL can be partially suppressed by introduction of a positively charged histidine (D2H3) or lysine (D2K3) at residue 3 of the mature protein. These results indicate that both the negative charge of the aspartate residue and some structural feature not present in a glutamate residue are required for sorting to the IM. The suppression of IM localization of the D2H3 LL double mutant can be eliminated by growing Escherichia coli at pH 8.4 to reduce the histidine partial positive charge. This result supports the essentiality of a negative charge in IM localization and indicates that the committed step in lipoprotein sorting is made in a cellular compartment, the periplasm, at equilibrium with the external pH.
Gennity
J M
JM
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine & Dentistry of New Jersey, Piscataway 08854-5635.
Inouye
M
M
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001483:Base Sequence; D004269:DNA, Bacterial; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D013489:Suppression, Genetic; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D008074:Lipoproteins; D001618:beta-Lactamases