The outer membrane protein PupB of Pseudomonas putida WCS358 facilitates transport of iron complexed to the siderophores pseudobactin BN8 and pseudobactin BN7 into the cell. Its synthesis is induced by the presence of these specific siderophores under iron limitation. The signal transduction pathway regulating siderophore‐dependent expression of pupB was shown to consist of two regulatory proteins, PupI and PupR, and the PupB receptor itself. Mutational analysis of the regulatory genes suggested that PupI acts as a positive regulator of pupB transcription, whereas PupR modifies PupI activity dependent on the presence of pseudobactin BN8. PupI and PupR do not share homology with the classical bacterial two‐component systems but display significant similarity to the FecI and FecR proteins of Escherichia coli involved in regulation of ferric dicitrate transport. The function of the PupB receptor in pupB regulation was studied by the use of chimeric receptor proteins composed of PupB and the ferric pseudobactin 358 receptor PupA. This experiment revealed that PupB is involved in the initiation of the signal transduction pathway, implying a so far unique role for an outer membrane protein in signal transduction.

The orientation of many membrane proteins is determined by the asymmetric distribution of positively charged amino acid residues in cytoplasmic and translocated loops. The positive-inside rule states that loops with large amounts of these residues tend to have cytoplasmic locations. Orientations of constructs derived from the inner membrane protein leader peptidase from Escherichia coli were found to depend on the anionic phospholipid content of the membrane. Lowering the contents of anionic phospholipids facilitated membrane passage of positively charged loops. On the other hand, elevated contents of acidic phospholipids in the membrane rendered translocation more sensitive to positively charged residues. The results demonstrate that anionic lipids are determinants of membrane protein topology and suggest that interactions between negatively charged phospholipids and positively charged amino acid residues contribute to the orientation of membrane proteins.

To determine the phospholipid requirement of the preprotein translocase in vitro, the Escherichia coli SecYEG complex was purified in a delipidated form using the detergent dodecyl maltoside. SecYEG was reconstituted into liposomes composed of defined synthetic phospholipids, and proteoliposomes were analyzed for their preprotein translocation and SecA translocation ATPase activity. The activity strictly required the presence of anionic phospholipids, whereas the non-bilayer lipid phosphatidylethanolamine was found stimulatory. The latter effect could also be induced by dioleoylglycerol, a lipid that adopts a non-bilayer conformation. Phosphatidylethanolamine derivatives that prefer the bilayer state were unable to stimulate translocation. In the absence of SecG, activity was reduced, but the phospholipid requirement was unaltered. Remarkably, nonbilayer lipids were found essential for the activity of the Bacillus subtilis SecYEG complex. Optimal activity required a mixture of anionic and non-bilayer lipids at concentrations that correspond to concentrations found in the natural membrane.

Leader peptidase is an integral membrane protein of E. coli and it catalyses the removal of most signal peptides from translocated precursor proteins. In this study it is shown that when the transmembrane anchors are removed in vivo, the remaining catalytic domain can bind to inner and outer membranes of E. coli. Furthermore, the purified catalytic domain binds to inner membrane vesicles and vesicles composed of purified inner membrane lipids with comparable efficiency. It is shown that the interaction is caused by penetration of a part of the catalytic domain between the lipids. Penetration is mediated by phosphatidylethanolamine, the most abundant lipid in E. coli, and does not seem to depend on electrostatic interactions. A hydrophobic segment around the catalytically important residue serine 90 is required for the interaction with membranes.z 1998 Federation of European Biochemical Societies.

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