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The lipoprotein PulS is a dedicated chaperone that is required to target the secretin PulD to the outer membrane in Klebsiella or Escherichia coli, and to protect it from proteolysis. Here, we present indirect evidence that PulD protomers do not assemble into the secretin dodecamer before they reach the outer membrane, and that PulS reaches the outer membrane in a soluble heterodimer with the general lipoprotein chaperone LolA. However, we could not find any direct evidence for PulD protomer association with the PulS-LolA heterodimer. Instead, in cells producing PulD and a permanently locked PulS-LolA dimer (in which LolA carries an R43L substitution that prevents lipoprotein transfer to LolB in the outer membrane), LolAR43L was found in the inner membrane, probably still associated with PulS bound to PulD that had been incorrectly targeted because of the LolAR43L substitution. It is speculated that PulD protomers normally cross the periplasm together with PulS bound to LolA but when the latter cannot be separated (due to the mutation in lolA), the PulD protomers form dodecamers that insert into the inner membrane., (© 2011 Blackwell Publishing Ltd.)

The ultimate membrane localization and function of most of the 185 predicted Pseudomonas aeruginosa PAO1 lipoproteins remain unknown. We constructed a fluorescent lipoprotein, CSFP(OmlA)-ChFP, by fusing the signal peptide and the first four amino acids of the P. aeruginosa outer membrane lipoprotein OmlA to the monomeric red fluorescent protein mCherry (ChFP). When cells were plasmolyzed with 0.5 M NaCl, the inner membrane separated from the outer membrane and formed plasmolysis bays. This permits the direct observation of fluorescence in either the outer or inner membrane. CSFP(OmlA)-ChFP was shown to localize in the outer membrane by fluorescence microscopy and immunoblotting analysis of inner and outer membrane fractions. The site-directed substitution of the amino acids at positions +2, +3, and +4 in CSFP(OmlA)-ChFP was performed to test the effects on lipoprotein localization of a series of amino acid sequences selected from a panel of predicted lipoproteins. We confirmed Asp(+2) and Lys(+3) Ser(+4) function as inner membrane retention signals and identified four novel inner membrane retention signals: CK(+2) V(+3) E(+4), CG(+2) G(+3) G(+4), CG(+2) D(+3) D(+4), and CQ(+2) G(+3) S(+4). These inner membrane retention signals are found in 5% of the 185 predicted P. aeruginosa lipoproteins. Full-length chimeras of predicted lipoproteins PA4370 and PA3262 fused to mCherry were shown to reside in the inner membrane and showed a nonuniform or patchy distribution in the membrane. The optical sectioning of cells producing PA4370(CGDD)-ChFP and PA3262(CDSQ)-ChFP by confocal microscopy improved the resolution and indicated a helix-like localization pattern in the inner membrane. The method described here permits the in situ visualization of lipoprotein localization and should work equally well for other membrane-associated proteins.

Chimeras created by fusing the monomeric red fluorescent protein (RFP) to a bacterial lipoprotein signal peptide (lipoRFPs) were visualized in the cell envelope by epifluorescence microscopy. Plasmolysis of the bacteria separated the inner and outer membranes, allowing the specific subcellular localization of lipoRFPs to be determined in situ. When equipped with the canonical inner membrane lipoprotein retention signal CDSR, lipoRFP was located in the inner membrane in Escherichia coli, whereas the outer membrane sorting signal CSSR caused lipoRFP to localize to the outer membrane. CFSR-RFP was also routed to the outer membrane, but CFNSR-RFP was located in the inner membrane, consistent with previous data showing that this sequence functions as an inner membrane retention signal. These four lipoproteins exhibited identical localization patterns in a panel of members of the family Enterobacteriaceae, showing that the lipoprotein sorting rules are conserved in these bacteria and validating the use of E. coli as a model system. Although most predicted inner membrane lipoproteins in these bacteria have an aspartate residue after the fatty acylated N-terminal cysteine residue, alternative signals such as CFN can and probably do function in parallel, as indicated by the existence of putative inner membrane lipoproteins with this sequence at their N termini.

Lipoproteins in Gram-negative Enterobacteriaceae carry three fatty acids on the N-terminal cysteine residue, two as a diacylglyceride and one through an N-linkage following signal peptide cleavage. Most lipoproteins are anchored in the outer membrane, facing the periplasm, but some lipoproteins remain in the plasma membrane, depending on the amino acid at position +2, immediately after the fatty-acylated cysteine. In vitro, the last step in lipoprotein maturation, N-acylation of apolipoproteins by the plasma membrane apolipoprotein N-acyltransferase (Lnt), is necessary for efficient recognition of outer membrane lipoproteins by the Lol system, which transports them from the plasma to the outer membrane (Fukuda, A., Matsuyama, S.-I., Hara, T., Nakayama, J., Nagasawa, H., and Tokuda, H. (2002) J. Biol. Chem. 277, 43512-43518). To study the role of Lnt in vivo, we constructed a conditional lnt mutant of Escherichia coli. The apo-form of peptidoglycan-anchored major lipoprotein (Lpp) and two other outer membrane lipoproteins accumulated in the plasma membrane when lnt expression was reduced. We also found that Lnt is an essential protein in E. coli and that the lethality is partially because of the retention of apoLpp in the plasma membrane. Topology mapping of Lnt with beta-galactosidase and alkaline phosphatase fusions indicated the presence of six membrane-spanning segments. The lnt gene in a mutant of Salmonella enterica displaying thermosensitive Lnt activity (Gupta, S. D., Gan, K., Schmid, M. B., and Wu, H. C. (1993) J. Biol. Chem. 268, 16551-16556) was found to carry a mutation causing a single glutamate to lysine substitution at a highly conserved position in the last predicted periplasmic loop of the protein.

To study lipoprotein sorting in Escherichia coli, we devised a novel screen in which sensitivity or resistance to bacteriophage T5 and colicin M reflects the membrane localization of the bacteriophage T5-encoded lipoprotein Llp, which inactivates the outer membrane (OM) T5 receptor (FhuA). When processed by lipoprotein signal peptidase, Llp has a serine at position +2, immediately after the fatty acylated N-terminal cysteine. As predicted by the '+2 lipoprotein sorting rule' that determines the localization of lipoproteins in the cell envelope, Llp is located in the OM. However, contrary to expectations, when serine +2 was replaced by aspartate, the canonical plasma membrane lipoprotein retention signal, Llp was still > or =40% targeted to the OM and protected cells against colicin M and phage T5. OM association of this Llp derivative was abolished when a peptide spacer was inserted between the aspartate and the rest of Llp or when the formation of an intramolecular disulphide bond in Llp was prevented by substituting one or other of the cysteines involved. Furthermore, analysis of a MalE-Llp hybrid protein with or without a lipid moiety demonstrated that fatty acylation of Llp is essential for its OM association and for protection against colicin M and bacteriophage T5. These data suggest (i) that phage-encoded Llp uses the endogenous E. coli Lol pathway for lipoprotein sorting to the OM and (ii) that the conformation of a lipoprotein can affect its sorting within the cell envelope.

Secretins are a unique class of bacterial multimeric outer membrane proteins that probably differ considerably from other, less complex outer membrane proteins in their overall structure and organization, and in their requirements for outer membrane targeting and assembly factors. In this MicroCommentary, we discuss these differences with respect to the role of a specific class of lipoproteins, often referred to as pilotins, in secretin complex assembly. We compare them with other lipoproteins that play a role in Omp85/YaeT-mediated assembly of more classical outer membrane proteins. One of the examples we have chosen is the Myxococcus Xanthus lipoprotein Tgl. Coculture of cells with and without Tgl allows secretin assembly (and, hence, type IV pilus assembly) in the cells without Tgl, indicating that it can act by cell-to-cell contact or can transfer between cells. [ABSTRACT FROM AUTHOR]

We report a novel strategy for selecting mutations that mislocalize lipoproteins within the Escherichia coli cell envelope and describe the mutants obtained. A strain carrying a deletion of the chromosomal malE gene, coding for the periplasmic maltose-binding protein (MalE), cannot use maltose unless a wild-type copy of malE is present in trans . Replacement of the natural signal peptide of preMalE by the signal peptide and the first four amino acids of a cytoplasmic membrane-anchored lipoprotein resulted in N-terminal fatty acylation of MalE (lipoMalE) and anchoring to the periplasmic face of the cytoplasmic membrane, where it could still function. When the aspartate at position +2 of this protein was replaced by a serine, lipoMalE was sorted to the outer membrane, where it could not function. Chemical mutagenesis followed by selection for maltose-using mutants resulted in the identification of two classes of mutations. The single class I mutant carried a plasmid-borne mutation that replaced the serine at position +2 by phenylalanine. Systematic substitutions of the amino acid at position +2 revealed that, besides phenylalanine, tryptophan, tyrosine, glycine and proline could all replace classical cytoplasmic membrane lipoprotein sorting signal (aspartate +2). Analysis of known and putative lipoproteins encoded by the E. coli K-12 genome indicated that these amino acids are rarely found at position +2. In the class II mutants, a chromosomal mutation caused small and variable amounts of lipoMalE to remain associated with the cytoplasmic membrane. Similar amounts of another, endogenous outer membrane lipoprotein, NlpD, were also present in the cytoplasmic membrane in these mutants, indicating a minor, general defect in the sorting of outer membrane lipoproteins. Four representative class II mutants analysed were shown not to carry mutations in the lolA or lolB genes, known to be involved in the sorting of lipoproteins to the outer membrane. [ABSTRACT FROM AUTHOR]

Related outer membrane proteins, termed secretins, participate in the secretion of macromolecules across the outer membrane of many Gram-negative bacteria. In the pullulanase-secretion system, PulS, an outer membrane-associated lipoprotein, is required both for the integrity and the proper outer membrane localization of the PulD secretin. Here we show that the PulS-binding site is located within the C-terminal 65 residues of PulD. Addition of this domain to the filamentous phage secretin, pIV, or to the unrelated maltose-binding protein rendered both proteins dependent on PulS for stability. A chimeric protein composed of bacteriophage f1 pIV and the C-terminal domain of PulD required properly localized PulS to support phage assembly. An in vivo complex formed between the pIV-PulD65 chimera and PulS was detected by co-immunoprecipitation and by affinity chromatography. [ABSTRACT FROM AUTHOR]

The chaperone-like protein of the main terminal branch of the general secretory pathway from Klebsiella oxytoca, the outer membrane lipoprotein PulS, protects the multimeric secretin PulD from degradation and promotes its correct localization to the outer membrane. To determine whether these are separable functions, or whether resistance to proteolysis results simply from correct localization of PulD, we replaced the lipoprotein-type signal peptide of PulS by the signal peptide of periplasmic maltose-binding protein. The resulting periplasmic PulS retained its ability to protect PulD, but not its ability to localize PulD to the outer membrane and to function in pullulanase secretion. Periplasmic PulS competed with wild-type PulS to prevent pullulanase secretion, presumably again by causing mislocalization of PulD. A hybrid protein comprising the mature part of PulS fused to the C-terminus of full-length maltose-binding protein (MalE-PulS) had similar properties to the periplasmic PulS protein. Moreover, MalE-PulS was shown to associate with PulD by amylose-affinity chromatography. The MalE-PulS hybrid was rendered completely functional (i.e. it restored pullulanase secretion in a pulS mutant) by replacing its signal peptide with a lipoprotein-type signal peptide. However, this fatty-acylated hybrid protein was only functional if it also carried a lipoprotein sorting signal that targeted it to the outer membrane. Thus, the two functions of PulS are separate and fully dissociable. Incorrect localization, rather than proteolysis, of PulD in the absence of PulS was shown to be the factor that causes high-level induction of the phage shock response. The Erwinia chrysanthemi PulS homologue, OutS, can substitute for PulS, and PulS can protect the secretin OutD from proteolysis in Escherichia coli, indicating the possible existence of a family of PulS-like chaperone proteins. [ABSTRACT FROM AUTHOR]

Abstract: The Klebsiella oxytoca lipoprotein PulS might function as either or both a pilot and a docking factor in the outer membrane targeting and assembly of the Type II secretion system secretin PulD. In the piloting model, PulS binds to PulD monomers and targets them to the outer membrane via the lipoprotein sorting pathway components LolA and LolB. In this model, PulS also protects the PulD monomers from proteolysis during transit through the periplasm. In the docking model, PulS is targeted alone to the outer membrane, where it acts as a receptor for PulD monomers, allowing them to accumulate and assemble specifically in this membrane. PulS was shown to dissociate from and/or re-associate freely with PulD multimers in zwitterionic detergent, making it difficult to determine whether PulS remains associated with PulD dodecamers in the outer membrane by co-purification. However, PulD protomers in the dodecamer were shown to be stable in the absence of PulS, indicating that PulS is only required to protect the protease-susceptible monomer. DegP was identified as one of the proteases that could contribute to PulD degradation in the absence of PulS. Studies on the in vitro assembly and targeting of PulD into Escherichia coli membrane vesicles demonstrated its strong preference to insert into the inner membrane, as is the case in vivo in the absence of PulS. However, PulD could be targeted to outer membrane fragments in vitro if they were preloaded with PulS, indicating the technical feasibility of the docking model. We conclude that both modes of action might contribute to efficient outer membrane targeting of PulD in vivo, although the piloting function is likely to predominate. [Copyright &y& Elsevier]