Structural determinants in addition to the amino-terminal sorting sequence influence membrane localization of Escherichia coli lipoproteins.

The lipid-modified nine-residue amino-terminal sequence of the mature form of the major outer membrane lipoprotein of Escherichia coli contains information that is responsible for sorting to either the inner or outer membrane. Fusion of this sorting sequence to beta-lactamase is sufficient for localization of the resultant lipo-beta-lactamase to the outer membrane (J. Ghrayeb and M. Inouye, J. Biol. Chem. 259:463-467, 1984). Substitution of the serine adjacent to the amino-terminal lipid-modified cysteine residue of the sorting sequence with the negatively charged residue aspartate causes inner membrane localization (K. Yamaguchi, F. Yu, and M. Inouye, Cell 53:423-432, 1988). Fusion of the aspartate-containing nine-residue inner membrane localization signal to the normally outer membrane lipoprotein bacteriocin release protein does cause partial localization to the inner membrane. However, a single replacement of the glutamine adjacent to the amino-terminal lipid-modified cysteine residue of bacteriocin release protein with aspartate causes no inner membrane localization. Therefore, an aspartate residue itself lacks the information necessary for inner membrane sorting when removed from the structural context provided by the additional eight residues of the sorting sequence. Although the aspartate-containing inner membrane sorting sequence causes an almost quantitative localization to the inner membrane when fused to the otherwise soluble protein beta-lactamase, this sequence cannot prevent significant outer membrane localization when fused to proteins (bacteriocin release protein and OmpA) normally found in the outer membrane. Therefore, structural determinants in addition to the amino-terminal sorting sequence influence the membrane localization of lipoproteins.     

Characterization of the formyl peptide chemotactic receptor appearing at the phagocytic cell surface after exposure to phorbol myristate acetate

We examined the biochemistry and subcellular source of new formyl peptide chemotactic receptor appearing at the human neutrophil and differentiated HL-60 (d-HL-60) cell surface after stimulation with phorbol myristate acetate (PMA). Formyl peptide receptor was analyzed by affinity labeling with formyl-norleu-leu-phe-norleu-[125I]iodotyr-lys and ethylene glycol bis(succinimidyl succinate) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometric analysis of autoradiographs. PMA, a specific granule secretagogue, increases affinity labeling of formyl peptide receptors on the neutrophil surface by 100%, and on d-HL-60, which lack specific granule markers, by 20%. Papain treatment markedly reduces surface labeling of formyl peptide receptor in both neutrophils and d-HL-60, and results in the appearance of a lower m.w. membrane-bound receptor fragment. PMA stimulation of papain-treated cells increases uncleaved surface receptor on neutrophils by 400%, and on d-HL-60 by only 45%. This newly appearing receptor is the same apparent m.w. (55,000 to 75,000 for neutrophils; 62,000 to 80,000 for d-HL-60) and yields the same papain cleavage product (Mr, 31,000 for neutrophils; Mr, 29,000 for d-HL-60) as receptor on the surface of unstimulated cells. Formyl peptide receptor detected by affinity labeling in neutrophil specific granule-enriched subcellular fractions is identical to receptor found on the surface of unstimulated cells appearing as equal amounts of two isoelectric forms (isoelectric points, 5.8 and 6.2) at Mr 55,000 to 70,000. There is twice as much receptor present in the specific granule-enriched fraction per cell equivalent compared with plasma membrane. Azurophil granules contain trace amounts of receptor. Similar analysis of neutrophils treated with papain before subcellular fractionation shows that papain cleaved receptor fragment is detectable almost exclusively in the plasma membrane-enriched fraction. Most of the affinity-labeled formyl peptide receptor present in specific granule enriched fraction is present in membranes other than plasma membrane or Golgi membrane, because specific granule-enriched fraction contains only a small amount of plasma membrane marker and an amount of Golgi membrane marker equal to that found in plasma membrane-enriched fraction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of phorbol myristate acetate on processing of formyl peptide receptors by human neutrophils

We examined the effect of phorbol myristate acetate on the ability of human neutrophils to process formyl peptide receptors. The receptor was affinity-labeled and its extracellular localization assessed over time, by cleavage of extracellular labeled receptor with papain. Neutrophils were capable of internalizing (and/or recycling) affinity labeled formyl peptide receptor in the absence of extracellular calcium. This phenomenon was dependent upon stimulation with phorbol myristate acetate, suggesting a role for protein kinase C in this process.     

The amino-terminal domain of the lamin B receptor is a nuclear envelope targeting signal

The lamin B receptor (LBR) is a polytopic protein of the inner nuclear membrane. It is synthesized without a cleavable amino-terminal signal sequence and composed of a nucleoplasmic amino-terminal domain of 204 amino acids followed by a hydrophobic domain with eight putative transmembrane segments. To identify a nuclear envelope targeting signal, we have examined the cellular localization by immunofluorescence microscopy of chicken LBR, its amino-terminal domain and chimeric proteins transiently expressed in transfected COS-7. Full- length LBR was targeted to the nuclear envelope. The amino-terminal domain, without any transmembrane segments, was transported to the nucleus but excluded from the nucleolus. When the amino-terminal domain of LBR was fused to the amino-terminal side of a transmembrane segment of a type II integral membrane protein of the ER/plasma membrane, the chimeric protein was targeted to the nuclear envelope, likely the inner nuclear membrane. When the amino-terminal domain was deleted from LBR and replaced by alpha-globin, the chimeric protein was retained in the ER. These findings demonstrate that the amino-terminal domain of LBR is targeted to the nucleus after synthesis in the cytoplasm and that this polypeptide can function as a nuclear envelope targeting signal when located at the amino terminus of a type II integral membrane protein synthesized on the ER.     

Peptides derived from HIV-1, HIV-2, Ebola virus, SARS coronavirus and coronavirus 229E exhibit high affinity binding to the formyl peptide receptor

Peptides derived from the membrane proximal region of fusion proteins of human immunodeficiency viruses 1 and 2, Coronavirus 229 E, severe acute respiratory syndrome coronavirus and Ebola virus were all potent antagonists of the formyl peptide receptor expressed in Chinese hamster ovary cells. Binding of viral peptides was affected by the naturally occurring polymorphisms at residues 190 and 192, which are located at second extracellular loop-transmembrane helix 5 interface. Substitution of R190 with W190 enhanced the affinity for a severe acute respiratory syndrome coronavirus peptide 6 fold but reduced the affinity for N-formyl-Nle-Leu-Phe by 2.5 fold. A 12 mer peptide derived from coronavirus 229E (ETYIKPWWVWL) was the most potent antagonist of the formyl peptide receptor W190 with a K(i) of 230 nM. Fluorescently labeled ETYIKPWWVWL was effectively internalized by all three variants with EC(50) of approximately 25 nM. An HKU-1 coronavirus peptide, MYVKWPWYVWL, was a potent antagonist but N-formyl-MYVKWPWYVWL was a potent agonist. ETYIKPWWVWL did not stimulate GTPgammaS binding but inhibited the stimulation by formyl-NleLeuPhe. It also blocked beta arrestin translocation and receptor downregulation induced by formyl-Nle-Leu-Phe. This indicates that formyl peptide receptor may be important in viral infections and that variations in its sequence among individuals may affect their likelihood of viral and bacterial infections.     

Membrane topology and mutational analysis of the TolQ protein of Escherichia coli required for the uptake of macromolecules and cell envelope integrity.

TolQ is a 230-amino-acid protein required to maintain the integrity of the bacterial envelope and to facilitate the import of both filamentous bacteriophage and group A colicins. Cellular fractionation experiments showed TolQ to be localized to the cytoplasmic membrane. Bacteria expressing a series of TolQ-beta-galactosidase and TolQ-alkaline phosphatase fusion proteins were analyzed for the appropriate enzyme activity, membrane location, and sensitivity to exogenously added protease. The results are consistent with TolQ being an integral cytoplasmic membrane protein with three membrane-spanning regions. The amino-terminal 19 residues as well as a small loop in the 155 to 170 residue region appear exposed in the periplasm, while the carboxy terminus and a large loop after the first transmembrane region are cytoplasmic. Amino-terminal sequence analysis of TolQ purified from the membrane revealed the presence of the initiating formyl methionine group, suggesting a rapid translocation of the amino-terminal region across the cytoplasmic membrane. Analysis of various tolQ mutant strains suggests that the third transmembrane region as well as parts of the large cytoplasmic loop are necessary for activity.     

Isolation of a cDNA that encodes a novel granulocyte N-formyl peptide receptor.

A cDNA of 1650 base pairs was isolated by screening an HL-60 granulocyte library with an N-formyl peptide receptor (NFPR) cDNA probe under low stringency conditions. The cDNA encodes a protein of 351 amino acids tentatively named FPR2, with a calculated molecular weight of 39 kDa. Sequence analysis revealed that FPR2 is 69% identical in sequence to the human NFPR and shares extensive homology to several other chemoattractant receptors. FPR2 expressed in transfected cells mediated formyl peptide-stimulated calcium mobilization at micromolar concentrations of ligand. FPR2 messenger is detected in granulocytic HL-60 cells, but not in undifferentiated HL-60 cells. These findings suggest that FPR2 is a novel receptor for formyl peptide ligand and a new member of the chemoattractant receptor gene family.     

A single amino acid determinant of the membrane localization of lipoproteins in E. coli

When beta-lactamase was fused with the signal peptide plus the amino-terminal 9 amino acid residues of the major outer membrane lipoprotein, the resultant lipo-beta-lactamase (LL-1) was shown to be localized to the outer membrane. However, when the 9 residue sequence was replaced with the amino-terminal 12 residue sequence of lipoprotein-28, an inner membrane protein, the resultant lipo-beta-lactamase (LL-2) was found exclusively in the inner membrane. The localization of LL-2 was shifted to the outer membrane simply by substituting the second amino acid residue (Asp) of LL-2 with Ser. Conversely, the alteration of the second residue (Ser) of LL-1 to Asp resulted in the localization of LL-1 to the inner membrane. These results suggest that the second amino acid residue of the lipoproteins plays a crucial role in determining their final locations in the E. coli envelope.     

Synthetic peptides as chemoattractants for bull spermatozoa structure activity correlations

The ability of various synthetic peptide analogs of. Formyl-Met-Leu-Phe to induce chemotaxis in bull sperm is compared using an inverted capillary assay. The formyl group is essential for chemotactic activity and corresponding t-butyloxycarbonyl tripeptides are inactive. Sequence analogs, Formyl-Met-Phe-Leu, Formyl-Leu-Met-Phe and Formyl-Leu-Phe-Met are active. Replacement of Met and Leu by Pro does not diminish activity. Formyl-Met-Leu-Phe-NH₂ is active suggesting that electrostatic interactions involving the carboxyl group may be unimportant in receptor interactions. The studies establish the importance of an amino terminal formyl group and a sequence of at least three hydrophobic residues, for inducing sperm chemotaxis.     

W-7 primes or inhibits the fMLP-stimulated respiratory burst in human neutrophil by concentration-dependent dual expression of the formyl peptide receptors on cell surface

It was investigated why the fMLP-stimulated respiratory burst in human neutrophils was enhanced by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a considered calmodulin antagonist, at lower concentration but inhibited at higher concentration. Flow cytometric analysis on binding of the receptor to the fluorescence-labeled formyl peptide and the polymerization of actin in cells showed that the drug inhibited actin polymerization and promoted expression of the fMLP receptors on cell membrane at lower concentration, while promoted the actin polymerization and depressed the receptor expression at higher concentration. As intracellular Ca(2+) ([Ca(2+)](i)) is elevated, polymerization of actin decreases and the receptor expression increases. At normal physiological and two moderately high intracellular calcium levels, the dual effect of W-7 became less significant as [Ca(2+)](i) was elevated indicating that the dual effect is calcium-dependent. Under two extreme conditions that the intracellular calcium was either depleted or highly elevated, the dual effect disappeared but only an inhibitory effect on actin polymerization was observed. Colchicine and taxol study showed that disruption or stabilization of microtubules had no effect on formyl peptide receptor expression. The results suggest that W-7 primes the fMLP stimulation by direct action on actin leading to breakdown of microfilaments and more expression of formyl peptide receptors, and inhibits the stimulation by indirect action on actin through inactivation of some Ca(2+)-dependent proteins resulting in assembly of actin into microfilaments. Which action is favorable depends on the drug concentration.     

Identification of a human neutrophil protein of Mr 24 000 that binds N-formyl peptides: co-sedimentation with specific granules

In assaying subcellular fractions of human neutrophils for N-formyl peptide binding sites using the photoaffinity ligand FMLPL-SASD-125I (125I-labelled N-formylmethionylleucylphenylalanyl-N epsilon- (2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionyl)-lysine) several molecular species were observed. We confirmed localization of the N-formyl peptide receptor of Mr 50 000-70 000 in the plasma membrane and specific granule fractions. A species of Mr 33 000-35 000 was detected in the light Golgi/endosomal fraction, whose size is consistent with the deglycosylated form of the receptor. In addition, a major binding species of Mr 24 000 was identified that co-localized on sucrose gradients with specific granule markers. This Mr 24 000 species, which was investigated further, was found to be released upon cell stimulation with phorbol myristate acetate or FMLP in the presence of dihydrocytochalasin B. It had an affinity for FMLPL-SASD of 145 nM (cf. 0.3 nM for the cell surface receptor). The specificity for the formyl group was lost as the nonformylated Met-Leu-Phe was as effective FMLPL in competing with FMLPL-SASD-125I for binding to th Mr 24 000 species. A structurally unrelated peptide, however, did not compete for the binding. The labelling of the Mr 24 000 species was dependent on the presence of Ca2+, as was its apparent Mr, which shifted from 24 000 to 50 000-70 000 in the presence of Ca2+. By incubating photoaffinity-labelled plasma membrane fractions with specific granule fractions, we could generate a receptor fragment of Mr 24 000, although the relationship to this fragment of the specific granule species is unknown at present. The N-terminal sequence of the Mr 24 000 species was determined and it appears to be a novel protein. Further work will allow its relationship to the receptor, if any, to be elucidated and allow assignment of a function to this potentially important molecule.     

Retro-all-D and retro isomers of a formyl-methionyl peptide chemoattractant: an insight into the mode of binding at the receptor on rabbit neutrophils

The retro-all-D analog and retro isomer of the formyl-methionyl-carboxamide-tripeptide chemoattractant, CHO-L-Met-L-Leu-L-Phe-NH2, namely CHO-D-Phe-D-Leu-D-Met-NH2 and CHO-L-Phe-L-Leu-L-Met-NH2, respectively, have been synthesized in solution by classical methods and fully characterized. The tetrapeptide CHO-L-Phe-Gly-L-Leu-L-Met-NH2, representing the C-terminal portion of the tachykinin, Substance P, and resembling the sequence of the retro isomer, has also been synthesized and characterized. The three N alpha-formylated tripeptide amides, prepared in order to obtain a deeper insight into the model of binding at the formyl peptide chemotactic receptor on rabbit neutrophils, have been tested for their ability to induce granule enzyme secretion from rabbit peritoneal neutrophils. The retro isomer, CHO-L-Phe-L-Leu-L-Met-NH2 is approximately 100-fold less active, the retro-all-D analog, CHO-D-Phe-D-Leu-D-Met-NH2 approximately 10,000-fold less active and the Substance P analog CHO-L-Phe-Gly-L-Leu-L-Met-NH2 1000-fold less active than the parent formyl peptide chemoattractant, CHO-L-Met-L-Leu-L-Phe-NH2. We interpret these results to indicate that a precise alignment of amino acid side chains as well as backbone amide bonds is an important factor involved in the receptor recognition of the formyl tripeptide chemoattractant.     

Protein sequence of endothelial glycoprotein IIIa derived from a cDNA clone. Identity with platelet glycoprotein IIIa and similarity to "integrin"

Platelet membrane glycoprotein (GP) IIIa forms a Ca2+-dependent heterodimer complex with GP IIb. The GP IIb-IIIa complex constitutes the fibrinogen and fibronectin receptor on stimulated platelets. A biochemically and immunologically similar membrane glycoprotein complex is present on endothelial cells. A human umbilical vein endothelial cell cDNA library was screened using oligonucleotide probes designed from peptide sequences obtained from platelet GP IIIa. A cDNA clone was sequenced and found to encode a protein of 84.5 kDa. The translated endothelial cDNA contained five sequences that corresponded to peptide sequences in platelet GP IIIa, including the amino-terminal 19 residues. Thus, the endothelial and platelet forms of GP IIIa are apparently identical. Glycoprotein IIIa consists of a long amino-terminal extracellular domain with several potential N-linked glycosylation sites and four cysteine-rich tandem repeats, a 29-residue hydrophobic transmembrane segment, and a short carboxyl-terminal cytoplasmic domain. Glycoprotein IIIa has a 47% amino acid sequence homology to "integrin," a fibronectin receptor from chicken embryo fibroblasts. This homology suggests that GP IIIa is a member of a family of cell-surface adhesion receptors.     

Deletion of the amino-terminal domain of asialoglycoprotein receptor H1 allows cleavage of the internal signal sequence

Human asialoglycoprotein receptor H1 is a single-spanning membrane protein with an amino-terminal domain of 40 residues exposed to the cytoplasm and the carboxyl-terminal domain translocated to the exoplasmic side of the membrane. It has been shown earlier that the transmembrane segment functions as an internal uncleaved signal sequence for insertion into the endoplasmic reticulum. In a deletion protein lacking almost the entire cytoplasmic domain, the signal sequence is cleaved at the carboxyl-terminal end of the transmembrane segment. All available criteria suggest that the protein is processed by signal peptidase. The cytoplasmic domain of the receptor does not directly inhibit signal cleavage since it does not detectably hinder cleavage of the normally amino-terminal signal sequence of influenza hemagglutinin in fusion proteins. We suggest that by its size or structure it affects the position of the receptor in the membrane and thus the accessibility of the potential cleavage site to signal peptidase.     

Recombinant expression of a secreted form of the atrial natriuretic peptide clearance receptor

A general structure for the atrial natriuretic peptide clearance receptor (ANP C-receptor) has been proposed based on hydropathicity analysis of the deduced amino acid sequence of this membrane protein (Fuller, F., Porter, J.G., Arfsten, A., Miller, J., Schilling, J., Scarborough, R.M., Lewicki, J.A., and Schenk, D.B. (1988) J. Biol. Chem. 263, 9395-9401). The ANP C-receptor is believed to possess a large amino-terminal extracellular domain (436 amino acids), a single hydrophobic transmembrane anchor (23 amino acids), and a short cytoplasmic tail (37 amino acids). As a means of testing the structure and proposed cellular orientation of this protein, we have employed the technique of in vitro mutagenesis to prepare a receptor mutant (anc-) lacking the transmembrane and cytoplasmic domains. Expression of this mutant in mammalian cells using a vaccinia virus vector results in secretion of a truncated soluble form of the ANP C-receptor which binds native ANP and synthetic ANP analogs with a specificity similar to that of the native ANP C-receptor. In contrast to the native ANP C-receptor that exists predominantly as a homodimer on the cell surface, the secreted receptor exists as a monomeric species. The results are consistent with the proposed structure of this receptor with the amino-terminal domain containing the ANP-binding site oriented extracellular to the plasma membrane. In addition, these data demonstrate that the receptor does not require association with the plasma membrane or its native dimeric configuration in order to bind ANP ligands with high affinity and specificity.     

Presence of a plasma membrane targeting sequence in the amino-terminal region of the rat somatostatin receptor 3

Although peptide hormone receptors commonly exert their actions at the plasma membrane the cellular mechanisms that route the receptor proteins to the cell surface during biosynthesis are not well characterized. Here we report on the identification of a plasma membrane targeting sequence of rat somatostatin receptor subtype 3. While type 3 somatostatin receptors are present almost exclusively at the cell surface, type 1 receptors localize in addition largely in intracellular vesicular compartments. Chimeric receptors were constructed between rat somatostatin receptors 3 and 1. They were tagged by recombinant DNA techniques with a herpes simplex virus glycoprotein D epitope at the carboxyl-termini to facilitate their detection using fluorescence microscopic methods. Following transfection of the constructs in human embryonic kidney and rat insulinoma cells the chimeric receptors were analyzed by indirect immunofluorescence using anti-epitope monoclonal antibody and confocal laser scanning microscopy. The results demonstrate that the amino-terminal domain of somatostatin receptor 3 suffices to guide chimeric receptors to the cell surface. In marked contrast, chimeric receptors that lack this sequence but contain instead the amino-terminus of somatostatin type 1 receptor localize in an intracellular vesicular compartment.     

Differential amplification of antagonistic receptor pathways in neutrophils.

In human neutrophils approximately 500 ligand-occupied beta-adrenergic receptors almost completely inhibit the superoxide production generated by at least 50,000 formyl peptide receptors, suggesting a massive amplification of the inhibitory receptor signals. We estimated two stages of amplification. In the first stage, we quantitated the ligand-dependent GTPase activities. For the formyl peptide receptor, the number of phosphates released from GTP in the presence of the saturating ligand is relatively modest, i.e. approximately 1/min/receptor, even though there are approximately 200 Gn (Gi type II) proteins/formyl peptide receptor in neutrophil membranes. In contrast, the number of GTPs cleaved in the presence of a beta-adrenergic agonist is approximately 100/min/beta-adrenergic receptor, and there are about 700 Gs/beta-adrenergic receptor in membranes. Thus the signal of the beta-adrenergic receptor is already massively amplified at the G protein, whereas the signal of the formyl peptide receptor is likely to be amplified at subsequent steps. New kinetic evidence from intact cells and biochemical evidence from permeabilized cells is provided that the second messenger of the inhibitory pathway is cAMP. To estimate the amplification of this step, we determined the cAMP concentration necessary to maximally inhibit superoxide anion production of formyl peptide-stimulated electropermeabilized cells, and we compare these concentrations to previously determined values of cAMP production in neutrophils. We conclude that each receptor may generate up to 10,000 molecules of cAMP.     

Amino-terminal alanine functions in a calcium-specific process essential for membrane binding by prothrombin fragment 1

Two acetylation sites on prothrombin fragment 1 (amino-terminal 156 amino acid residues of bovine prothrombin) are essential for the tight calcium and membrane binding functions of the protein; calcium protects both of these sites from acetylation [Welsch, D. J., Pletcher, C. H., & Nelsestuen, G. L. (1988) Biochemistry (first of three papers in this issue)]. The epsilon-amino groups of the lysine residues (positions 3, 11, 44, 57, and 97) were not critical to protein function and were acetylated in the calcium-protected protein. The most reactive of the two essential acetylation sites was identified as amino-terminal alanine. To identify this site, fragment 1 was first acetylated in the presence of calcium to derivatize the nonessential sites. Removal of calcium and partial acetylation with radioactive reagent produced a single major radioactive peptide. Isolation and characterization of this peptide showed that the radioactivity was associated with amino-terminal alanine. In addition, sequence analysis of calcium-protected protein showed the presence of underivatized amino-terminal alanine. Surprisingly, covalent modification with a trinitrophenyl group did not alter membrane binding activity. Thus, the positive charge on the amino terminus did not appear critical to its function. Acetylation of amino-terminal alanine without acetylation of the second essential site produced a fragment 1 derivative which had a high requirement for calcium and which had lost most membrane binding function. However, this protein had only slightly altered affinity for magnesium ion. In agreement with this metal ion selectivity, protection of amino-terminal alanine was calcium specific, and magnesium ion did not protect this site from acetylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

An intramolecular disulphide bond reduces the efficacy of a lipoprotein plasma membrane sorting signal

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.