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L-type amino acid transporter 1 (LAT1; SLC7A5), which preferentially transports large neutral amino acids, is highly upregulated in various cancers. LAT1 supplies cancer cells with amino acids as substrates for enhanced biosynthetic and bioenergetic reactions and stimulates signalling networks involved in the regulation of survival, growth and proliferation. LAT1 inhibitors show anti-cancer effects and a representative compound, JPH203, is under clinical evaluation. However, pharmacological impacts of LAT1 inhibition on the cellular amino acid transport and the translational activity in cancer cells that are conceptually pivotal for its anti-proliferative effect have not been elucidated yet. Here, we demonstrated that JPH203 drastically inhibits the transport of all the large neutral amino acids in pancreatic ductal adenocarcinoma cells. The inhibitory effects of JPH203 were observed even in competition with high concentrations of amino acids in a cell culture medium. The analyses of the nutrient-sensing mTORC1 and GAAC pathways and the protein synthesis activity revealed that JPH203 downregulates the global translation. This study demonstrates a predominant contribution of LAT1 to the transport of large neutral amino acids in cancer cells and the suppression of protein synthesis by JPH203 supposed to underly its broad anti-proliferative effects across various types of cancer cells.

L‐type amino acid transporter 1 (LAT1) is highly expressed in various cancers and plays important roles not only in the amino acid uptake necessary for cancer growth but also in cellular signaling. Recent research studies have reported anticancer effects of LAT1 inhibitors and demonstrated their potential for cancer therapy. Here, we characterized the proteome and phosphoproteome in LAT1‐inhibited cancer cells. We used JPH203, a selective LAT1 inhibitor, and performed tandem mass tag–based quantitative proteomics and phosphoproteomics on four biliary tract cancer cell lines sensitive to JPH203. Our analysis identified hundreds to thousands of differentially expressed proteins and phosphorylated sites, demonstrating the broad influence of LAT1 inhibition. Our findings showed various functional pathways altered by LAT1 inhibition, and provided possible regulators and key kinases in LAT1‐inhibited cells. Comparison of these changes among cell lines provides insights into general pathways and regulators associated with LAT1 inhibition and particularly suggests the importance of cell cycle–related pathways and kinases. Moreover, we evaluated the anticancer effects of the combinations of JPH203 with cell cycle–related kinase inhibitors and demonstrated their potential for cancer therapy. This is the first study providing the proteome‐wide scope of both protein expression and phosphorylation signaling perturbed by LAT1 inhibition in cancer cells., Our study investigated anticancer effects caused by inhibition of L‐type amino acid transporter 1 (LAT1), which is highly expressed in cancers and plays important roles in the amino acid uptake as well as cellular signaling. The integrated proteomics and phosphoproteomics on four biliary tract cancer cell lines revealed altered biological pathways, possible regulators, and key kinases in LAT1‐inhibited cells. We found the importance of cell cycle–related pathways in LAT1‐inhibited cells and demonstrated the therapeutic potential of LAT1 inhibitor in combination with cell cycle–related kinase inhibitor in cancer treatment.

Halogenated tyrosine/phenylalanine derivatives have been developed for use in tumor imaging and targeted alpha therapy. 3-Fluoro-α-methyl-l-tyrosine (FAMT), targeting amino acid transporter LAT1 (SLC7A5), is a cancer-specific positron emission tomography probe that exhibits high renal accumulation, which is supposed to be mediated by organic anion transporter OAT1 (SLC22A6). In the present study, we investigated the structural requirements of FAMT essential for interaction with OAT1. OAT1 transported FAMT with a K m of 171.9 μM. In structure-activity relationship analyses, removal of either the 3-halogen or 4-hydroxyl group from FAMT or its structural analog 3-iodo-α-methyl-l-tyrosine greatly decreased the interaction with OAT1, reducing the [14C]p-aminohippurate uptake inhibition and the efflux induction. By contrast, the α-methyl group, which is essential for LAT1 specificity, contributed to a lesser degree. In fluorinated tyrosine derivatives, fluorine at any position was accepted by OAT1 when there was a hydroxyl group at the ortho-position, whereas ortho-fluorine was less interactive when a hydroxyl group was at meta- or para-positions. The replacement of the ortho-fluorine with a bulky iodine atom greatly increased the interaction. In in vivo studies, probenecid decreased the renal accumulation (P < 0.001) and urinary excretion (P = 0.0012) of FAMT, whereas the plasma concentration was increased, suggesting the involvement of OAT1-mediated transepithelial organic anion excretion. LAT1-specific 2-fluoro-α-methyltyrosine, which had lower affinity for OAT1, exhibited lower renal accumulation (P = 0.0142) and higher tumor uptake (P = 0.0192) compared with FAMT. These results would provide a basis to design tumor-specific compounds that can avoid renal accumulation for tumor imaging and targeted alpha therapy. SIGNIFICANCE STATEMENT: We revealed the structural characteristics of halogenated tyrosine derivatives essential for interaction with the organic anion transporter responsible for their renal accumulation. We have confirmed that such interactions are important for renal handling and tumor uptake. The critical contribution of hydroxyl and halogen groups and their positions as well as the role of α-methyl group found in the present study may facilitate the development of tumor-specific compounds while avoiding renal accumulation for use in tumor imaging and targeted alpha therapy.

Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity, that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. This is the first characterization of molecular features of EAAT2 and the selective inhibition mechanism, underlying structure-based drug design for EAAT2.

SummaryThe L-type amino acid transporter 1 (LAT1) transports large neutral amino acids and drugs across the plasma membrane and is crucial for nutrient uptake, brain drug delivery and tumor growth. LAT1 is a unique solute carrier that forms a disulfide-linked heterodimer with the cell-surface glycoprotein CD98 heavy chain (CD98hc), but the mechanisms of its molecular assembly and amino acid transport are poorly understood. Here we report the cryo-EM structure of the human LAT1-CD98hc heterodimer at 3.4 Å resolution, revealing the hitherto unprecedented architecture of a solute carrier-glycoprotein heterocomplex. LAT1 features a canonical LeuT-fold while exhibiting an unusual loop structure on transmembrane helix 6, creating an extended cavity to accommodate bulky hydrophobic amino acids and drugs. CD98hc engages with LAT1 through multiple interactions, not only in the extracellular and transmembrane domains but also in the interdomain linker. The heterodimer interface features multiple sterol molecules, corroborating previous biochemical data on the role of cholesterols in heterodimer stabilization. We also visualized the binding modes of two anti-CD98 antibodies and show that they recognize distinct, multiple epitopes on CD98hc but not its glycans, explaining their robust reactivities despite the glycan heterogeneity. Furthermore, we mapped disease-causing mutations onto the structure and homology models, which rationalized some of the phenotypes of SLC3- and SLC7-related congenital disorders. Together, these results shed light on the principles of the structural assembly between a glycoprotein and a solute carrier, and provide a template for improving preclinical drugs and therapeutic antibodies targeting LAT1 and CD98.

Background Tumor angiogenesis is regarded as a rational anti-cancer target. The efficacy and indications of anti-angiogenic therapies in clinical practice, however, are relatively limited. Therefore, there still exists a demand for revealing the distinct characteristics of tumor endothelium that is crucial for the pathological angiogenesis. L-type amino acid transporter 1 (LAT1) is well known to be highly and broadly upregulated in tumor cells to support their growth and proliferation. In this study, we aimed to establish the upregulation of LAT1 as a novel general characteristic of tumor-associated endothelial cells as well, and to explore the functional relevance in tumor angiogenesis. Methods Expression of LAT1 in tumor-associated endothelial cells was immunohistologically investigated in human pancreatic ductal adenocarcinoma (PDA) and xenograft- and syngeneic mouse tumor models. The effects of pharmacological and genetic ablation of endothelial LAT1 were examined in aortic ring assay, Matrigel plug assay, and mouse tumor models. The effects of LAT1 inhibitors and gene knockdown on cell proliferation, regulation of translation, as well as on the VEGF-A-dependent angiogenic processes and intracellular signaling were investigated in in vitro by using human umbilical vein endothelial cells. Results LAT1 was highly expressed in vascular endothelial cells of human PDA but not in normal pancreas. Similarly, high endothelial LAT1 expression was observed in mouse tumor models. The angiogenesis in ex/in vivo assays was suppressed by abrogating the function or expression of LAT1. Tumor growth in mice was significantly impaired through the inhibition of angiogenesis by targeting endothelial LAT1. LAT1-mediated amino acid transport was fundamental to support endothelial cell proliferation and translation initiation in vitro. Furthermore, LAT1 was required for the VEGF-A-dependent migration, invasion, tube formation, and activation of mTORC1, suggesting a novel cross-talk between pro-angiogenic signaling and nutrient-sensing in endothelial cells. Conclusions These results demonstrate that the endothelial LAT1 is a novel key player in tumor angiogenesis, which regulates proliferation, translation, and pro-angiogenic VEGF-A signaling. This study furthermore indicates a new insight into the dual functioning of LAT1 in tumor progression both in tumor cells and stromal endothelium. Therapeutic inhibition of LAT1 may offer an ideal option to potentiate anti-angiogenic therapies.

Background: New strategies are needed to combat multidrug-resistant bacteria. The restriction of iron uptake by bacteria is a promising way to inhibit their growth. We aimed to suppress the growth of Vibrio bacterial species by inhibiting their ferric ion-binding protein (FbpA) using food components. Methods: Twenty spices were selected for the screening of FbpA inhibitors. The candidate was applied to antibacterial tests, and the mechanism was further studied. Results: An active compound, rosmarinic acid (RA), was screened out. RA binds competitively and more tightly than Fe3+ to VmFbpA, the FbpA from V. metschnikovii, with apparent KD values of 8 μM vs. 17 μM. Moreover, RA can inhibit the growth of V. metschnikovii to one-third of the control at 1000 μM. Interestingly, sodium citrate (SC) enhances the growth inhibition effect of RA, although SC only does not inhibit the growth. The combination of RA/SC completely inhibits the growth of not only V. metschnikovii at 100/100 μM but also the vibriosis-causative pathogens V. vulnificus and V. parahaemolyticus, at 100/100 and 1000/100 μM, respectively. However, RA/SC does not affect the growth of Escherichia coli. Conclusions: RA/SC is a potential bacteriostatic agent against Vibrio species while causing little damage to indigenous gastrointestinal bacteria.

Novobiocin is an orally active antibiotic that inhibits DNA gyrase by binding the ATP-binding site in the ATPase subunit. Although effective against Gram-positive pathogens, novobiocin has limited activity against Gram-negative organisms due to the presence of the lipopolysaccharide-containing outer membrane, which acts as a permeability barrier. Using a novobiocin-sensitive Escherichia coli strain with a leaky outer membrane, we identified a mutant with increased resistance to novobiocin. Unexpectedly, the mutation that increases novobiocin resistance was not found to alter gyrase, but the ATPase that powers lipopolysaccharide (LPS) transport. Co-crystal structures, biochemical, and genetic evidence show novobiocin directly binds this ATPase. Novobiocin does not bind the ATP binding site but rather the interface between the ATPase subunits and the transmembrane subunits of the LPS transporter. This interaction increases the activity of the LPS transporter, which in turn alters the permeability of the outer membrane. We propose that novobiocin will be a useful tool for understanding how ATP hydrolysis is coupled to LPS transport.

The L-type amino acid transporter 1 (LAT1 or SLC7A5) transports large neutral amino acids across the membrane and is crucial for brain drug delivery and tumor growth. LAT1 forms a disulfide-linked heterodimer with CD98 heavy chain (CD98hc, 4F2hc or SLC3A2), but the mechanism of assembly and amino acid transport are poorly understood. Here we report the cryo-EM structure of the human LAT1-CD98hc heterodimer at 3.3-Å resolution. LAT1 features a canonical Leu T-fold and exhibits an unusual loop structure on transmembrane helix 6, creating an extended cavity that might accommodate bulky amino acids and drugs. CD98hc engages with LAT1 through the extracellular, transmembrane and putative cholesterol-mediated interactions. We also show that two anti-CD98 antibodies recognize distinct, multiple epitopes on CD98hc but not its glycans, explaining their robust reactivities. These results reveal the principles of glycoprotein-solute carrier assembly and provide templates for improving preclinical drugs and antibodies targeting LAT1 or CD98hc.

Canagliflozin, a selective sodium/glucose cotransporter (SGLT) 2 inhibitor, suppresses the renal reabsorption of glucose and decreases blood glucose level in patients with type 2 diabetes. A characteristic of canagliflozin is its modest SGLT1 inhibitory action in the intestine at clinical dosage. To reveal its mechanism of action, we investigated the interaction of canagliflozin with SGLT1 and SGLT2. Inhibition kinetics and transporter-mediated uptake were examined in human SGLT1- or SGLT2-expressing cells. Whole-cell patch-clamp recording was conducted to examine the sidedness of drug action. Canagliflozin competitively inhibited SGLT1 and SGLT2, with high potency and selectivity for SGLT2. Inhibition constant (Ki) values for SGLT1 and SGLT2 were 770.5 and 4.0 nM, respectively. (14)C-canagliflozin was suggested to be transported by SGLT2; however, the transport rate was less than that of α-methyl-d-glucopyranoside. Canagliflozin inhibited α-methyl-d-glucopyranoside-induced SGLT1- and SGLT2-mediated inward currents preferentially from the extracellular side and not from the intracellular side. Based on the Ki value, canagliflozin is estimated to sufficiently inhibit SGLT2 from the urinary side in renal proximal tubules. The Ki value for SGLT1 suggests that canagliflozin suppresses SGLT1 in the small intestine from the luminal side, whereas it does not affect SGLT1 in the heart and skeletal muscle, considering the maximal concentration of plasma-unbound canagliflozin. Similarly, SGLT1 in the kidney would not be inhibited, thereby aiding in the prevention of hypoglycemia. After binding to SGLT2, canagliflozin may be reabsorbed by SGLT2, which leads to the low urinary excretion and prolonged drug action of canagliflozin.

3-(18)F-l-α-methyl-tyrosine ([18F]FAMT), a PET probe for tumor imaging, has advantages of high cancer-specificity and lower physiologic background. FAMT-PET has been proved useful in clinical studies for the prediction of prognosis, the assessment of therapy response and the differentiation of malignant tumors from inflammation and benign lesions. The tumor uptake of [18F]FAMT in PET is strongly correlated with the expression of L-type amino acid transporter 1 (LAT1), an isoform of system L upregulated in cancers. In this study, to assess the transporter-mediated mechanisms in FAMT uptake by tumors, we examined amino acid transporters for FAMT transport. We synthesized [14C]FAMT and measured its transport by human amino acid transporters expressed in Xenopus oocytes. The transport of FAMT was compared with that of l-methionine, a well-studied amino acid PET probe. The significance of LAT1 in FAMT uptake by tumor cells was confirmed by siRNA knockdown. Among amino acid transporters, [14C]FAMT was specifically transported by LAT1, whereas l-[14C]methionine was taken up by most of the transporters. Km of LAT1-mediated [14C]FAMT transport was 72.7 μM, similar to that for endogenous substrates. Knockdown of LAT1 resulted in the marked reduction of [14C]FAMT transport in HeLa S3 cells, confirming the contribution of LAT1 in FAMT uptake by tumor cells. FAMT is highly specific to cancer-type amino acid transporter LAT1, which explains the cancer-specific accumulation of [18F]FAMT in PET. This, vice versa, further supports the cancer-specific expression of LAT1. This study has established FAMT as a LAT1-specific molecular probe to monitor the expression of a potential tumor biomarker LAT1.

A PET tracer for tumor imaging, 3- 18 F- l - α -methyl-tyrosine ([ 18 F]FAMT), has advantages of high cancer-specificity and low physiological background. In clinical studies, FAMT-PET has been proved useful for the detection of malignant tumors and their differentiation from inflammation and benign lesions. The tumor specific uptake of FAMT is due to its high-selectivity to cancer-type amino acid transporter LAT1 among amino acid transporters. In [ 18 F]FAMT PET, kidney is the only organ that shows high physiological background. To reveal transporters involved in renal accumulation of FAMT, we have examined [ 14 C]FAMT uptake on the organic ion transporters responsible for the uptake into tubular epithelial cells. We have found that OAT1, OAT10 and OCTN2 transport [ 14 C]FAMT. The [ 14 C]FAMT uptake was inhibited by probenecid, furosemide and ethacrynic acid, consistent with the properties of the transporters. The amino acid uptake inhibitor, 2-amino-2-norbornanecarboxylic acid (BCH), also inhibited the [ 14 C]FAMT uptake, whereas OCTN2-mediated [ 14 C]FAMT uptake was Na + -dependent. We propose that FAMT uptake by OAT1, OAT10 and OCTN2 into tubular epithelial cells could contribute to the renal accumulation of FAMT. The results from this study would provide clues to the treatments to reduce renal background and enhance tumor uptake as well as to designing PET tracers with less renal accumulation.

Among amino acids, leucine is a potential signaling molecule to regulate cell growth and metabolism by activating mechanistic target of rapamycin complex 1 (mTORC1). To reveal the critical structures of leucine molecule to activate mTORC1, we examined the structure-activity relationships of leucine derivatives in HeLa S3 cells for cellular uptake and for the induction of phosphorylation of p70 ribosomal S6 kinase 1 (p70S6K), a downstream effector of mTORC1. The activation of mTORC1 by leucine and its derivatives was the consequence of two successive events: the cellular uptake by L-type amino acid transporter 1 (LAT1) responsible for leucine uptake in HeLa S3 cells and the activation of mTORC1 following the transport. The structural requirement for the recognition by LAT1 was to have carbonyl oxygen, alkoxy oxygen of carboxyl group, amino group and hydrophobic side chain. In contrast, the requirement for mTORC1 activation was more rigorous. It additionally required fixed distance between carbonyl oxygen and alkoxy oxygen of carboxyl group, and amino group positioned at α-carbon. L-Configuration in chirality and appropriate length of side chain with a terminal isopropyl group were also important. This confirmed that LAT1 itself is not a leucine sensor. Some specialized leucine sensing mechanism with rigorous requirement for agonistic structures should exist inside the cells because leucine derivatives not transported by LAT1 did not activate mTORC1. Because LAT1-mTOR axis is involved in the regulation of cell growth and cancer progression, the results from this study may provide a new insight into therapeutics targeting both LAT1 and leucine sensor.

Various physiological and pathological processes are accompanied with the alteration of pH at extracellular juxtamembrane region. Accordingly, the methods to analyze the cell surface pH have been demanded in biological and medical sciences. In this study, we have established a novel methodology for cell surface pH imaging using poly(ethylene glycol)-phospholipid (PEG-lipid) as a core structure of ratiometric fluorescent probes. PEG-lipid is a synthetic amphiphilic polymer originally developed for the cell surface modification in transplantation therapy. Via its hydrophobic alkyl chains of the phospholipid moiety, PEG-lipid is, when applied extracellularly, spontaneously inserted into the plasma membrane and retained at the surface of the cells. We have demonstrated that the PEG-lipid conjugated with fluorescein isothiocyanate (FITC-PEG-lipid) can be used as a sensitive and reversible cell-surface-anchored pH probe between weakly alkaline and acidic pH with an excellent spatiotemporal resolution. The remarkably simple procedure for cell-surface labeling with FITC-PEG-lipid would also be advantageous when considering its application to high-throughput in vitro assay. This study further indicates that various probes useful for the investigation of juxtamembrane environments could also be developed by using PEG-lipid as the core structure for bio-membrane anchoring.

L-type amino acid transporter 1 (LAT1) is known as a cancer-type amino acid transporter. In cancer cells, LAT1 is responsible for the cellular uptake of many essential amino acids including leucine that activates mechanistic/mammalian target of rapamycin (mTOR), regulating cancer cell growth. In this study, we designed a novel series of LAT1 inhibitors, SKN101–105, based on the structure of triiodothyronine (T3), a known LAT1 blocker. The compounds consist of core structure of 2-amino-3-[3,5-dichloro-4-(naphthalene-1-methoxy)-phenyl]-propanoic acid and different modifications on the naphthalene. Among them, the compounds including SKN103 with a modified phenyl group at C-7 position of naphthalene inhibited LAT1-mediated leucine transport, whereas SKN102 with a phenyl group at C-6 position did not, indicating the importance of the position of substituents on the naphthalene for the interaction with LAT1. SKN103 was suggested to be a non-transportable blocker rather than a substrate of LAT1 and inhibited LAT1 in a competitive manner with the K i value of 2.1 μM. SKN103 suppressed mTOR activity and the growth of cancer cells. Moreover, SKN103 in combination with cisplatin additively enhanced the growth inhibition in cancer cells. This study provides an additional insight into the structure-activity relationship of LAT1 ligands, which could lead to designing desirable LAT1 inhibitors.

Heterodimeric amino acid transporters play crucial roles in epithelial transport, as well as in cellular nutrition. Among them, the heterodimer of a membrane protein b(0,+)AT/SLC7A9 and its auxiliary subunit rBAT/SLC3A1 is responsible for cystine reabsorption in renal proximal tubules. The mutations in either subunit cause cystinuria, an inherited amino aciduria with impaired renal reabsorption of cystine and dibasic amino acids. However, an unsolved paradox is that rBAT is highly expressed in the S3 segment, the late proximal tubules, whereas b(0,+)AT expression is highest in the S1 segment, the early proximal tubules, so that the presence of an unknown partner of rBAT in the S3 segment has been proposed. In this study, by means of coimmunoprecipitation followed by mass spectrometry, we have found that a membrane protein AGT1/SLC7A13 is the second partner of rBAT. AGT1 is localized in the apical membrane of the S3 segment, where it forms a heterodimer with rBAT. Depletion of rBAT in mice eliminates the expression of AGT1 in the renal apical membrane. We have reconstituted the purified AGT1-rBAT heterodimer into proteoliposomes and showed that AGT1 transports cystine, aspartate, and glutamate. In the apical membrane of the S3 segment, AGT1 is suggested to locate itself in close proximity to sodium-dependent acidic amino acid transporter EAAC1 for efficient functional coupling. EAAC1 is proposed to take up aspartate and glutamate released into luminal fluid by AGT1 due to its countertransport so that preventing the urinary loss of aspartate and glutamate. Taken all together, AGT1 is the long-postulated second cystine transporter in the S3 segment of proximal tubules and a possible candidate to be involved in isolated cystinuria.

Powering LPS Export The surface of Gram-negative bacteria such as Escherichia coli is covered in lipopolysaccharide (LPS), which must be synthesized within the cytoplasm and then exported across the periplasm to the outer leaflet of the outer membrane. Okuda et al. (p. 1214 , published online 8 November) describe the detection of LPS molecules bound to specific components of the E. coli lipopolysaccharide exporter during transport in vivo. In vitro reconstitution of LPS transport revealed the energetic requirements for the first two steps of transport, release from the membrane and transit along the periplasmic bridge of the exporter that connects the inner and outer membrane components. The findings suggest that adenosine triphosphate hydrolysis by a cytoplasmic component of the machine provides energy to push a continuous stream of LPS against its concentration gradient to the cell surface.

Lipoproteins that play critical roles in various cellular functions of Gram-negative bacteria are localized in the cells inner and outer membranes. Lol proteins (LolA, LolB, LolC, LolD, and LolE) are involved in the transportation of outer membrane-directed lipoproteins from the inner to the outer membrane. LolA is a periplasmic chaperone that transports lipoproteins, and LolB is an outer membrane receptor that accepts lipoproteins. To clarify the structural basis for the lipoprotein transfer from LolA to LolB, we examined the interaction between LolA and mLolB, a soluble mutant of LolB, using solution NMR spectroscopy. We determined the interaction mode between LolA and mLolB with conformational changes of LolA. Based upon the observations, we propose that the LolA·LolB complex forms a tunnel-like structure, where the hydrophobic insides of LolA and LolB are connected, which enables lipoproteins to transfer from LolA to LolB.

Escherichia coli cells express at least 90 species of lipoprotein. LolB is one of the essential outer membrane lipoproteins, being involved in the last step of lipoprotein sorting. It accepts lipoproteins from a periplasmic molecular chaperone, LolA, and mediates the outer membrane anchoring of lipoproteins through a largely unknown mechanism. It has been shown previously that a LolB derivative, mLolB, lacking an N-terminal acyl chain, can bind lipoproteins. We examined how the lack of an N-terminal anchor affects the outer membrane anchoring of lipoproteins. Surprisingly, mLolB compensates for LolB function and supports E. coli growth, indicating that the N-terminal anchor is not essential for its function. Indeed, mLolB correctly localizes lipoproteins to either the inner or outer membrane depending on the sorting signal at the steady state. Furthermore, periplasmic mLolB enables the dissection of LolB function, namely lipoprotein binding, membrane targeting and lipoprotein anchoring. It mediates the transfer of lipoproteins from LolA to the outer membrane, but also the inner membrane and liposomes, indicating that mLolB exhibits no membrane preference and targets to phospholipids. Consequently, an outer membrane-specific lipoprotein is transiently mislocalized to the inner membrane when cells express only mLolB. LolB anchored to the outer membrane does not cause such mislocalization and is more active than mLolB. Phosphatidylethanolamine has been found to stimulate the mLolB-dependent membrane anchoring of lipoproteins. Taken together, these results indicate that lipoprotein binding, membrane targeting and membrane incorporation of lipoproteins are intrinsic functions of LolB.

The structures of a lipoprotein carrier, LolA, and a lipoprotein receptor, LolB, are similar except for an extra C-terminal loop containing a 3(10) helix and beta-strand 12 in LolA. Lipoprotein release was significantly reduced when beta-12 was deleted. Deletion of the 3(10) helix also inhibited the lipoprotein release. Furthermore, lipoproteins were non-specifically localized to membranes when LolA lacked the 3(10) helix. Thus, the membrane localization of lipoproteins with the LolA derivative lacking the 3(10) helix was independent of LolB whereas LolB was essential for the outer membrane localization of lipoproteins with the wild-type LolA.

Lipopolysaccharide is a major glycolipid component in the outer leaflet of the outer membrane (OM), a peculiar permeability barrier of Gram-negative bacteria that prevents many toxic compounds from entering the cell. Lipopolysaccharide transport (Lpt) across the periplasmic space and its assembly at the Escherichia coli cell surface are carried out by a transenvelope complex of seven essential Lpt proteins spanning the inner membrane (LptBCFG), the periplasm (LptA), and the OM (LptDE), which appears to operate as a unique machinery. LptC is an essential inner membrane-anchored protein with a large periplasm-protruding domain. LptC binds the inner membrane LptBFG ABC transporter and interacts with the periplasmic protein LptA. However, its role in lipopolysaccharide transport is unclear. Here we show that LptC lacking the transmembrane region is viable and can bind the LptBFG inner membrane complex; thus, the essential LptC functions are located in the periplasmic domain. In addition, we characterize two previously described inactive single mutations at two conserved glycines (G56V and G153R, respectively) of the LptC periplasmic domain, showing that neither mutant is able to assemble the transenvelope machinery. However, while LptCG56V failed to copurify any Lpt component, LptCG153R was able to interact with the inner membrane protein complex LptBFG. Overall, our data further support the model whereby the bridge connecting the inner and outer membranes would be based on the conserved structurally homologous jellyroll domain shared by five out of the seven Lpt components.

A photo-sensitive amino acid analogue was introduced into an outer membrane lipoprotein, Pal, and then subjected to photo-crosslinking with the lipoprotein-specific ABC transporter LolCDE. Pal crosslinked to LolE but not LolC in vivo despite that both are structurally similar membrane subunits. LolCDE liganded with Pal containing the photo-sensitive amino acid analogue was isolated and subjected to in vitro photo-crosslinking. LolE was found to be the binding site for Pal. ATP binding to LolD decreased the LolE–Pal crosslinking by decreasing their hydrophobic interaction. ATP hydrolysis in the presence of LolA completely abolished the LolE–Pal crosslinking and, concomitantly, generated a new LolA–Pal crosslinked product. Structured summary of protein interactions LolE and Pal physically interact by cross-linking study ( View interaction ) LolE and Pal bind by cross-linking study ( View interaction ) Pal and Pal bind by cross-linking study ( View interaction ) Pal and LolA physically interact by competition binding ( View interaction )

The presence of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) of Gram-negative bacteria creates a permeability barrier that prevents the entry of most currently available antibiotics. The seven lipopolysaccharide transport (Lpt) proteins involved in transporting and assembling this glycolipid are essential for growth and division in Escherichia coli; therefore, inhibiting their functions leads to cell death. LptB, the ATPase that provides energy for LPS transport and assembly, forms a complex with three other inner membrane (IM) components, LptC, F, and G. We demonstrate that inhibitors of pure LptB can also inhibit the full IM complex, LptBFGC, purified in detergent. We also compare inhibition of LptB and the LptBFGC complex with the antibiotic activity of these compounds. Our long-term goal is to develop tools to study inhibitors of LPS biogenesis that could serve as potentiators by disrupting the OM permeability barrier, facilitating entry of clinically used antibiotics not normally used to treat Gram-negative infections, or that can serve as antibiotics themselves.

Gram-negative bacteria are impervious to many drugs and environmental stresses because they possess an outer membrane (OM) containing lipopolysaccharide (LPS). LPS is biosynthesized at the cytoplasmic (inner) membrane (IM) and is transported to the OM by an unknown mechanism involving the LPS transport proteins, LptA-G. These proteins have been proposed to form a bridge between the two membranes; however, it is not known how this bridge is assembled to prevent mistargeting of LPS. We use in vivo photocrosslinking to reveal the specific protein-protein interaction sites that give rise to the Lpt bridge. We also show that the formation of this transenvelope bridge cannot proceed before the correct assembly of the LPS translocon in the OM. This ordered sequence of events may ensure that LPS is never transported to the OM if it cannot be translocated across it to the cell surface.