The Quorum Sensing Auto-Inducer 2 (AI-2) Stimulates Nitrogen Fixation and Favors Ethanol Production over Biomass Accumulation in Zymomonas mobilis.

Autoinducer 2 (or AI-2) is one of the molecules used by bacteria to trigger the Quorum Sensing (QS) response, which activates expression of genes involved in a series of alternative mechanisms, when cells reach high population densities (including bioluminescence, motility, biofilm formation, stress resistance, and production of public goods, or pathogenicity factors, among others). Contrary to most autoinducers, AI-2 can induce QS responses in both Gram-negative and Gram-positive bacteria, and has been suggested to constitute a trans-specific system of bacterial communication, capable of affecting even bacteria that cannot produce this autoinducer. In this work, we demonstrate that the ethanologenic Gram-negative bacterium Zymomonas mobilis (a non-AI-2 producer) responds to exogenous AI-2 by modulating expression of genes involved in mechanisms typically associated with QS in other bacteria, such as motility, DNA repair, and nitrogen fixation. Interestingly, the metabolism of AI-2-induced Z. mobilis cells seems to favor ethanol production over biomass accumulation, probably as an adaptation to the high-energy demand of N2 fixation. This opens the possibility of employing AI-2 during the industrial production of second-generation ethanol, as a way to boost N2 fixation by these bacteria, which could reduce costs associated with the use of nitrogen-based fertilizers, without compromising ethanol production in industrial plants.

Zmo0994, a novel LEA-like protein from Zymomonas mobilis, increases multi-abiotic stress tolerance in Escherichia coli.

BACKGROUND: Pretreatment processes and subsequent enzymatic hydrolysis are prerequisites to utilize lignocellulosic sugar for fermentation. However, the resulting hydrolysate frequently hinders fermentation processes due to the presence of inhibitors and toxic products (e.g., ethanol). Thus, it is crucial to develop robust microbes conferring multi-stress tolerance. RESULTS: Zmo0994, a functionally uncharacterized protein from Zymomonas mobilis, was identified and characterized for the first time. A major effect of Zmo0994 was a significant enhancement in the tolerance to abiotic stresses such as ethanol, furfural, 5'-hydroxymethylfurfural and high temperature, when expressed in Escherichia coli. Through transcriptome analysis and in vivo experiments, the cellular mechanism of this protein was revealed as due to its ability to trigger genes, involved in aerobic respiration for ATP synthesis. CONCLUSIONS: These findings have significant implications that might lead to the development of robust microbes for the highly efficient industrial fermentation processes.

A Gß protein and the TupA Co-Regulator Bind to Protein Kinase A Tpk2 to Act as Antagonistic Molecular Switches of Fungal Morphological Changes.

The human pathogenic fungus Paracoccidioides brasiliensis (Pb) undergoes a morphological transition from a saprobic mycelium to pathogenic yeast that is controlled by the cAMP-signaling pathway. There is a change in the expression of the Gß-protein PbGpb1, which interacts with adenylate cyclase, during this morphological transition. We exploited the fact that the cAMP-signaling pathway of Saccharomyces cerevisiae does not include a Gß-protein to probe the functional role of PbGpb1. We present data that indicates that PbGpb1 and the transcriptional regulator PbTupA both bind to the PKA protein PbTpk2. PbTPK2 was able to complement a TPK2Δ strain of S. cerevisiae, XPY5a/α, which was defective in pseudohyphal growth. Whilst PbGPB1 had no effect on the parent S. cerevisiae strain, MLY61a/α, it repressed the filamentous growth of XPY5a/α transformed with PbTPK2, behaviour that correlated with a reduced expression of the floculin FLO11. In vitro, PbGpb1 reduced the kinase activity of PbTpk2, suggesting that inhibition of PbTpk2 by PbGpb1 reduces the level of expression of Flo11, antagonizing the filamentous growth of the cells. In contrast, expressing the co-regulator PbTUPA in XPY5a/α cells transformed with PbTPK2, but not untransformed cells, induced hyperfilamentous growth, which could be antagonized by co-transforming the cells with PbGPB1. PbTUPA was unable to induce the hyperfilamentous growth of a FLO8Δ strain, suggesting that PbTupA functions in conjunction with the transcription factor Flo8 to control Flo11 expression. Our data indicates that P. brasiliensis PbGpb1 and PbTupA, both of which have WD/ß-propeller structures, bind to PbTpk2 to act as antagonistic molecular switches of cell morphology, with PbTupA and PbGpb1 inducing and repressing filamentous growth, respectively. Our findings define a potential mechanism for controlling the morphological switch that underpins the virulence of dimorphic fungi.

Tripartite efflux pumps: energy is required for dissociation, but not assembly or opening of the outer membrane channel of the pump.

The MtrCDE multidrug pump, from Neisseria gonorrhoeae, is assembled from the inner and outer membrane proteins MtrD and MtrE, which are connected by the periplasmic membrane fusion protein MtrC. Although it is clear that MtrD delivers drugs to the channel of MtrE, it remains unclear how drug delivery and channel opening are connected. We used a vancomycin sensitivity assay to test for opening of the MtrE channel. Cells expressing MtrE or MtrE-E434K were insensitive to vancomycin; but became moderately and highly sensitive to vancomycin respectively, when coexpressed with MtrC, suggesting that the MtrE channel opening requires MtrC binding and is energy-independent. Cells expressing wild-type MtrD, in an MtrCE background, were vancomycin-insensitive, but moderately sensitive in an MtrCE-E434K background. The mutation of residues involved in proton translocation inactivated MtrD and abolished drug efflux, rendered both MtrE and MtrE-E434K vancomycin-insensitive; imply that the pump-component interactions are preserved, and that the complex is stable in the absence of proton flux, thus sealing the open end of MtrE. Following the energy-dependent dissociation of the tripartite complex, the MtrE channel is able to reseal, while MtrE-E434K is unable to do so, resulting in the vancomycin-sensitive phenotype. Thus, our findings suggest that opening of the OMP via interaction with the MFP is energy-independent, while both drug export and complex dissociation require active proton flux.

Evidence for the assembly of a bacterial tripartite multidrug pump with a stoichiometry of 3:6:3.

The multiple transferable resistance (mTR) pump from Neisseria gonorrhoeae MtrCDE multidrug pump is assembled from the inner and outer membrane proteins MtrD and MtrE and the periplasmic membrane fusion protein MtrC. Previously we established that while there is a weak interaction of MtrD and MtrE, MtrC binds with relatively high affinity to both MtrD and MtrE. MtrD conferred antibiotic resistance only when it was expressed with MtrE and MtrC, suggesting that these proteins form a functional tripartite complex in which MtrC bridges MtrD and MtrE. Furthermore, we demonstrated that MtrC interacts with an intraprotomer groove on the surface of MtrE, inducing channel opening. However, a second groove is apparent at the interface of the MtrE subunits, which might also be capable of engaging MtrC. We have now established that MtrC can be cross-linked to cysteines placed in this interprotomer groove and that mutation of residues in the groove impair the ability of the pump to confer antibiotic resistance by locking MtrE in the closed channel conformation. Moreover, MtrE K390C forms an intermolecular disulfide bond with MtrC E149C locking MtrE in the open channel conformation, suggesting that a functional salt bridge forms between these residues during the transition from closed to open channel conformations. MtrC forms dimers that assemble into hexamers, and electron microscopy studies of single particles revealed that these hexamers are arranged into ring-like structures with an internal aperture sufficiently large to accommodate the MtrE trimer. Cross-linking of single cysteine mutants of MtrC to stabilize the dimer interface in the presence of MtrE, trapped an MtrC-MtrE complex with a molecular mass consistent with a stoichiometry of 3:6 (MtrE(3)MtrC(6)), suggesting that dimers of MtrC interact with MtrE, presumably by binding to the two grooves. As both MtrE and MtrD are trimeric, our studies suggest that the functional pump is assembled with a stoichiometry of 3:6:3.

The C-terminus of the phage λ Orf recombinase is involved in DNA binding.

Phage λ Orf substitutes for the activities of the Escherichia coli RecFOR proteins in vivo and is therefore implicated as a recombination mediator, encouraging the assembly of bacterial RecA onto single-stranded DNA (ssDNA) coated with SSB. Orf exists as a dimer in solution, associates with E. coli SSB and binds preferentially to ssDNA. To help identify interacting domains we analysed Orf and SSB proteins carrying mutations or truncations in the C-terminal region. A cluster of acidic residues at the carboxy-terminus of SSB is known to attract multiple protein partners to assist in DNA replication and repair. In this case an alternative domain must be utilized since Orf association with SSB was unaffected by an SSB113 point mutant (P176S) or removal of the last ten residues (ΔC10). Structurally the Orf C-terminus consists of a helix with a flexible tail that protrudes from each side of the dimer and could serve as a binding site for either SSB or DNA. Eliminating the six residue flexible tail (ΔC6) or the entire helix (ΔC19) had no significant impact on the Orf-SSB interaction. However, the OrfΔC6 protein exhibited reduced DNA binding, a feature shared by single amino acid substitutions within (W141F) or adjacent (R140A) to this region. The OrfΔC19 mutant bound poorly to DNA and secondary structure analysis in solution revealed that this truncation induces protein misfolding and aggregation. The results show that the carboxy-terminus of Orf is involved in nucleic acid recognition and also plays an unexpected role in maintaining structural integrity.

Opening of the outer membrane protein channel in tripartite efflux pumps is induced by interaction with the membrane fusion partner.

The multiple transferable resistance (MTR) pump, from Neisseria gonorrhoeae, is typical of the specialized machinery used to translocate drugs across the inner and outer membranes of Gram-negative bacteria. It consists of a tripartite complex composed of an inner-membrane transporter, MtrD, a periplasmic membrane fusion protein, MtrC, and an outer-membrane channel, MtrE. We have expressed the components of the pump in Escherichia coli and used the antibiotic vancomycin, which is too large to cross the outer-membrane by passive diffusion, to test for opening of the MtrE channel. Cells expressing MtrCDE are not susceptible to vancomycin, indicating that the channel is closed; but become susceptible to vancomycin in the presence of transported substrates, consistent with drug-induced opening of the MtrE channel. A mutational analysis identified residues Asn-198, Glu-434, and Gln-441, lining an intraprotomer groove on the surface of MtrE, to be important for pump function; mutation of these residues yielded cells that were sensitive to vancomycin. Pull-down assays and micro-calorimetry measurements indicated that this functional impairment is not due to the inability of MtrC to interact with the MtrE mutants; nor was it due to the MtrE mutants adopting an open conformation, because cells expressing these MtrE mutants alone are relatively insensitive to vancomycin. However, cells expressing the MtrE mutants with MtrC are sensitive to vancomycin, indicating that residues lining the intra-protomer groove control opening of the MtrE channel in response to binding of MtrC.

Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions.

We describe a general mass spectrometry approach to determine subunit stoichiometry and lipid binding in intact membrane protein complexes. By exploring conditions for preserving interactions during transmission into the gas phase and for optimally stripping away detergent, by subjecting the complex to multiple collisions, we released the intact complex largely devoid of detergent. This enabled us to characterize both subunit stoichiometry and lipid binding in 4 membrane protein complexes.

alpha-cleavage of the prion protein occurs in a late compartment of the secretory pathway and is independent of lipid rafts.

Endoproteolysis of the cellular prion protein (PrP(C)) modulates both the normal function of the protein and the pathogenesis of the neurodegenerative prion diseases. PrP(C) undergoes alpha-cleavage to generate the N-terminally truncated fragment C1. Utilizing various constructs of PrP(C) expressed in human neuroblastoma cells we investigated the subcellular compartment where alpha-cleavage occurs. C1 was detected at the cell surface and the generation of C1 occurred in mutants of PrP(C) incapable of Cu2+-mediated endocytosis. A transmembrane-anchored form that is not lipid raft-localised, as well as a secreted construct lacking the glycosyl-phosphatidylinositol membrane anchor, were also subject to alpha-cleavage. However, when this transmembrane-anchored form was modified with an endoplasmic reticulum retention motif, C1 was not formed. Inhibition of protein export from the Golgi by temperature block increased the amount of C1. Our data thus demonstrate that the alpha-cleavage of PrP(C) occurs predominantly in a raft-independent manner in a late compartment of the secretory pathway.

MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA.

Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled.

The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a human pathogenic fungus that switches from a saprobic mycelium to a pathogenic yeast. Consistent with the morphological transition being regulated by the cAMP-signalling pathway, there is an increase in cellular cAMP levels both transiently at the onset (< 24 h) and progressively in the later stages (> 120 h) of the transition to the yeast form, and this transition can be modulated by exogenous cAMP. We have cloned the cyr1 gene encoding adenylate cyclase (AC) and established that its transcript levels correlate with cAMP levels. In addition, we have cloned the genes encoding three Galpha (Gpa1-3), Gbeta (Gpb1) and Ggamma (Gpg1) G proteins. Gpa1 and Gpb1 interact with one another and the N-terminus of AC, but neither Gpa2 nor Gpa3 interacted with Gpb1 or AC. The interaction of Gpa1 with Gpb1 was blocked by GTP, but its interaction with AC was independent of bound nucleotide. The transcript levels for gpa1, gpb1 and gpg1 were similar in mycelium, but there was a transient excess of gpb1 during the transition, and an excess of gpa1 in yeast. We have interpreted our findings in terms of a novel signalling mechanism in which the activity of AC is differentially modulated by Gpa1 and Gpb1 to maintain the signal over the 10 days needed for the morphological switch.

An arsenic metallochaperone for an arsenic detoxification pump.

Environmental arsenic is a world-wide health issue, making it imperative for us to understand mechanisms of metalloid uptake and detoxification. The predominant intracellular form is the highly mephitic arsenite, which is detoxified by removal from cytosol. What prevents arsenite toxicity as it diffuses through cytosol to efflux systems? Although intracellular copper is regulated by metallochaperones, no chaperones involved in conferring resistance to other metals have been identified. In this article, we report identification of an arsenic chaperone, ArsD, encoded by the arsRDABC operon of Escherichia coli. ArsD transfers trivalent metalloids to ArsA, the catalytic subunit of an As(III)/Sb(III) efflux pump. Interaction with ArsD increases the affinity of ArsA for arsenite, thus increasing its ATPase activity at lower concentrations of arsenite and enhancing the rate of arsenite extrusion. Cells are consequently resistant to environmental concentrations of arsenic. This report of an arsenic chaperone suggests that cells regulate the intracellular concentration of arsenite to prevent toxicity.

Elucidation of the hrp clusters of Xanthomonas oryzae pv. oryzicola that control the hypersensitive response in nonhost tobacco and pathogenicity in susceptible host rice.

Xanthomonas oryzae pv. oryzicola, the cause of bacterial leaf streak in rice, possesses clusters of hrp genes that determine its ability to elicit a hypersensitive response (HR) in nonhost tobacco and pathogenicity in host rice. A 27-kb region of the genome of X. oryzae pv. oryzicola (RS105) was identified and sequenced, revealing 10 hrp, 9 hrc (hrp conserved), and 8 hpa (hrp-associated) genes and 7 regulatory plant-inducible promoter boxes. While the region from hpa2 to hpaB and the hrpF operon resembled the corresponding genes of other xanthomonads, the hpaB-hrpF region incorporated an hrpE3 gene that was not present in X. oryzae pv. oryzae. We found that an hrpF mutant had lost the ability to elicit the HR in tobacco and pathogenicity in adult rice plants but still caused water-soaking symptoms in rice seedlings and that Hpa1 is an HR elicitor in nonhost tobacco whose expression is controlled by an hrp regulator, HrpX. Using an Hrp phenotype complementation test, we identified a small hrp cluster containing the hrpG and hrpX regulatory genes, which is separated from the core hrp cluster. In addition, we identified a gene, prhA (plant-regulated hrp), that played a key role in the Hrp phenotype of X. oryzae pv. oryzicola but was neither in the core hrp cluster nor in the hrp regulatory cluster. A prhA mutant failed to reduce the HR in tobacco and pathogenicity in rice but caused water-soaking symptoms in rice. This is the first report that X. oryzae pv. oryzicola possesses three separate DNA regions for HR induction in nonhost tobacco and pathogenicity in host rice, which will provide a fundamental base to understand pathogenicity determinants of X. oryzae pv. oryzicola compared with those of X. oryzae pv. oryzae.

Functional similarities between phage lambda Orf and Escherichia coli RecFOR in initiation of genetic exchange.

Genetic recombination in bacteriophage lambda relies on DNA end processing by Exo to expose 3'-tailed strands for annealing and exchange by beta protein. Phage lambda encodes an additional recombinase, Orf, which participates in the early stages of recombination by supplying a function equivalent to the Escherichia coli RecFOR complex. These host enzymes assist loading of the RecA strand exchange protein onto ssDNA coated with ssDNA-binding protein. In this study, we purified the Orf protein, analyzed its biochemical properties, and determined its crystal structure at 2.5 angstroms. The homodimeric Orf protein is arranged as a toroid with a shallow U-shaped cleft, lined with basic residues, running perpendicular to the central cavity. Orf binds DNA, favoring single-stranded over duplex and with no obvious preference for gapped, 3'-tailed, or 5'-tailed substrates. An interaction between Orf and ssDNA-binding protein was indicated by far Western analysis. The functional similarities between Orf and RecFOR are discussed in relation to the early steps of recombinational exchange and the interplay between phage and bacterial recombinases.

VceR regulates the vceCAB drug efflux pump operon of Vibrio cholerae by alternating between mutually exclusive conformations that bind either drugs or promoter DNA.

VceR, a member of the TetR family of transcriptional regulators, is a repressor of the vceCAB operon, which encodes a multidrug efflux pump in Vibrio cholerae. VceR binds to a 28 bp inverted-repeat within the vceR-vceC intergenic region and is dissociated from this site with CCCP, a pump substrate. The rate of the CCCP-induced conformational change in VceR was determined by stopped-flow fluorescence spectroscopy, revealing a highly co-operative process that occurs with a Hill coefficient of approximately 4. The apparent affinity for CCCP decreased in a linear manner with increasing concentrations of DNA, indicative of competition between the CCCP and DNA for binding to VceR. These data are consistent with an equilibrium between mutually exclusive conformations that are supported by the binding of DNA and CCCP to the N and C termini of VceR, respectively. Size-exclusion chromatography and dynamic light-scattering studies indicate that VceR exists predominantly as a dimer; however, a pair of dimers binds to the DNA. In order to account for the fact that VceR is a dimer in the absence of DNA but binds CCCP with a Hill co-efficient of 4, implying that it has at least four binding-sites, we propose that the VceR monomer possesses a pair of binding sites that can be simultaneously occupied by CCCP. Using a gene-reporter system and stopped-flow spectroscopy, we established that the equilibrium between free VceR and VceR-CCCP plays a critical role in controlling expression of the pump. The co-operative transition between these states allows the repressor to respond to relatively small changes in drug concentration. Thus, repression and induction can be readily switched about a critical drug concentration which will prove toxic to the cell.

The crystal structure of the outer membrane protein VceC from the bacterial pathogen Vibrio cholerae at 1.8 A resolution.

Multidrug resistance in Gram-negative bacteria arises in part from the activities of tripartite drug efflux pumps. In the pathogen Vibrio cholerae, one such pump comprises the inner membrane proton antiporter VceB, the periplasmic adaptor VceA, and the outer membrane channel VceC. Here, we report the crystal structure of VceC at 1.8 A resolution. The trimeric VceC is organized in the crystal lattice within laminar arrays that resemble membranes. A well resolved detergent molecule within this array interacts with the transmembrane beta-barrel domain in a fashion that may mimic protein-lipopolysaccharide contacts. Our analyses of the external surfaces of VceC and other channel proteins suggest that different classes of efflux pumps have distinct architectures. We discuss the implications of these findings for mechanisms of drug and protein export.

Ectodomain shedding of human Nogo-66 receptor homologue-1 by zinc metalloproteinases.

The Nogo-66 receptor (NgR) plays a pivotal role in the inhibition of neuroregeneration as the receptor for multiple neurite outgrowth inhibitors such as Nogo-A. We have previously shown that NgR undergoes zinc metalloproteinase-mediated ectodomain shedding in neuroblastoma cells. Here, we demonstrate that the NgR-related protein NgR homologue-1 is released from neuroblastoma cells as a full-length ectodomain (NgRH1-ecto) and an N-terminal fragment (NTF-NgRH1) containing the leucine-rich repeat region of the protein. Inhibitors of the major protease classes failed to block the release of NgRH1-ecto, suggesting that this occurs via a protease-independent mechanism, presumably by a phospholipase-like enzyme. The release of NTF-NgRH1 was blocked by a hydroxamate-based zinc metalloproteinase inhibitor and tissue inhibitor of metalloproteinases-2 and -3, but not -1, implicating the involvement of membrane-type matrix metalloproteinases in this process. Our findings thus highlight the parallels between the ectodomain shedding of NgRH1 and that previously described for NgR.

Versican V2 and the central inhibitory domain of Nogo-A inhibit neurite growth via p75NTR/NgR-independent pathways that converge at RhoA.

Myelin is a major obstacle for regenerating nerve fibers of the adult mammalian central nervous system (CNS). Several proteins including Nogo-A, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp) and the chondroitin-sulfate proteoglycan (CSPG) Versican V2 have been identified as inhibitory components present in CNS myelin. MAG, OMgp as well as the Nogo specific domain Nogo-66 exert their inhibitory activity by binding to a neuronal receptor complex containing the Nogo-66 receptor NgR and the neurotrophin receptor p75(NTR). While this suggests a converging role of the p75(NTR)/NgR receptor complex for myelin-derived neurite growth inhibitors, we show here that NgR/p75(NTR) is not required for mediating the inhibitory activity of the two myelin components NiG, unlike Nogo-66 a distinct domain of Nogo-A, and Versican V2. Primary neurons derived from a complete null mutant of p75(NTR) are still sensitive to NiG and Versican V2. In line with this result, neurite growth of p75(NTR) deficient neurons is still significantly blocked on total bovine CNS myelin. Furthermore, modulation of RhoA and Rac1 in p75(NTR)-/- neurons persists with NiG and Versican V2. Finally, we demonstrate that neither NiG nor Versican V2 interact with the p75(NTR)/NgR receptor complex and provide evidence that the binding sites of NiG and Nogo-66 are physically distinct from each other on neural tissue. These results indicate not only the existence of neuronal receptors for myelin inhibitors independent from the p75(NTR)/NgR receptor complex but also establish Rho GTPases as a common point of signal convergence of diverse myelin-induced regeneration inhibitory pathways.

Structural understanding of efflux-mediated drug resistance: potential routes to efflux inhibition.

The active efflux of cytotoxic drugs mediated by multidrug transporters is the basis of multidrug resistance in prokaryotic and eukaryotic cells. Individual multidrug transporters can be extremely versatile, often exhibiting a staggering range of substrate specificity that can negate the effects of clinically relevant therapies. The effective treatment of bacterial, fungal and protozoan infections, along with certain cancer treatments, has been compromised by the presence of multidrug transporters. Traditionally, advances in the understanding of multidrug transporters have been made through biochemical analyses; more recently, however, fundamental advances have been made with the elucidation of several three dimensional structures of representative multidrug pumps. Biochemical and structural analysis of multidrug pumps could lead to the development of novel 'anti-efflux' therapies.

Zinc metalloproteinase-mediated cleavage of the human Nogo-66 receptor.

The central nervous system myelin components oligodendrocyte-myelin glycoprotein, myelin-associated glycoprotein and the Nogo-66 domain of Nogo-A inhibit neurite outgrowth by binding the neuronal glycosyl-phosphatidylinositol-anchored Nogo-66 receptor (NgR) that transduces the inhibitory signal to the cell interior via a transmembrane co-receptor, p75NTR. Here, we demonstrate that human NgR expressed in human neuroblastoma cells is constitutively cleaved in a post-ER compartment to generate a lipid-raft associated C-terminal fragment that is present on the cell surface and a soluble N-terminal fragment that is released into the medium. Mass spectrometric analysis demonstrated that the N-terminal fragment terminated just after the C-terminus of the ligand-binding domain of NgR. In common with other shedding mechanisms, the release of this fragment was blocked by a hydroxamate-based inhibitor of zinc metalloproteinases, but not by inhibitors of other protease classes and up-regulated by treatment with the cellular cholesterol depleting agent methyl-beta-cyclodextrin. The N-terminal fragment bound Nogo-66 and blocked Nogo-66 binding to cell surface NgR but failed to associate with p75NTR, indicative of a role as a Nogo-66 antagonist. Furthermore, the N- and C-terminal fragments of NgR were detectable in human brain cortex and the N-terminal fragment was also present in human cerebrospinal fluid, demonstrating that NgR proteolysis occurs within the human nervous system. Our findings thus identify a potential cellular mechanism for the regulation of NgR function at the level of the receptor.