Probing the solution structure of the E. coli multidrug transporter MdfA using DEER distance measurements with nitroxide and Gd(III) spin labels(2019) Scientific Reports. 9, 12528. Abstract
Methodological and technological advances in EPR spectroscopy have enabled novel insight into the structural and dynamic aspects of integral membrane proteins. In addition to an extensive toolkit of EPR methods, multiple spin labels have been developed and utilized, among them Gd(III)-chelates which offer high sensitivity at high magnetic fields. Here, we applied a dual labeling approach, employing nitroxide and Gd(III) spin labels, in conjunction with Q-band and W-band double electron-electron resonance (DEER) measurements to characterize the solution structure of the detergent-solubilized multidrug transporter MdfA from E. coli. Our results identify highly flexible regions of MdfA, which may play an important role in its functional dynamics. Comparison of distance distribution of spin label pairs on the periplasm with those calculated using inward- and outward-facing crystal structures of MdfA, show that in detergent micelles, the protein adopts a predominantly outward-facing conformation, although more closed than the crystal structure. The cytoplasmic pairs suggest a small preference to the outward-facing crystal structure, with a somewhat more open conformation than the crystal structure. Parallel DEER measurements with the two types of labels led to similar distance distributions, demonstrating the feasibility of using W-band spectroscopy with a Gd(III) label for investigation of the structural dynamics of membrane proteins.[All authors]
The fascinating but mysterious mechanistic aspects of multidrug transport by MdfA from Escherichia coli(2018) Research in Microbiology. 169, 7-8, p. 455-460 Abstract
MdfA is an interesting member of a large group of secondary multidrug (Mdr) transporters. Through genetic, biochemical and biophysical studies of MdfA, many challenging aspects of the multidrug transport phenomenon have been addressed. This includes its ability to interact with chemically unrelated drugs and how it utilizes energy to drive efflux of compounds that are not only structurally, but also electrically, different. Admittedly, however, despite all efforts and a recent pioneering structural contribution, several important mechanistic issues of the promiscuous capabilities of MdfA still seek better molecular and dynamic understanding.
Co-translational folding intermediate dictates membrane targeting of the signal recognition particle-receptor(2018) Journal of Molecular Biology. 430, 11, p. 1607-1620 Abstract
Much of our knowledge on the function of proteins is deduced from their mature, folded states. However, it is unknown whether partially synthesized nascent protein segments can execute biological functions during translation and whether their premature folding states matter. A recent observation that a nascent chain performs a distinct function, co-translational targeting in vivo, has been made with the Escherichia coli signal recognition particle receptor FtsY, a major player in the conserved pathway of membrane protein biogenesis. FtsY functions as a membrane-associated entity, but very little is known about the mode of its targeting to the membrane. Here we investigated the underlying structural mechanism of the co-translational FtsY targeting to the membrane. Our results show that helices N 2–4, which mediate membrane targeting, form a stable folding intermediate co-translationally that greatly differs from its fold in the mature FtsY. These results thus resolve a long-standing mystery of how the receptor targets the membrane even when deleted of its alleged membrane targeting sequence. The structurally distinct targeting determinant of FtsY exists only co-translationally. Our studies will facilitate further efforts to seek cellular factors required for proper targeting and association of FtsY with the membrane. Moreover, the results offer a hallmark example for how co-translational nascent intermediates may dictate biological functions.
(2018) Journal of Molecular Biology. 430, 9, p. 1368-1385 Abstract
Secondary multidrug (Mdr) transporters utilize ion concentration gradients to actively remove antibiotics and other toxic compounds from cells. The model Mdr transporter MdfA from Escherichia coli exchanges dissimilar drugs for protons. The transporter should open at the cytoplasmic side to enable access of drugs into the Mdr recognition pocket. Here we show that the cytoplasmic rim around the Mdr recognition pocket represents a previously overlooked important regulatory determinant in MdfA. We demonstrate that increasing the positive charge of the electrically asymmetric rim dramatically inhibits MdfA activity and sometimes even leads to influx of planar, positively charged compounds, resulting in drug sensitivity. Our results suggest that unlike the mutants with the electrically modified rim, the membrane-embedded wild-type MdfA exhibits a significant probability of an inward-closed conformation, which is further increased by drug binding. Since MdfA binds drugs from its inward-facing environment, these results are intriguing and raise the possibility that the transporter has a sensitive, drug-induced conformational switch, which favors an inward-closed state.[All authors]
Evidence for a cytoplasmic pool of ribosome-free mRNAs encoding inner membrane proteins in Escherichia coli(2017) PLoS ONE. 12, 8, e0183862. Abstract
Translation-independent mRNA localization represents an emerging concept in cell biology. In Escherichia coli, mRNAs encoding integral membrane proteins (MPRs) are targeted to the membrane where they are translated by membrane associated ribosomes and the produced proteins are inserted into the membrane co-translationally. In order to better understand aspects of the biogenesis and localization of MPRs, we investigated their subcellular distribution using cell fractionation, RNA-seq and qPCR. The results show that MPRs are overrepresented in the membrane fraction, as expected, and depletion of the signal recognition particle-receptor, FtsY reduced the amounts of all mRNAs on the membrane. Surprisingly, however, MPRs were also found relatively abundant in the soluble ribosome-free fraction and their amount in this fraction is increased upon overexpression of CspE, which was recently shown to interact with MPRs. CspE also conferred a positive effect on the membrane-expression of integral membrane proteins. We discuss the possibility that the effects of CspE overexpression may link the intriguing subcellular localization of MPRs to the cytosolic ribosome-free fraction with their translation into membrane proteins and that the ribosome-free pool of MPRs may represent a stage during their targeting to the membrane, which precedes translation.
Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane.(2016) eLife. 5, Abstract
Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers.
Model Uracil-Rich RNAs and Membrane Protein mRNAs Interact Specifically with Cold Shock Proteins in Escherichia coli(2015) PLoS ONE. 10, 7, e0134413. Abstract
Are integral membrane protein-encoding mRNAs (MPRs) different from other mRNAs such as those encoding cytosolic mRNAs (CPRs)? This is implied from the emerging concept that MPRs are specifically recognized and delivered to membrane-bound ribosomes in a translation-independent manner. MPRs might be recognized through uracil-rich segments that encode hydrophobic transmembrane helices. To investigate this hypothesis, we designed DNA sequences encoding model untranslatable transcripts that mimic MPRs or CPRs. By utilizing in vitro-synthesized biotinylated RNAs mixed with Escherichia coli extracts, we identified a highly specific interaction that takes place between transcripts that mimic MPRs and the cold shock proteins CspE and CspC, which are normally expressed under physiological conditions. Co-purification studies with E. coli expressing 6His-tagged CspE or CspC confirmed that the specific interaction occurs in vivo not only with the model uracil-rich untranslatable transcripts but also with endogenous MPRs. Our results suggest that the evolutionarily conserved cold shock proteins may have a role, possibly as promiscuous chaperons, in the biogenesis of MPRs.
(2015) Journal of Cell Science. 128, p. 1444-1452 Abstract
The signal recognition particle (SRP) receptor is a major player in the pathway of membrane protein biogenesis in all organisms. The receptor functions as a membrane-bound entity but very little is known about its targeting to the membrane. Here, we demonstrate in vivo that the Escherichia coli SRP receptor targets the membrane co-translationally. This requires emergence from the ribosome of the four-helix-long N-domain of the receptor, of which only helices 2-4 are required for co-translational membrane attachment. The results also suggest that the targeting might be regulated co-translationally. Taken together, our in vivo studies shed light on the biogenesis of the SRP receptor and its hypothetical role in targeting ribosomes to the E. coli membrane.
(2014) Microbial Cell. 1, 10, p. 349-351 Abstract
Multidrug transporters are membrane proteins that catalyze efflux of antibiotics and other toxic compounds from cells, thereby conferring drug resistance on various organisms. Unlike most solute transporters that transport a single type of compound or similar analogues, multidrug transporters are extremely promiscuous. They transport a broad spectrum of dissimilar drugs and represent a serious obstacle to antimicrobial or anticancer chemotherapy. Many challenging aspects of multidrug transporters, which are unique, have been studied in detail, including their ability to interact with chemically unrelated drugs, and how they utilize energy to drive efflux of compounds that are not only structurally but electrically different. A new and surprising dimension of the promiscuous nature of multidrug transporters has been described recently: they can move long molecules through the membrane in a processive manner.
(2014) Nature Communications. 5, 4615. Abstract
Secondary multidrug transporters use ion concentration gradients to energize the removal from cells of various antibiotics. The Escherichia coli multidrug transporter MdfA exchanges a single proton with a single monovalent cationic drug molecule. This stoichiometry renders the efflux of divalent cationic drugs energetically unfavourable, as it requires exchange with at least two protons. Here we show that surprisingly, MdfA catalyses efflux of divalent cations, provided that they have a unique architecture: the two charged moieties must be separated by a long linker. These drugs are exchanged for two protons despite the apparent inability of MdfA to exchange two protons for a single drug molecule. Our results suggest that these drugs are transported in two consecutive transport cycles, where each cationic moiety is transported as if it were a separate substrate. We propose that secondary transport can adopt a processive-like mode of action, thus expanding the substrate spectrum of multidrug transporters.
(2014) eLife. 3, 03440. Abstract
In all living organisms, ribosomes translating membrane proteins are targeted to membrane translocons early in translation, by the ubiquitous Signal Recognition Particle (SRP) system. In eukaryotes, the SRP Alu domain arrests translation elongation of membrane proteins until targeting is complete. Curiously however, the Alu domain is lacking in most eubacteria. Here, by analyzing genome-wide data on translation rates, we identified a potential compensatory mechanism in E. coli that serves to slow down translation during membrane protein targeting. The underlying mechanism is likely programmed into the coding sequence, where Shine-Dalgarno-like elements trigger elongation pauses at strategic positions during early stages of translation. We provide experimental evidence that slow translation during targeting improves membrane protein production fidelity, as it correlates with better folding of overexpressed membrane proteins. Thus, slow elongation is important for membrane protein targeting in E. coli, which utilizes mechanisms different from the eukaryotic one to control translation speed.
Translation- and SRP-independent mRNA targeting to the endoplasmic reticulum in the yeast Saccharomyces cerevisiae(2013) Molecular Biology of the Cell. 24, 19, p. 3069-3084 Abstract
mRNAs encoding secreted/membrane proteins (mSMPs) are believed to reach the endoplasmic reticulum (ER) in a translation-dependent manner to confer protein translocation. Evidence exists, however, for translation-and signal recognition particle (SRP)-independent mRNA localization to the ER, suggesting that there are alternate paths for RNA delivery. We localized endogenously expressed mSMPs in yeast using an aptamer-based RNA-tagging procedure and fluorescence microscopy. Unlike mRNAs encoding polarity and secretion factors that colocalize with cortical ER at the bud tip, mSMPs and mRNAs encoding soluble, nonsecreted, nonpolarized proteins localized mainly to ER peripheral to the nucleus (nER). Synthetic nontranslatable uracil-rich mRNAs were also demonstrated to colocalize with nER in yeast. This mRNA-ER association was verified by subcellular fractionation and reverse transcription-PCR, single-molecule fluorescence in situ hybridization, and was not inhibited upon SRP inactivation. To better understand mSMP targeting, we examined aptamer-tagged USE1, which encodes a tail-anchored membrane protein, and SUC2, which encodes a soluble secreted enzyme. USE1 and SUC2 mRNA targeting was not abolished by the inhibition of translation or removal of elements involved in translational control. Overall we show that mSMP targeting to the ER is both translation-and SRP-independent, and regulated by cis elements contained within the message and trans-acting RNA-binding proteins (e. g., She2, Puf2).
(2012) Molecular Cell. 47, 5, p. 777-787 Abstract
Multidrug transporters are ubiquitous efflux pumps that provide cells with defense against various toxic compounds. In bacteria, which typically harbor numerous multidrug transporter genes, the majority function as secondary multidrug/proton antiporters. Proton-coupled secondary transport is a fundamental process that is not fully understood, largely owing to the obscure nature of proton-transporter interactions. Here we analyzed the substrate/proton coupling mechanism in MdfA, a model multidrug/proton antiporter. By measuring the effect of protons on substrate binding and by directly measuring proton binding and release, we show that substrates and protons compete for binding to MdfA. Our studies strongly suggest that competition is an integral feature of secondary multidrug transport. We identified the proton-binding acidic residue and show that, surprisingly, the substrate binds at a different site. Together, the results suggest an interesting mode of indirect competition as a mechanism of multidrug/proton antiport.
(2012) Proceedings of the National Academy of Sciences of the United States of America. 109, 31, p. 12473-12478 Abstract
Multidrug transporters are integral membrane proteins that use cellular energy to actively extrude antibiotics and other toxic compounds from cells. The multidrug/proton antiporter MdfA from Escherichia coli exchanges monovalent cationic substrates for protons with a stoichiometry of 1, meaning that it translocates only one proton per antiport cycle. This may explain why transport of divalent cationic drugs by MdfA is energetically unfavorable. Remarkably, however, we show that MdfA can be easily converted into a divalent cationic drug/>= 2 proton-antiporter, either by random mutagenesis or by rational design. The results suggest that exchange of divalent cationic drugs with two (or more) protons requires an additional acidic residue in the multidrug recognition pocket of MdfA. This outcome further illustrates the exceptional promiscuous capabilities of multidrug transporters.
(2012) Trends in Biochemical Sciences. 37, 1, p. 1-6 Abstract
Integral membrane proteins (IMPs) are usually synthesized by membrane-bound ribosomes, and this process requires proper localization of ribosomes and IMP-encoding transcripts. However, the underlying molecular mechanism of the pathway has not yet been fully established in vivo. The prevailing hypothesis is that ribosomes and transcripts are delivered to the membrane together during IMP translation by the signal recognition particle (SRP) and its receptor. Here, I discuss an alternative hypothesis that posits that ribosomes and transcripts are targeted separately. Ribosome targeting to the membrane might be mediated by the SRP receptor, rather than by SRP, and IMP-encoding transcripts might be targeted to the membrane in a translation-independent manner. According to this scenario, the SRP executes its essential function on the membrane at a later stage of the targeting pathway.
(2011) Biochimica Et Biophysica Acta-Biomembranes. 1808, 3, p. 841-850 Abstract
All living cells have co-translational pathways for targeting membrane proteins. Co-translation pathways for secretory proteins also exist but mostly in eukaryotes. Unlike secretory proteins, the biosynthetic pathway of most membrane proteins is conserved through evolution and these proteins are usually synthesized by membrane-bound ribosomes. Translation on the membrane requires that both the ribosomes and the mRNAs be properly localized. Theoretically, this can be achieved by several means. (i) The current view is that the targeting of cytosolic mRNA-ribosome-nascent chain complexes (RNCs) to the membrane is initiated by information in the emerging hydrophobic nascent polypeptides. ( ii) The alternative model suggests that ribosomes may be targeted to the membrane also constitutively, whereas the appropriate mRNAs may be carried on small ribosomal subunits or targeted by other cellular factors to the membrane-bound ribosomes. Importantly, the available experimental data do not rule out the possibility that cells may also utilize both pathways in parallel. In any case, it is well documented that a major player in the targeting pathway is the signal recognition particle (SRP) system composed of the SRP and its receptor (SR). Although the functional core of the SRP system is evolutionarily conserved, its composition and biological practice come with different flavors in various organisms. This review is dedicated mainly to the Escherichia (E.) coli SRP, where the biochemical and structural properties of components of the SRP system have been relatively characterized, yielding essential information about various aspects of the pathway. In addition, several cellular interactions of the SRP and its receptor have been described in E. coli, providing insights into their spatial function. Collectively, these in vitro studies have led to the current view of the targeting pathway [see (i) above]. Interestingly, however, in vivo studies of the role of the SRP and its recep
Genetic Evidence for Functional Interaction of the Escherichia coli Signal Recognition Particle Receptor with Acidic Lipids in Vivo(2010) Journal of Biological Chemistry. 285, 52, p. 40508-40514 Abstract
The mechanism underlying the interaction of the Escherichia coli signal recognition particle receptor FtsY with the cytoplasmic membrane has been studied in detail. Recently, we proposed that FtsY requires functional interaction with inner membrane lipids at a late stage of the signal recognition particle pathway. In addition, an essential lipid-binding alpha-helix was identified in FtsY of various origins. Theoretical considerations and in vitro studies have suggested that it interacts with acidic lipids, but this notion is not yet fully supported by in vivo experimental evidence. Here, we present an unbiased genetic clue, obtained by serendipity, supporting the involvement of acidic lipids. Utilizing a dominant negative mutant of FtsY (termed NG), which is defective in its functional interaction with lipids, we screened for E. coli genes that suppress the negative dominant phenotype. In addition to several unrelated phenotype-suppressor genes, we identified pgsA, which encodes the enzyme phosphatidylglycerophosphate synthase (PgsA). PgsA is an integral membrane protein that catalyzes the committed step to acidic phospholipid synthesis, and we show that its overexpression increases the contents of cardiolipin and phosphatidylglycerol. Remarkably, expression of PgsA also stabilizes NG and restores its biological function. Collectively, our results strongly support the notion that FtsY functionally interacts with acidic lipids.
Escherichia coli SRP, Its Protein Subunit Ffh, and the Ffh M Domain Are Able To Selectively Limit Membrane Protein Expression When Overexpressed(2010) mBio. 1, 2, Abstract
The Escherichia coli signal recognition particle (SRP) system plays an important role in membrane protein biogenesis. Previous studies have suggested indirectly that in addition to its role during the targeting of ribosomes translating membrane proteins to translocons, the SRP might also have a quality control role in preventing premature synthesis of membrane proteins in the cytoplasm. This proposal was studied here using cells simultaneously overexpressing various membrane proteins and either SRP, the SRP protein Ffh, its 4.5S RNA, or the FfhMdomain. The results show that SRP, Ffh, and the M domain are all able to selectively inhibit the expression of membrane proteins. We observed no apparent changes in the steady-state mRNA levels or membrane protein stability, suggesting that inhibition may occur at the level of translation, possibly through the interaction between Ffh and ribosome-hydrophobic nascent chain complexes. Since E. coli SRP does not have a eukaryote-like translation arrest domain, we discuss other possible mechanisms by which this SRP might regulate membrane protein translation when overexpressed. IMPORTANCE The eukaryotic SRP slows down translation of SRP substrates by cytoplasmic ribosomes. This activity is important for preventing premature synthesis of secretory and membrane proteins in the cytoplasm. It is likely that an analogous quality control step would be required in all living cells. However, on the basis of its composition and domain structure and limited in vitro studies, it is believed that the E. coli SRP is unable to regulate ribosomes translating membrane proteins. Nevertheless, several in vivo studies have suggested otherwise. To address this issue further in vivo, we utilized unbalanced conditions under which E. coli simultaneously overexpresses SRP and each of several membrane or cytosolic proteins. Surprisingly, our results clearly show that the E. coli SRP is capable of regulating membrane protein synthesis and demonstrate t
(2010) Science and Engineering Ethics. 16, 1, p. 85-97 Abstract
Israel has a long history of concern with chemical and biological threats, since several hostile states in the Middle East are likely to possess such weapons. The Twin-Tower terrorist attacks and Anthrax envelope scares of 2001 were a watershed for public perceptions of the threat of unconventional terror in general and of biological terror in particular. New advances in biotechnology will only increase the ability of terrorists to exploit the burgeoning availability of related information to develop ever-more destructive bioweapons. Many areas of modern biological research are unavoidably dual-use by nature. They thus have a great potential for both help and harm; and facilitating the former while preventing the latter remains a serious challenge to researchers and governments alike. This article addresses how Israel might best ( 1) prevent hostile elements from obtaining, from Israel's biological research system, materials, information and technologies that might facilitate their carrying out a biological attack, while ( 2) continuing to promote academic openness, excellence and other hallmarks of that system. This important and sensitive issue was assessed by a special national committee, and their recommendations are presented and discussed. One particularly innovative element is the restructuring and use of Israel's extensive biosafety system to also address biosecurity goals, with minimal disruption or delay.
(2010) PLoS ONE. 5, 2, e9130. Abstract
Background: The Escherichia coli version of the mammalian signal recognition particle (SRP) system is required for biogenesis of membrane proteins and contains two essential proteins: the SRP subunit Ffh and the SRP-receptor FtsY. Scattered in vivo studies have raised the possibility that expression of membrane proteins is inhibited in cells depleted of FtsY, whereas Ffh-depletion only affects their assembly. These differential results are surprising in light of the proposed model that FtsY and Ffh play a role in the same pathway of ribosome targeting to the membrane. Therefore, we decided to evaluate these unexpected results systematically. Methodology/Principal Findings: We characterized the following aspects of membrane protein biogenesis under conditions of either FtsY- or Ffh-depletion: (i) Protein expression, stability and localization; (ii) mRNA levels; (iii) folding and activity. With FtsY, we show that it is specifically required for expression of membrane proteins. Since no changes in mRNA levels or membrane protein stability were detected in cells depleted of FtsY, we propose that its depletion may lead to specific inhibition of translation of membrane proteins. Surprisingly, although FtsY and Ffh function in the same pathway, depletion of Ffh did not affect membrane protein expression or localization. Conclusions: Our results suggest that indeed, while FtsY-depletion affects earlier steps in the pathway (possibly translation), Ffh-depletion disrupts membrane protein biogenesis later during the targeting pathway by preventing their functional assembly in the membrane.
(2009) Biochemistry. 48, 51, p. 12314-12322 Abstract
All intramembrane proteases are known to cleave membrane proteins with a single transmembrane helix. Such cleavages often release anchored soluble domains, which play a role in physiologically important inter- and intracellular processes. However, in many cases the physiological roles/substrates of intramembrane proteases are not known. It is interesting that no multispanning substrates were identified so far, despite the Fact that intramembrane proteases have promiscuous substrate recognition and cleavage capabilities. Here we determined whether, in a synthetic experimental system, intramembrane proteases have the capability to interact with and cleave multispanning membrane proteins. We utilized the Escherichia coli rhomboid GlpG, an intramembrane serine protease, and truncated versions of the E. coli multidrug transporter MdfA as model multispanning membrane proteins. On the basis of in vivo and in vitro studies on the association of GlpG with various MdfA constructs and their cleavage, we conclude that GlpG is able to recognize and cleave truncated forms of MdfA but not the intact protein. We propose that GlpG has the capacity to act on unfolded multispanning membrane proteins, thus providing an Incentive for investigating possible physiological consequences.
(2009) Journal of Biological Chemistry. 284, 47, p. 32296-32304 Abstract
Multidrug (Mdr) transporters are membrane proteins that actively export structurally dissimilar drugs from the cell, thereby rendering the cell resistant to toxic compounds. Similar to substrate-specific transporters, Mdr transporters also undergo substrate-induced conformational changes. However, the mechanism by which a variety of dissimilar substrates are able to induce similar transport-compatible conformational responses in a single transporter remains unclear. To address this major aspect of Mdr transport, we studied the conformational behavior of the Escherichia coli Mdr transporter MdfA. Our results show that indeed, different substrates induce similar conformational changes in the transporter. Intriguingly, in addition, we observed that compounds other than substrates are able to confer similar conformational changes when covalently attached at the putative Mdr recognition pocket of MdfA. Taken together, the results suggest that the Mdr-binding pocket of MdfA is conformationally sensitive. We speculate that the same conformational switch that usually drives active transport is triggered promiscuously by merely occupying the Mdr-binding site.
(2009) Biochimica Et Biophysica Acta-Proteins And Proteomics. 1794, 5, p. 738-747 Abstract
Multidrug transporters are membrane proteins that expel a wide spectrum of cytotoxic compounds from the cell. Through this function, they render cells resistant to multiple drugs. These transporters are found in many different families of transport proteins, of which the largest is the major facilitator superfamily. Multidrug transporters from this family are highly represented in bacteria and studies of them have provided important insight into the mechanism underlying multidrug transport. This review summarizes the work carried out on these interesting proteins and underscores the differences and similarities to other transport systems. (C) 2008 Elsevier B.V. All rights reserved.
(2009) Nature. 459, 7245, p. 371-378 Abstract
Intramembrane proteolysis is increasingly seen as a regulatory step in a range of diverse processes, including development, organelle shaping, metabolism, pathogenicity and degenerative disease. Initial scepticism over the existence of intramembrane proteases was soon replaced by intense exploration of their catalytic mechanisms, substrate specificities, regulation and structures. Crystal structures of metal-dependent and serine intramembrane proteases have revealed active sites embedded in the plane of the membrane but accessible by water, a requirement for hydrolytic reactions. Efforts to understand how these membrane-bound proteases carry out their reactions have started to yield results.
Studying membrane proteins through the eyes of the genetic code revealed a strong uracil bias in their coding mRNAs(2009) Proceedings of the National Academy of Sciences of the United States of America. 106, 16, p. 6662-6666 Abstract
Posttranscriptional processes often involve specific signals in mRNAs. Because mRNAs of integral membrane proteins across evolution are usually translated at distinct locations, we searched for universally conserved specific features in this group of mRNAs. Our analysis revealed that codons of very hydrophobic amino acids, highly represented in integral membrane proteins, are composed of 50% uracils (U). As expected from such a strong U bias, the calculated U profiles of mRNAs closely resemble the hydrophobicity profiles of their encoded proteins and may designate genes encoding integral membrane proteins, even in the absence of information on ORFs. We also show that, unexpectedly, the U-richness phenomenon is not merely a consequence of the codon composition of very hydrophobic amino acids, because counterintuitively, the relatively hydrophilic serine and tyrosine, also encoded by U-rich codons, are overrepresented in integral membrane proteins. Interestingly, although the U-richness phenomenon is conserved, there is an evolutionary trend that minimizes usage of U-rich codons. Taken together, the results suggest that U-richness is an evolutionarily ancient feature of mRNAs encoding integral membrane proteins, which might serve as a physiologically relevant distinctive signature to this group of mRNAs.
The Secondary Multidrug/Proton Antiporter MdfA Tolerates Displacements of an Essential Negatively Charged Side Chain(2009) Journal of Biological Chemistry. 284, 11, p. 6966-6971 Abstract
The largest family of solute transporters includes ion motive force-driven secondary transporters. Several well characterized solute-specific transport systems in this group have at least one irreplaceable acidic residue that plays a critical role in energy coupling during transport. Previous studies have established the importance of acidic residues in substrate recognition by major facilitator superfamily secondary multidrug transporters, but their role in the transport mechanism remained unknown. We have been investigating the involvement of acidic residues in the mechanism of MdfA, an Escherichia coli secondary multidrug/proton antiporter. We demonstrated that no single negatively charged side chain plays an irreplaceable role in MdfA. Accordingly, we hypothesized that MdfA might be able to utilize at least two acidic residues alternatively. In this study, we present evidence that indeed, unlike solute-specific secondary transporters, MdfA tolerates displacements of an essential negative charge to various locations in the putative drug translocation pathway. The results suggest that MdfA utilizes a proton translocation strategy that is less sensitive to perturbations in the geometry of the proton-binding site, further illustrating the exceptional structural promiscuity of multidrug transporters.
Escherichia coli signal recognition particle receptor FtsY contains an essential and autonomous membrane-binding amphipathic helix(2007) Journal of Biological Chemistry. 282, 44, p. 32176-32184 Abstract
Escherichia coli membrane protein biogenesis is mediated by a signal recognition particle and its membrane-associated receptor ( FtsY). Although crucial for its function, it is still not clear how FtsY interacts with the membrane. Analysis of the structure/function differences between severely truncated active ( NG + 1) and inactive ( NG) mutants of FtsY enabled us to identify an essential membrane-interacting determinant. Comparison of the three-dimensional structures of the mutants, combined with site-directed mutagenesis, modeling, and liposome-binding assays, revealed that FtsY contains a conserved autonomous lipid-binding amphipathic alpha-helix at the N-terminal end of the N domain. Deletion experiments showed that this helix is essential for FtsY function in vivo, thus offering, for the first time, clear evidence for the functionally important, physiologically relevant interaction of FtsY with lipids.
(2007) Journal of Biological Chemistry. 282, 44, p. 32168-32175 Abstract
The mechanism underlying the interaction of the Escherichia coli signal recognition particle ( SRP) receptor FtsY with the cytoplasmic membrane is not fully understood. We investigated this issue by utilizing active ( NG + 1) and inactive ( NG) mutants of FtsY. In solution, the mutants comparably bind and hydrolyze nucleotides and associate with SRP. In contrast, a major difference was observed in the cellular distribution of NG and NG + 1. Unlike NG + 1, which distributes almost as the wild-type receptor, the inactive NG mutant accumulates on the membrane, together with ribosomes and SRP. The results suggest that NG function is compromised only at a later stage of the targeting pathway and that despite their identical behavior in solution, the membrane-bound NG-SRP complex is less active than NG + 1-SRP. This notion is strongly supported by the observation that lipids stimulate the GTPase activity of NG + 1-SRP, whereas no stimulation is observed with NG-SRP. In conclusion, we propose that the SRP receptor has two distinct and separable roles in ( i) mediating membrane targeting and docking of ribosomes and ( ii) promoting their productive release from the docking site.
(2007) Biochemistry. 46, 17, p. 5200-5208 Abstract
MdfA is a 410-residue-long secondary multidrug transporter from E. coli. Cells expressing MdfA from a multicopy plasmid exhibit resistance against a diverse group of toxic compounds, including neutral and cationic ones, because of active multidrug export. As a prerequisite for high-resolution structural studies and a better understanding of the mechanism of substrate recognition and translocation by MdfA, we investigated its biochemical properties and overall structural characteristics. To this end, we purified the beta-dodecyl maltopyranoside (DDM)-solubilized protein using a 6-His tag and metal affinity chromatography, and size exclusion chromatography (SE-HPLC). Purified MdfA was analyzed for its DDM and phospholipid (PL) content, and tetraphenylphosphonium (TPP+)-binding activity. The results are consistent with MdfA being an active monomer in DDM solution. Furthermore, an investigation of two-dimensional crystals by electron crystallography and 3D reconstruction lent support to the notion that MdfA may also be monomeric in reconstituted proteoliposomes.
(2007) EMBO Journal. 26, 5, p. 1211-1220 Abstract
Intracellular trafficking of the precursor of Spitz (Spi), the major Drosophila EGF receptor ( EGFR) ligand, is facilitated by the chaperone Star, a type II transmembrane protein. This study identifies a novel mechanism for modulating the activity of Star, thereby influencing the levels of active Spi ligand produced. We demonstrate that Star can efficiently traffic Spi even when present at sub-stoichiometric levels, and that in Drosophila S2R+ cells, Spi is trafficked from the endoplasmic reticulum to the late endosome compartment, also enriched for Rhomboid, an intramembrane protease. Rhomboid, which cleaves the Spi precursor, is now shown to also cleave Star within its transmembrane domain both in cell culture and in flies, expanding the repertoire of known Rhomboid substrates to include both type I and type II transmembrane proteins. Cleavage of Star restricts the amount of Spi that is trafficked, and may explain the exceptional dosage sensitivity of the Star locus in flies.
(2007) Proceedings of the National Academy of Sciences of the United States of America. 104, 2, p. 462-466 Abstract
Intramembrane proteases catalyze peptide bond cleavage of integral membrane protein substrates. This activity is crucial for many biological and pathological processes. Rhomboids are evolutionarily widespread intramembrane serine proteases. Here, we present the 2.3-angstrom-resolution crystal structure of a rhomboid from Escherichia coli. The enzyme has six transmembrane helices, five of which surround a short TM4, which starts deep within the membrane at the catalytic serine residue. Thus, the catalytic serine is in an externally exposed cavity, which provides a hydrophilic environment for proteolysis. Our results reveal a mechanism to enable water-dependent catalysis at the depth of the hydrophobic milieu of the membrane and suggest how substrates gain access to the sequestered rhomboid active site.
No single irreplaceable acidic residues in the Escherichia coli secondary multidrug transporter MdfA(2006) Journal of Bacteriology. 188, 15, p. 5635-5639 Abstract
The largest family of solute transporters (major facilitator superfamily [MFS]) includes proton-motive-force-driven secondary transporters. Several characterized MFS transporters utilize essential acidic residues that play a critical role in the energy-coupling mechanism during transport. Surprisingly, we show here that no single acidic residue plays an irreplaceable role in the Escherichia coli secondary multidrug transporter MdfA.
(2006) Molecular Microbiology. 61, 2, p. 277-284 Abstract
Multidrug (Mdr) transport is an obstacle to the successful treatment of cancer and infectious diseases, and it is mediated by Mdr transporters that recognize and export an unusually broad spectrum of chemically dissimilar toxic compounds. Therefore, in addition to its clinical significance, the Mdr transport phenomenon presents intriguing and challenging mechanistic queries. Recent studies of secondary Mdr transporters of the major facilitator superfamily (MFS) have revealed that they are promiscuous not only regarding their substrate recognition profile, but also with respect to matters of energy utilization, electrical and chemical flexibility in the Mdr recognition pocket, and surprisingly, also in their physiological functions.
(2006) Journal of Molecular Microbiology and Biotechnology. 11, 6, p. 308-317 Abstract
MdfA is a prototypic secondary multidrug transporter from Escherichia coli, which recognizes and exports a broad spectrum of structurally and electrically dissimilar toxic compounds. Here we review recent studies of MdfA, which, on the one hand, provide advanced understanding of certain aspects of secondary multidrug transport, and, on the other, address major mechanistic questions, some of which remain to be elucidated. Using biochemical, genetic, and physiological approaches, we have revealed several surprisingly promiscuous properties of MdfA including its multidrug recognition capacity, proton recognition determinants, aspects of energy utilization, and physiological role. Copyright (c) 2006 S. Karger AG, Basel.
(2005) Biochimica Et Biophysica Acta-Biomembranes. 1717, 2, p. 67-88 Abstract
The capacity of bacteria to survive and grow at alkaline pH values is of widespread importance in the epidemiology of pathogenic bacteria, in remediation and industrial settings, as well as in marine, plant-associated and extremely alkaline ecological niches. Alkali-tolerance and alkaliphily, in turn, strongly depend upon mechanisms for alkaline pH homeostasis, as shown in pH shift experiments and growth experiments in chemostats at different external pH values. Transcriptome and proteome analyses have recently complemented physiological and genetic studies, revealing numerous adaptations that contribute to alkaline pH homeostasis. These include elevated levels of transporters and enzymes that promote proton capture and retention (e.g., the ATP synthase and monovalent cation/proton antiporters), metabolic changes that lead to increased acid production, and changes in the cell surface layers that contribute to cytoplasmic proton retention. Targeted studies over the past decade have followed up the long-recognized importance of monovalent cations in active pH homeostasis. These studies show the centrality of monovalent cation/proton antiporters in this process while microbial genomics provides information about the constellation of such antiporters in individual strains. A comprehensive phylogenetic analysis of both eukaryotic and prokaryotic genome databases has identified orthologs from bacteria to humans that allow better understanding of the specific functions and physiological roles of the antiporters. Detailed information about the properties of multiple antiporters in individual strains is starting to explain how specific monovalent cation/proton antiporters play dominant roles in alkaline pH homeostasis in cells that have several additional antiporters catalyzing ostensibly similar reactions. New insights into the pH-dependent Na+/H+ antiporter NhaA that plays an important role in Escherichia coli have recently emerged from the determination of the structur
3D model of the Escherichia coli multidrug transporter MdfA reveals an essential membrane-embedded positive charge(2005) Biochemistry. 44, 45, p. 14870-14880 Abstract
MdfA is an Escherichia coli multidrug transporter of the major facilitator superfamily (MFS) of secondary transporters. Although several aspects of multidrug recognition by MdfA have been characterized, better understanding the detailed mechanism of its function requires structural information. Previous studies have modeled the 3D structures of MFS proteins, based on the X-ray structure of LacY and G1pT. However, because of poor sequence homology, between LacY, G1pT, and MdfA additional constraints were required for a reliable homology modeling. Using an algorithm that predicts the angular orientation of each transmembrane helix (TM) (kPROT), we obtained a remarkably similar pattern for the 12 TMs of MdfA and those of G1pT and LacY, suggesting that they all have similar helix packing. Consequently, a 3D model was constructed for MdfA by structural alignment with LacY and G1pT, using the kPROT results as an additional constraint. Further refinement and a preliminary evaluation of the model were achieved by correlated mutation analysis and the available experimental data. Surprisingly, in addition to the previously characterized membrane-embedded glutamate at position 26, the model suggests that Asp34 and Arg112 are located within the membrane, on the same face of the cavity as Glu26. Importantly, Arg112 is evolutionarily conserved in secondary drug transporters, and here we show that a positive charge at this position is absolutely essential for multidrug transport by MdfA.
(2005) Nature Reviews Microbiology. 3, 7, p. 566-572 Abstract
Drug and multidrug resistance have greatly compromised the compounds that were once the mainstays of antibiotic therapy. This resistance often persists despite reductions in the use of antibiotics, indicating that the proteins encoded by antibiotic-resistance genes have alternative physiological roles that can foster such persistence in the absence of selective pressure by antibiotics. The recent observations that Tet(L), a tetracycline-efflux transporter, and MdfA, a multidrug-efflux transporter, both confer alkali tolerance offer a striking case study in support of this hypothesis.
(2005) Proceedings of the National Academy of Sciences of the United States of America. 102, 12, p. 4246-4251 Abstract
Target directed proteolysis allows specific processing of proteins in vivo. This method uses tobacco etch virus (TEV) Nla protease that recognizes a seven-residue consensus sequence. Because of its specificity, proteins engineered to contain a cleavage site are proteolysed, whereas other proteins remain unaffected. Therefore, this approach can be used to study the structure and function of target proteins in their natural environment within living cells. One application is the conditional inactivation of essential proteins, which is based on the concept that a target containing a recognition site can be inactivated by coexpressed TEV protease. We have previously identified one site in the secretion factor SecA that tolerated a TEV protease site insert. Coexpression of TEV protease in the cytoplasm led to incomplete cleavage and a mild secretion defect. To improve the efficiency of proteolysis, TEV protease was attached to the ribosome. We show here that cleaving SecA under these conditions is one way of increasing the efficiency of target directed proteolysis. The implications of recruiting novel biological activities to ribosomes are discussed.
Promiscuity in the geometry of electrostatic interactions between the Escherichia coli multidrug resistance transporter MdfA and cationic substrates(2005) Journal of Biological Chemistry. 280, 4, p. 2721-2729 Abstract
The Escherichia coli multidrug transporter MdfA contains a single membrane-embedded charged residue (Glu-26) that plays a critical role in the recognition of cationic substrates (Edgar, M, and Bibi, E. (1999) EMBO J. 18, 822-832). Using an inactive mutant (MdfA-E26T), we isolated a spontaneous second-site mutation (MdfA-E26T/V335E) that re-established the recognition of cationic drugs by the transporter. Only a negative charge at position 335 was able to restore the functioning of the inactive mutant MdfA-E26T. Intriguingly, the two genetically interacting residues are located at remote and distinct regions along the secondary structure of MdfA. Glu-26 is located in the periplasmic half of transmembrane helix 1, and as shown here, the complementing charge at position 335 resides within the cytoplasmic loop connecting transmembrane helices 10 and 11. The spatial relation between the two residues was investigated by cross-linking. A functional split version of MdfA devoid of cysteines was constructed and introduced with a cysteine pair at positions 26 and 335. Strikingly, the results indicate that residues 26 and 335 are spatially adjacent, suggesting that they both constitute parts of the multidrug recognition pocket of MdfA. The fact that electrostatic interactions are preserved with cationic substrates even if the critical acidic residue is placed on another face of the pocket reveals an additional dimension of promiscuity in multidrug recognition and transport.
(2004) Proceedings of the National Academy of Sciences of the United States of America. 101, 39, p. 14073-14078 Abstract
MdfA is an Escherichia coli multidrug-resistance transporter. Cells expressing MdfA from a multicopy plasmid exhibit multidrug resistance against a diverse group of toxic compounds. In this article, we show that, in addition to its role in multidrug resistance, MdfA confers extreme alkaline pH resistance and allows the growth of transformed cells under conditions that are close to those used normally by alkaliphiles (up to pH 10) by maintaining a physiological internal pH. MdfA-deleted E. coli cells are sensitive even to mild alkaline conditions, and the wild-type phenotype is restored fully by MdfA expressed from a plasmid. This activity of MdfA requires Na+ or K+. Fluorescence studies with inverted membrane vesicles demonstrate that MdfA catalyzes Na+- or K+-dependent proton transport, and experiments with reconstituted proteoliposomes confirm that MdfA is solely responsible for this phenomenon. Studies with multidrug resistance-defective MdfA mutants and competitive transport assays suggest that these activities of MdfA are related. Together, the results demonstrate that a single protein has an unprecedented capacity to turn E. coli from an obligatory neutrophile into an alkalitolerant bacterium, and they suggest a previously uncharacterized physiological role for MdfA in pH homeostasis.
The core Escherichia coli signal recognition particle receptor contains only the N and G domains of FtsY(2004) Journal of Bacteriology. 186, 8, p. 2492-2494 Abstract
Previous studies have proposed that the N-terminal A domain (similar to200 amino acid residues) of the Escherichia coli signal recognition particle (SRP) receptor, FtsY, is required for membrane targeting. In contrast to this suggestion, we show that A domain-truncated versions of FtsY, harboring only domains N and G, are functional. Therefore, we propose that N and G domains constitute the core SRP receptor.
Determinants of substrate recognition by the Escherichia coli multidrug transporter MdfA identified on both sides of the membrane(2004) Journal of Biological Chemistry. 279, 10, p. 8957-8965 Abstract
The Escherichia coli multidrug transporter MdfA contains a membrane-embedded charged residue (Glu-26) that was shown to play an important role in substrate recognition. To identify additional determinants of multidrug recognition we isolated 58 intragenic second-site mutations that restored the function of inactive MdfA E26X mutants. In addition, two single-site mutations that enhanced the activity of wild-type MdfA were identified. Most of the mutations were found in two regions, the cytoplasmic half of transmembrane segments (TMs) 4, 5, and 6 (cluster 1) and the periplasmic half of TM 1 and 2 ( cluster 2). The identified residues were mutated to cysteines in the background of a functional cysteineless MdfA, and substrate protection against alkylation was analyzed. The results support the suggestion that the two clusters are involved in substrate recognition. Using inverted membrane vesicles we observed that a proton electrochemical gradient (Delta(μ) over tilde (H+), inside positive and acidic) enhanced the substrate-protective effect in the cytoplasmic region, whereas it largely reduced this effect in the periplasmic side of MdfA. Therefore, we propose that substrates interact with two sites in MdfA, one in the cytoplasmic leaflet of the membrane and the other in the periplasmic leaflet. Theoretically, these domains could constitute a large part of the multidrug pathway through MdfA.
(2004) Biochemistry. 43, 2, p. 518-525 Abstract
According to the current topology model of the Escherichia coli multidrug transporter MdfA, it contains a membrane-embedded negatively charged residue, Glu26, which was shown to play an important role in substrate recognition. To further elucidate the role of this substrate recognition determinant, various Glu26 replacements were characterized. Surprisingly, studies with neutral MdfA substrates showed that, unlike many enzymatic systems where the size and chemical properties of binding site residues are relatively defined, MdfA tolerates a variety of changes at position 26, including size, hydrophobicity, and charge. Moreover, although efficient transport of positively charged substrates requires a negative charge at position 26 (Glu or Asp), neutralization of this charge does not always abrogate the interaction of MdfA with cationic drugs, thus demonstrating that the negative charge does not play an essential role in the multidrug transport mechanism. Collectively, these results suggest a link between the broad substrate specificity profile of multidrug transporters and the structural and chemical promiscuity at their substrate recognition pockets.
Expression of Na+,K+-ATPpase in Pichia pastoris analysis of wild type and D369N mutant proteins by Fe2+-catalyzed oxidative cleavage and molecular modeling(2003) Journal of Biological Chemistry. 278, 46, p. 46064-46073 Abstract
Na+,K+-ATPase (pig alpha1,beta1) has been expressed in the methylotrophic yeast Pichia pastoris. A protease-deficient strain was used, recombinant clones were screened for multicopy genomic integrants, and protein expression, and time and temperature of methanol induction were optimized. A 3-liter culture provides 300-500 mg of membrane protein with ouabain binding capacity of 30-50 pmol mg(-1). Turnover numbers of recombinant and renal Na+,K+-ATPase are similar, as are specific chymotryptic cleavages. Wild type (WT) and a D369N mutant have been analyzed by Fe2+- and ATP-Fe2+-catalyzed oxidative cleavage, described for renal Na+,K+-ATPase. Cleavage of the D369N mutant provides strong evidence for two Fe2+ sites: site 1 composed of residues in P and A cytoplasmic domains, and site 2 near trans-membrane segments M3/M1. The D369N mutation suppresses cleavages at site 1, which appears to be a normal Mg2+ site in E-2 conformations. The results suggest a possible role of the charge of Asp(369) on the E-1 E-2 conformational equilibrium. 5'-Adenylyl-beta,gamma-imidodiphosphate(AMP-PNP)-Fe2+-catalyzed cleavage of the D369N mutant produces fragments in P ((VNDS)-V-712) and N (near (440)VAGDA) domains, described for WT, but only at high AMP-PNP-Fe2+ concentrations, and a new fragment in the P domain (near (367)CSDKTGT) resulting from cleavage. Thus, the mutation distorts the active site. A molecular dynamic simulation of ATP-Mg2+ binding to WT and D351N structures of Ca2+-ATPase (analogous to Asp(369) of Na+,K+-ATPase) supplies possible explanations for the new cleavage and for a high ATP affinity, which was observed previously for the mutant. The Asn(351) structure with bound ATP-Mg2+ may resemble the transition state of the WT poised for phosphorylation.
The Escherichia coli multidrug transporter MdfA catalyzes both electrogenic and electroneutral transport reactions(2003) Proceedings of the National Academy of Sciences of the United States of America. 100, 4, p. 1667-1672 Abstract
The resistance of cells to many drugs simultaneously (multidrug resistance) often involves the expression of membrane transporters (Mdrs); each recognizes and expels a broad spectrum of chemically unrelated drugs from the cell. The Escherichia coli Mdr transporter MdfA is able to transport differentially charged substrates in exchange for protons. This includes neutral compounds, namely chloramphenicol and thiamphenicol, and lipophilic cations such as tetraphenylphosphonium and ethidium. Here we show that the chloramphenicol and thiamphenicol transport reactions are electrogenic, whereas the transport of several monovalent cationic substrates is electroneutral. Therefore, unlike with positively charged substrates, the transmembrane electrical potential (negative inside) constitutes a major part of the driving force for the transport of electroneutral substrates by MdfA. These results demonstrate an unprecedented ability of a single secondary transporter to catalyze discrete transport reactions that differ in their electrogenicity and are governed by different components of the proton motive force.
Accumulation of endoplasmic membranes and novel membrane-bound ribosome-signal recognition particle receptor complexes in Escherichia coli(2002) Journal of Cell Biology. 159, 3, p. 403-410 Abstract
In Escherichia coli, ribosomes must interact with translocons on the membrane for the proper integration of newly synthesized membrane proteins, cotranslationally. Previous in vivo studies indicated that unlike the E coli signal recognition particle (SRP), the SRP receptor FtsY is required for membrane targeting of ribosomes. Accordingly, a putative SRP-independent, FtsY-mediated ribosomal targeting pathway has been suggested (Herskovits, A.A., E.S. Bochkareva, and E. Bibi. 2000. Mol. Microbiol. 38:927-939). However, the nature of the early contact of ribosomes with the membrane, and the involvement of FtsY in this interaction are unknown. Here we show that in cells depleted of the SRP protein, Ffh or the translocon component SecE, the ribosomal targeting pathway is blocked downstream and unprecedented, membrane-bound FtsY-ribosomal complexes are captured. Concurrently, under these conditions, novel, ribosome-loaded intracellular membrane structures are formed. We propose that in the absence of a functional SRP or translocon, ribosomes remain jammed at their primary membrane docking site, whereas FtsY-dependent ribosomal targeting to the membrane continues. The accumulation of FtsY-ribosome complexes induces the formation of intracellular membranes needed for their quantitative accommodation. Our results with E. coli, in conjunction with recent observations made with the yeast Saccharomyces cerevisiae, raise the possibility that the SRP receptor-mediated formation of intracellular membrane networks is governed by evolutionarily conserved principles.
Identification and characterization of the Escherichia coli stress protein UP12, a putative in vivo substrate of GroEL(2002) FEBS Journal. 269, 12, p. 3032-3040 Abstract
Many groups of proteins play important roles in the cell's response to various stresses. The molecular chaperone GroEL of Escherichia coli represents one such highly conserved family of stress proteins. We have observed that isolated GroEL complexes from stationary cultures contain various polypeptides that can be released from the chaperonin by GroES and/or ATP, and identified two such polypeptides as the proteins GatY and UP12. Whereas GatY had been isolated previously, as an in vivo substrate of GroEL, the isolation of UP12 in a complex with GroEL was intriguing, because based on sequence similarity it was suggested that UP12 might also be a functional stress protein. UP12 belongs to a family of universal stress proteins (UspA family), of which UspA itself, and three additional paralogues, have been characterized previously. Here we show that UP12 accumulates under various growth inhibitory conditions and induced by heat shock. Furthermore, unlike wild-type cells, a UP12 deletion mutant recovers slowly from late stationary growth conditions, and has a marked sensitivity to the toxic agent carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Finally, coimmunoprecipitation experiments confirmed the initial observation that UP12 interacts with GroEL. Therefore, we suggest that UP12 may function as a universal stress protein, interaction of which with GroEL possibly ensures its proper folding state.
Membrane topology of the multidrug transporter MdfA: Complementary gene fusion studies reveal a nonessential C-terminal domain(2002) Journal of Bacteriology. 184, 12, p. 3313-3320 Abstract
The hydrophobicity profile and sequence alignment of the Escherichia coli multidrug transporter MdfA indicate that it belongs to the 12-transmembrane-domain family of transporters. According to this prediction, MdfA contains a single membrane-embedded charged residue (Glu26), which was shown to play an important role in substrate recognition. To test the predicted secondary structure of MdfA, we analyzed complementary pairs of hybrids of MdfA-PhoA (alkaline phosphatase, functional in the periplasm) and MdfA-Cat (chloramphenicol acetyltransferase, functional in the cytoplasm), generated in all the putative cytoplasmic and periplasmic loops of MdfA. Our results support the 12-transmembrane topology model and the suggestion that except for Glu26, no other charged residues are present in the membrane domain of MdfA. Surprisingly, by testing the ability of the truncated MdfA-Cat and MdfA-PhoA hybrids to confer multidrug resistance, we demonstrate that the entire C-terminal transmembrane domain and the cytoplasmic C terminus are not essential for MdfA-mediated drug resistance and transport.
Evidence for coupling of membrane targeting and function of the signal recognition particle (SRP) receptor FtsY(2001) EMBO Reports. 2, 11, p. 1040-1046 Abstract
Recent studies have indicated that FtsY, the signal recognition particle receptor of Escherichia coli, plays a central role in membrane protein biogenesis. For proper function, FtsY must be targeted to the membrane, but its membrane-targeting pathway is unknown. We investigated the relationship between targeting and function of FtsY in vivo, by separating its catalytic domain (NG) from its putative targeting domain (A) by three means: expression of split ftsY, insertion of various spacers between A and NG, and separation of A and NG by in vivo proteolysis. Proteolytic separation of A and NG does not abolish function, whereas separation by long linkers or expression of split ftsY is detrimental. We propose that proteolytic cleavage of FtsY occurs after completion of co-translational targeting and assembly of NG. In contrast, separation by other means may interrupt proper synchronization of co-translational targeting and membrane assembly of NG. The co-translational interaction of FtsY with the membrane was confirmed by in vitro experiments.[All authors]
Evidence for simultaneous binding of dissimilar substrates by the Escherichia coli multidrug transporter MdfA(2001) Biochemistry. 40, 42, p. 12612-12618 Abstract
The mechanism by which multidrug transporters interact with structurally unrelated substrates remains enigmatic. Based on transport competition experiments, photoaffinity labeling, and effects on enzymatic activities, it was proposed in the past that multidrug transporters can interact simultaneously with a number of dissimilar substrate molecules. To study this phenomenon, we applied a direct binding approach and transport assays using the Escherichia coli multidrug transporter MdfA, which exports both positively charged (e.g., tetraphenylphosphonium, TPP+), zwitterionic (e.g., ciprofloxacin), and neutral (e.g., chloramphenicol) drugs. The interaction of MdfA with various substrates was examined by direct binding assays with the purified transporter. The immobilized MdfA binds TPP+ in a specific manner, and all the tested positively charged substrates inhibit TPP+ binding. Surprisingly, although TPP+ binding C is not affected by zwitterionic substrates, the neutral substrate chloramphenicol stimulates TPP+ binding by enhancing its affinity to MdfA. In contrast, transport competition assays show inhibition of TPP+ transport by chloramphenicol. We suggest that MdfA binds TPP+ and chloramphenicol simultaneously to distinct but interacting binding sites, and the interaction between these two substrates during transport is discussed.
MdfA, an interesting model protein for studying multidrug transport(2001) Journal of Molecular Microbiology and Biotechnology. 3, 2, p. 171-177 Abstract
The resistance of cells to many drugs simultaneously (multidrug resistance) often involves the expression of membrane transporters (Mdrs); each can recognize and expel a broad spectrum of chemically unrelated drugs from the cell. Despite extensive research for many years, the actual mechanism of multidrug transport is still largely unknown. In addition to general questions dealing with energy coupling, the molecular view of substrate recognition by Mdrs is generally obscure. This mini-review describes structural and functional properties of the Escherichia coli Mdr, MdfA, and discusses the possibility that this transporter may serve as a model for studying the multidrug recognition phenomenon and the mechanism of multidrug transport.
(2000) Molecular Microbiology. 38, 5, p. 927-939 Abstract
In vivo and in vitro studies have suggested that the bacterial version of the mammalian signal recognition particle (SRP) system plays an essential and selective role in protein biogenesis. The bacterial SRP system consists of at least two proteins and an RNA molecule (termed Ffh, FtsY and 4.5S RNA, respectively, in Escherichia coli). Recent evidence suggests that other putative bacterial-specific SRP components may also exist. In vitro experiments confirmed the expected basic features of the bacterial SRP system by demonstrating interactions among the SRP components themselves, between them and ribosomes, ribosome-linked hydrophobic nascent polypeptides or inner membranes. The availability of a conserved (and essential) bacterial SRP version has facilitated the implementation of powerful genetic and biochemical approaches for studying the cascade of events during the SRP-mediated targeting process in vivo and in vitro as well as the three-dimensional structures and the properties of each SRP component and complex.
Association of Escherichia coli ribosomes with the inner membrane requires the signal recognition particle receptor but is independent of the signal recognition particle(2000) Proceedings of the National Academy of Sciences of the United States of America. 97, 9, p. 4621-4626 Abstract
In mammalian cells, as well as Escherichia coil, ribosomes translating membrane proteins interact cotranslationally with translocons in the membrane, and this interaction is essential for proper insertion of nascent polypeptides into the membrane. Both the signal recognition particle (SRP) and its receptor (SR) are required for functional association of ribosomes translating integral membrane proteins with the translocon. Herein, we confirm that membrane targeting of E. coil ribosomes requires the prokaryotic SR alpha homolog FtsY in vivo. Surprisingly, however, depletion of the E. coli SRP54 homolog (Ffh) has no significant effect on binding of ribosomes to the membrane, although Ffh depletion is detrimental to growth. These and other observations suggest that, in E. coli. SRP may operate downstream of SR-mediated targeting of ribosomes to the plasma membrane.
(2000) Trends in Biochemical Sciences. 25, 2, p. 39-43 Abstract
Multidrug transporters bind chemically dissimilar, potentially cytotoxic compounds and remove them from the cell. How these transporters carry out either of these functions is unknown. On the basis of crystal structures of the multidrug-binding domain of the transcription activator BmrR and mutagenesis studies on the bacterial multidrug transporter MdfA, we propose a possible mechanism for the binding of cationic lipophilic drugs by multidrug transporters, The key element of this mechanism includes a conformational change in the transporter that exposes a buried charged residue in the substrate-binding pocket and allows access to this site by only those drugs that are its steric and electrostatic complements.
Resistance to bacitracin as modulated by an Escherichia coli homologue of the bacitracin ABC transporter BcrC subunit from Bacillus licheniformis(1999) Journal of Bacteriology. 181, 19, p. 6176-6178 Abstract
A small open reading frame from the Escherichia coli chromosome, bcrC(EC), encodes a homologue to the BcrC subunit of the bacitracin permease from Bacillus licheniformis. We show that disruption of the chromosomal bcrC(EC) gene causes bacitracin sensitivity and, conversely, that BcrC(EC) confers bacitracin resistance when expressed from a multicopy plasmid.
A single membrane-embedded negative charge is critical for recognizing positively charged drugs by the Escherichia coli multidrug resistance protein MdfA(1999) EMBO Journal. 18, 4, p. 822-832 Abstract
The nature of the broad substrate specificity phenomenon, as manifested by multidrug resistance proteins, is not yet understood. In the Escherichia coli multidrug transporter MdfA, the hydrophobicity profile and PhoA fusion analysis have so far identified only one membrane-embedded charged amino acid residue (E26), In order to determine whether this negatively charged residue may play a role in multidrug recognition, we evaluated the expression and function of MdfA constructs mutated at this position. Replacing E26 with the positively charged residue lysine abolished the multidrug resistance activity against positively charged drugs, but retained chloramphenicol efflux and resistance, In contrast, when the negative charge was preserved in a mutant with aspartate instead of E26, chloramphenicol recognition and transport were drastically inhibited; however, the mutant exhibited almost wild-type multidrug resistance activity against lipophilic cations, These results suggest that although the negative charge at position 26 is not essential for active transport, it dictates the multidrug resistance character of MdfA, We show that such a negative charge is also found in other drug resistance transporters, and its possible significance regarding multidrug resistance is discussed.
Multidrug resistance transporters in Escherichia coli(1999) Microbial Ecology And Infectious Disease. p. 276-283 Abstract
Keywords: GRAM-NEGATIVE BACTERIA; EFFLUX PUMPS; ACTIVE EFFLUX; FUNCTIONAL EXPRESSION; NUCLEOTIDE-SEQUENCE; MEMBRANE TOPOLOGY; OUTER-MEMBRANE; P-GLYCOPROTEIN; PROTEIN; GENE
The role of the ribosome-translocon complex in translation and assembly of polytopic membrane proteins(1998) Trends in Biochemical Sciences. 23, 2, p. 51-55 Abstract
Newly synthesized polytopic membrane proteins and secretory proteins often share the same target membrane as their primary destination, and in some cases, the cellular machinery that targets and transfers them into or across the membrane. Unlike secretory proteins, which are localized to the external compartment, each polytopic membrane protein molecule must be partitioned among the cytoplasm, the membrane and the external milieu. How does the ribosome-translocon complex cope with the different domains of polytopic membrane proteins?
The NG domain of the prokaryotic signal recognition particle receptor, FtsY, is fully functional when fused to an unrelated integral membrane polypeptide(1997) Proceedings of the National Academy of Sciences of the United States of America. 94, 12, p. 6025-6029 Abstract
Recent studies have revealed that Escherichia coli possesses an essential targeting system for integral membrane proteins, similar to the mammalian signal recognition particle (SRP) machinery, One essential protein in this system is FtsY, a homologue of the alpha-subunit of the mammalian SRP-receptor (SR-alpha). However, E. coli does not possess a close homologue of the integral membrane protein SR-beta, which anchors SR-alpha to the membrane. Moreover, although FtsY can be found as a peripheral membrane protein, the majority is found soluble in the cytoplasm. In this study, we obtained genetic and biochemical evidence that FtsY must be targeted to the membrane for proper function, We demonstrate that the essential membrane targeting activity of FtsY is mediated by a 198-residue-long acidic N-terminal domain, This domain can be functionally replaced by unrelated integral membrane polypeptides, thus avoiding the need for specific FtsY membrane targeting factors, Therefore, the N terminus of FtsY constitutes an independent domain, which is required only for the targeting of the C-terminal NG domain of FtsY to the membrane.
MdfA, an Escherichia coli multidrug resistance protein with an extraordinarily broad spectrum of drug recognition(1997) Journal of Bacteriology. 179, 7, p. 2274-2280 Abstract
Multidrug resistance (MDR) translocators recently identified in bacteria constitute an excellent model system for studying the MDR phenomenon and its clinical relevance, Here we describe the identification and characterization of an unusual MDR gene (mdfA) from Escherichia coli, mdfA encodes a putative membrane protein (MdfA) of 410 amino acid residues which belongs to the major facilitator superfamily of transport proteins, Cells expressing MdfA from a multicopy plasmid are substantially more resistant to a diverse group of cationic or zwitterionic lipophilic compounds such as ethidium bromide, tetraphenylphosphonium, rhodamine, daunomycin, benzalkonium, rifampin, tetracycline, and puromycin. Surprisingly, however, MdfA also confers resistance to chemically unrelated, clinically important antibiotics such as chloramphenicol, erythromycin, and certain aminoglycosides and fluoroquinolones. Transport experiments with an E. coli strain lacking F-1-F-0 proton ATPase activity indicate that MdfA is a multidrug transporter that is driven by the proton electrochemical gradient.
FtsY, the prokaryotic signal recognition particle receptor homologue, is essential for biogenesis of membrane proteins(1997) Journal of Biological Chemistry. 272, 4, p. 2053-2055 Abstract
In mammalian cells, many secretory proteins are targeted to the endoplasmic reticulum co-translationally, by the signal recognition particle (SRP) and its receptor. In Escherichia coli, the targeting of secretory proteins to the inner membrane can be accomplished post-translationally. Unexpectedly, despite this variance, E. coli contains essential genes encoding Ffh and FtsY with a significant similarity to proteins of the eukaryotic SRP machinery. In this study, we investigated the possibility that the prokaryotic SRP-like machinery is involved in biogenesis of membrane proteins in E. coli. The data presented here demonstrate that the SRP-receptor homologue, FtsY, is indeed essential for expression of integral membrane proteins in E. coli, indicating that, in the case of this group of proteins, FtsY and the mammalian SRP receptor have similar functions.
Chaperonin-promoted Post-translational Membrane Insertion of a Multispanning Membrane Protein Lactose Permease(1996) Journal of Biological Chemistry. 271, 36, p. 22256-22261 Abstract
Using an in vitro membrane-free translation system from Escherichia coli, it is shown that chaperonin GroEL added cotranslationally interacts with newly synthesized lactose permease (LacY), a polytopic membrane protein, thereby preventing aggregation, Subsequently, when the isolated GroEL-LacY complex is incubated with inverted membrane vesicles, the permease is inserted into the membrane in a MgATP-dependent manner. Post-translational membrane insertion is also observed when aggregation of newly synthesized LacY is prevented by addition of the nonionic detergent n-dodecyl-beta,D-maltoside during translation in place of GroEL. No membrane integration occurs with right-side-out vesicles, indicating that LacY interacts specifically only with the cytosolic face of the membrane. Ligand thiodigalactoside protection against alkylation of the Cys-148 residue in the permease shows proper posttranslational insertion. Moreover, limited proteolysis of soluble LacY either complexed with GroEL or in detergent indicates that the newly synthesized protein assumes a conformation that is comparable to that of native, membrane-embedded permease prior to insertion into the membrane.
(1996) Proceedings of the National Academy of Sciences of the United States of America. 93, 18, p. 9612-9617 Abstract
The assembly of functional proteins from fragments in vivo has been recently described for several proteins, including the secreted maltose binding protein in Escherichia coil. Here we demonstrate for the first time that split gene products can function within the eukaryotic secretory system. Saccharomyces cerevisiae strains able to use sucrose produce the enzyme invertase, which is targeted by a signal peptide to the central secretory pathway and the periplasmic space. Using this enzyme as a model we find the following: (i) Polypeptide fragments of invertase, each containing a signal peptide, are independently translocated into the endoplasmic reticulum (ER) are modified by glycosylation, and travel the entire secretory pathway reaching the yeast periplasm. (ii) Simultaneous expression of independently translated and translocated overlapping fragments of invertase leads to the formation of an enzymatically active complex, whereas individually expressed fragments exhibit no activity. (iii) An active invertase complex is assembled in the ER, is targeted to the yeast periplasm, and is biologically functional, as judged by its ability to facilitate growth on sucrose as a single carbon source. These observations are discussed in relation to protein folding and assembly in the ER and to the trafficking of proteins through the secretory pathway.
Biogenesis and topology of integral membrane proteins: Characterization of lactose Permease-chloramphenicol acetyltransferase hybrids(1996) Biochemistry. 35, 33, p. 10872-10878 Abstract
Use of beta-lactamase in gene fusions to study membrane protein topology permits exploitation of its biological activity to select for positive (external) hybrids on ampicillin agar plates. When the enzyme is attached to cytoplasmic loops of a membrane protein, it is not secreted and is therefore unable to confer ampicillin resistance. In this study, we examine the use of the cytoplasmic enzyme chloramphenicol acetyltransferase (Cat) as a complement to the use of periplasmic beta-lactamase, in gene fusion studies. This enzyme is responsible for chloramphenicol resistance in Escherichia coil. We show that Cat confers substantial antibiotic resistance when fused to cytoplasmic loops of lactose permease. As expected, periplasmically exposed Cat is enzymatically active in vitro but unable to confer significant chloramphenicol resistance, presumably because of the absence of acetylcoenzyme A in the periplasm. Therefore, Cat may serve as a topogenic sensor in gene fusion studies. The new Cat fusion approach is discussed with regard to its potential use for selecting E. coli mutants which are defective in the assembly of membrane proteins.
Comparative topology studies in Saccharomyces cerevisiae and in Escherichia coli - The N-terminal half of the yeast ABC protein Ste6(1996) Journal of Biological Chemistry. 271, 23, p. 13746-13753 Abstract
Gene fusions have provided a strategy for determining the topology of polytopic membrane proteins in Escherichia coli. To valuate whether this highly effective approach is applicable to heterologously expressed eukaryotic integral membrane proteins, we have carried out a comparative topological study of the eukaryotic membrane protein Ste6 both in bacteria and in yeast, Ste6, is an ATP binding cassette (ABC) protein essential for export of the a-factor mating pheromone in Saccharomyces cerevisiae, The topogenic reporters, invertase in S. cerevisiae and alkaline phosphatase in E. coli, were fused to Ste6 at identical sites and the fusions were expressed in yeast and bacteria, respectively. The results obtained in both systems are similar, although more definitive in E. coli, and support the predicted six-transmembrane spans organization of the N-terminal half of Ste6, Thus, the topological determinants for membrane insertion of polytopic proteins in prokaryotic and in eukaryotic systems appear to be highly similar, In this study we also demonstrate that Ste6 does not contain a cleaved signal sequence.
(1996) Proceedings of the National Academy of Sciences of the United States of America. 93, 12, p. 5969-5974 Abstract
Functional expression of the multidrug resistance protein P-glycoprotein (P-gp) in Escherichia coli is providing an appropriate system for structure/function studies and might provide an invaluable tool to screen potential P-gp substrates and inhibitors. The major problem encountered in such studies, however, is the impermeability of the outer membrane of Gram-negative bacteria, which protects microorganisms against the cytotoxic effects of many lipophilic cancer drugs and blocks accessibility of P-gp reversal agents. In the present study we have constructed, by mutagenesis, a ''leaky'' (containing a permeable outer membrane) strain off. coli, which is significantly more susceptible to the toxic effect of known P-gp substrates and cytotoxic agents. Expression of mouse Mdr1 in the mutant confers cross-resistance to daunomycin, quinidine, chloroquine, rhodamine 6G, and puromycin. Most importantly, reserpine and doxorubicin completely abolish Mdr1-mediated rhodamine resistance. The results provide strong support for previous observations, suggesting that Mdr1 can be expressed functionally in E. coli and indicate that the leaky mutant will be useful for further structure/function studies of the heterologously expressed eukaryotic drug efflux protein.
MEMBRANE TOPOLOGY OF HELICE-VII AND HELICE-XI IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI STUDIED BY LACY-PHOA FUSION ANALYSIS AND SITE-DIRECTED SPECTROSCOPY(1995) Biochemistry. 34, 45, p. 14909-14917 Abstract
The use of lactose permease-alkaline phosphatase fusions (lacY-phoA) demonstrates that the lactose permease of Escherichia coli contains 12 transmembrane domains and that approximately half of a transmembrane domain is required to translocate alkaline phosphatase to the periplasmic surface of the membrane [Calamia, J., & Manoil, C. (1990) Proc. Natl. Acad. Sci U.S.A. 87, 4937-4941]. We have now used fusion analysis in combination with site-directed spectroscopy to examine more precisely the topology of putative helices VII and XI which contain the interacting residues Asp237 and Lys358, respectively. For this purpose, alkaline phosphatase was fused to alternate amino acid residues in transmembrane domains VII and XI. A sharp increase in alkaline phosphatase activity is observed as the fusion junction proceeds from Tyr228 to Ile230 in helix VII and from Phe354 to Phe356 in helix XI, suggesting that these residues approximate the middle of the corresponding transmembrane helices. Analysis of fluorescence quenching of the pyrene-labeled single-Cys mutants Asp237-->Cys or Lys358-->Cys, as well as measurement of collision frequencies between freely diffusing paramagnetic probes and a nitroxide spin-label at these sites, also indicates that Asp237 and also Asp240, which interacts with Lys319 (helix X), are located in transmembrane domains. However, Asp237 and Asp240 are accessible both from the aqueous phase and from within the membrane. The results provide more direct evidence that the three residues are located within transmembrane helices and suggest that Asp237 and Asp240 are either located near the periplasmic surface of the membrane or exposed within a solvent-filled cleft in the permease.
MULTIDRUG-RESISTANCE PROTEIN (MDR)-ALKALINE PHOSPHATASE HYBRIDS IN ESCHERICHIA-COLI SUGGEST A MAJOR REVISION IN THE TOPOLOGY OF THE C-TERMINAL HALF OF MDR(1995) Journal of Biological Chemistry. 270, 21, p. 12351-12354 Abstract
Recent studies reveal that the organization of the multidrug resistance protein (Mdr) in the membrane is probably not exactly as predicted from hydropathy profiling, When expressed in Escherichia coli, phoA gene fusions can be utilized to study the membrane topology of Mdr. Using this approach, it was proposed recently that the N-terminal hydrophobic domain of Mdr spans the membrane six times, in a different fashion from that predicted by hydropathy analysis (Bibi, E. and Beja, O. (1994) J. Biol. Chem. 269, 19910-19915). In this study, we analyze mdr-phoA fusions constructed in the C-terminal half of Mdr. Overall, the results presented here lead to a significant revision in the membrane topology model of the C-terminal half of Mdr. The new topology is discussed with regard to the hydropathy profiles of the well characterized ABC proteins MalG and MalF, which are strikingly similar to those of the N- and C-terminal halves of Mdr, respectively.
THE ROLE OF TRANSMEMBRANE DOMAIN-III IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI(1994) Protein Science. 3, 12, p. 2302-2310 Abstract
Deletion of putative transmembrane helix III from the lactose permease of Escherichia coli results in complete loss of transport activity. Similarly, replacement of this region en bloc with 23 contiguous Ala, Leu, or Phe residues abolishes active lactose transport. The observations suggest that helix III may contain functionally important residues; therefore, this region was subjected to Cys-scanning mutagenesis. Using a functional mutant devoid of Cys residues (C-less permease) each residue from Tyr 75 to Leu 99 was individually replaced with Cys. Twenty-one of the 25 mutants accumulate lactose to >70% of the steady-state exhibited by C-less permease, and an additional 3 mutants transport to lower, but significant levels (40-60% of C-less). Cys replacement for Leu 76 results in low transport activity (18% of C-less). However, when placed in the wild-type background, mutant Leu 76 --> Cys exhibits highly significant rates of transport (55% of wild type) and steady-state levels of lactose accumulation (65% of wild type). Immunoblots reveal that the mutants are inserted into the membrane at concentrations comparable to wild type. Studies with N-ethylmaleimide show that mutant Gly 96 --> Cys is rapidly inactivated, whereas the other single-Cys mutants are not altered significantly by the alkylating agent, Moreover, the rate of inactivation of Gly 96 --> Cys permease is enhanced at least 2-fold in the presence of P-galactopyranosyl 1-thio-beta,D-galactopyranoside. The observations demonstrate that although no residue per se appears to be essential, structural properties of helix III are important for active lactose transport.
Membrane topology of multidrug resistance protein expressed in Escherichia coli. N-terminal domain(1994) Journal of Biological Chemistry. 269, 31, p. 19910-19915 Abstract
Expression of eukaryotic polytopic membrane proteins in Escherichia coli could provide an invaluable system for structure-function studies. Recently, the functional expression of a mouse multidrug resistance protein (Mdr1) in E. coli was described (Bibi, E., Gros, P., and Kaback, H. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9209-9213). In the present study, the phoA gene fusion approach has been utilized to determine the membrane topology of the N-terminal domain of Mdr. The results support the idea that the N-terminal half of Mdr contains six transmembrane helices (TMs). However, our observations suggest that the previously proposed TM4 (amino acid residues Thr(214)-Ala(232)) is located at the periplasmic face of the membrane. Alternatively, we propose that another stretch of amino acid residues (Leu(243) (out) to Ile(260) (in)) traverses the membrane. This assignment is indicated also by the following observations: 1) deletion of a segment containing the originally predicted TM4 (Delta T214-K241) does not reverse the orientation of the Mdr-alkaline phosphatase hybrid that is located in the following cytoplasmic loop; 2) a ''sandwich'' chimera, in which alkaline phosphatase is inserted in-frame between amino acid residues Leu(226) and Ser(227), exhibits high alkaline phosphatase activity. The existence of an externally exposed hydrophobic domain in Mdr may have special structural and functional implications, and these may also be relevant to other members of the ABC superfamily.
(1994) Biochemistry. 33, 27, p. 8198-8206 Abstract
The lactose permease of Escherichia coli is a membrane transport protein containing 12 transmembrane hydrophobic domains connected by hydrophilic loops. Coexpression of lacY gene fragments encoding contiguous polypeptides corresponding to the first and second halves of the permease [Bibi, E., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4325-4329] or the first two transmembrane domains and the remainder of the molecule [Wrubel, W., Stochaj, U., Sonnewald, U., Theres, C., & Ehring, R. (1990) J. Bacteriol. 172, 5374-5381] leads to active lactose transport. It is shown here that contiguous permease fragments with discontinuities in loop 1 (periplasmic), loop 6 (cytoplasmic), or loop 7 (periplasmic) exhibit transport activity; however, fragments with discontinuities in transmembrane domains III or VII fail to do so. The results are consistent with the interpretation that contiguous permease fragments with discontinuities in hydrophilic loops form functional duplexes, while fragments with discontinuities in transmembrane alpha-helical domains do not. On the basis of this notion, a series of contiguous, nonoverlapping permease fragments with discontinuities at various positions in loop 6, putative helix VII, and loop 7 were coexpressed to approximate the boundaries of putative transmembrane domain VII. Contiguous fragments with a discontinuity between Leu222 and Trp223 or between Gly254 and Glu255 are functional, but fragments with a discontinuity between Cys234 and Thr235, between Gln241 and Gln242, or between Phe247 and Thr248 are inactive. Therefore, it is likely that Leu222 and Gly254 are located in hydrophilic loops 6 and 7, respectively, while Cys234, Gln241, and Phe247 are probably located within transmembrane domain VII. These and other results are consistent with a modified secondary-structure model of lactose permease in which Asp237 and Asp240 are contained within transmembrane domain VII rather than periplasmic loop 7.
(1993) Proceedings of the National Academy of Sciences of the United States of America. 90, 19, p. 9209-9213 Abstract
We describe functional expression of the mouse multidrug-resistance protein (P-glycoprotein; P-gp) in an Escherichia coli mutant defective in the outer membrane protease ompT. Heterologously expressed mdr1 appears as an unglycosylated species with an apparent molecular mass of 140 kDa in the membrane of the mutant. Unglycosylated mdr1 retains the ability to bind the photoactivatable drug analog [I-125]iodoarylazidoprazosin and confers resistance to tetraphenylphosphonium (TPP+) and tetraphenylarsonium (TPA+), known mdr1 substrates. In vivo resistance is linked to reduced cellular accumulation and energy-dependent efflux of the lipophilic cations. Surprisingly, discrete mutations in the predicted nucleotide binding folds of mdr1 that abolish drug resistance in mammalian cells have no apparent effect on TPA+ efflux via mdr1 in E. coli.
THE N-TERMINAL 22 AMINO-ACID-RESIDUES IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI ARE NOT OBLIGATORY FOR MEMBRANE INSERTION OR TRANSPORT ACTIVITY(1992) Proceedings of the National Academy of Sciences of the United States of America. 89, 8, p. 3180-3184 Abstract
When the lactose (lac) permease of Escherichia coli is expressed from the lac promoter at relatively low rates, deletion of amino acid residues 2-8 (DELTA-7) or 2-9 (DELTA-8) from the hydrophilic N terminus has a relatively minor effect on the ability of the permease to catalyze active lactose transport. Activity is essentially abolished, however, and the permease is hardly detected in the membrane when two additional amino acid residues are deleted (DELTA-10), and mutants deleted of residues 2-23 (DELTA-22) or 2-39 (DELTA-38) also exhibit no activity and are not inserted into the membrane. Dramatically, when the defective deletion mutants are overexpressed at high rates via the T7 promoter, DELTA-10 and DELTA-22 are inserted into the membrane in a stable form and catalyze active lactose transport in a highly significant manner, whereas DELTA-38 is hardly detected in the membrane and exhibits no activity. Interestingly, a fusion protein consisting of DELTA-38 and the ompA leader peptide is inserted into the membrane but exhibits no transport activity. The results indicate that the N-terminal hydrophilic domain of lac permease and the N-terminal half of the first putative transmembrane alpha-helix are not mandatory for either membrane insertion or transport activity.
(1992) Proceedings of the National Academy of Sciences of the United States of America. 89, 5, p. 1524-1528 Abstract
Using the lactose permease of Escherichia coli, a well-characterized membrane protein with 12 trans-membrane domains, we demonstrated that certain paired in-frame deletion constructs complement each other functionally. Although cells expressing the deletion mutants individually are unable to catalyze active lactose accumulation, cells simultaneously expressing specific pairs of deletions catalyze transport up to 60% as do cells expressing wild-type permease. Moreover, complementation clearly does not occur at the level of DNA but probably occurs at the protein level. Remarkably, functional complementation is observed only with pairs of permease molecules containing large deletions and is not observed with missense mutations or point deletions. Although the mechanism of functional complementation is obscure, the findings indicate that certain pairs of permease molecules containing specific internal deletions can interact to form a functional complex in the same way phenomenologically as do independently expressed polypeptides corresponding to different N- and C-terminal portions of the permease.
(1992) Biochemistry. 31, 7, p. 1992-1998 Abstract
The possibility that simple lipophilic cations such as tetraphenylphosphonium (TPP+), tetraphenylarsonium (TPA+), triphenylmethylphosphonium (TPMP+), and diphenyldimethylphosphonium (DDP+) are substrates for the multidrug-resistance transport protein, P-glycoprotein, was tested. Hamster cells transfected with and overexpressing mouse mdr1 or mouse mdr3 exhibit high levels of resistance to TPP+ and TPA+ (20-fold) and somewhat lower levels of resistance to TPMP+ and DDP+ (3-12-fold). Transfected cell clones expressing mdr1 or mdr3 mutants with decreased activity against drugs of the MDR spectrum (e.g., Vinca alkaloids and anthracyclines) also show reduced resistance to lipophilic cations. Studies with radiolabeled TPP+ and TPA+ demonstrate that increased resistance to cytotoxic concentrations of these lipophilic cations is correlated quantitatively with a decrease in intracellular accumulation in mdr1- and mdr3-transfected cells. This decreased intracellular accumulation is shown to be strictly dependent on intact intracellular nucleotide triphosphate pools and is reversed by verapamil, a known competitive inhibitor of P-glycoprotein. Taken together, these results demonstrate that lipophilic cations are a new class of substrates for P-glycoprotein and can be used to study its mechanism of action in homologous and heterologous systems.
ORGANIZATION AND STABILITY OF A POLYTOPIC MEMBRANE-PROTEIN - DELETION ANALYSIS OF THE LACTOSE PERMEASE OF ESCHERICHIA-COLI(1991) Proceedings of the National Academy of Sciences of the United States of America. 88, 16, p. 7271-7275 Abstract
The overall topology of polytopic membrane proteins is thought to result from either the oriented insertion of the N-terminal alpha-helical domain followed by passive insertion of subsequent helices or from the function of independent topogenic determinants dispersed throughout the molecules. By using the lactose permease of Escherichia coli, a well-characterized membrane protein with 12 transmembrane domains and the N and C termini on the cytoplasmic surface of the membrane, we have studied the insertion and stability of in-frame deletion mutants. So long as the first N-terminal and the last four C-terminal putative alpha-helical domains are retained, stable polypeptides are inserted into the membrane, even when an odd number of helical domains is deleted. Moreover, even when an odd number of helices is deleted, the C terminus remains on the cytoplasmic surface of the membrane, as judged by lacY-phoA fusion analysis. In addition, permease molecules devoid of even or odd numbers of putative transmembrane helices retain a specific pathway for downhill lactose translocation. The findings imply that relatively short C-terminal domains of the permease contain topological information sufficient for insertion in the native orientation regardless of the orientation of the N terminus.