Publications

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 ₂₄, 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.

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. In this study, by analyzing genome-wide data on translation rates, we identified a potential compensatory mechanism in E. coli that serves to slow down the translation during membrane protein targeting. The underlying mechanism is likely programmed into the coding sequence, where ShineDalgarnolike elements trigger elongation pauses at strategic positions during the early stages of translation. We provide experimental evidence that slow translation during targeting and 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 the translation speed.

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.

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.

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.

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.

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.

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.

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.

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 structure of NhaA. This review highlights the approaches, major findings and unresolved problems in alkaline pH homeostasis, focusing on the small number of well-characterized alkali-tolerant and extremely alkaliphilic bacteria.

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.

Previous studies have proposed that the N-terminal A domain (∼200 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.

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.

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 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.

In mammalian cells, as well as Escherichia coli, 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. coli ribosomes requires the prokaryotic SRα 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.

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.

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.

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.

Use of β-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 β-lactamase, in gene fusion studies. This enzyme is responsible for chloramphenicol resistance in Escherichia coli. We show that Cat confers substantial antibiotic resistance when fused to cytoplasmic loops of lactose permcase. 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.

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 (Mdrl) 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 Thr²¹⁴-Ala²³²) is located at the periplasmic face of the membrane. Alternatively, we propose that another stretch of amino acid residues (Leu²⁴³ (out) to He²⁶⁰ (in)) traverses the membrane. This assignment is indicated also by the following observations: 1) deletion of a segment containing the originally predicted TM4 (ΔT214-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 inframe between amino acid residues Leu²²⁶ and Ser²²⁷, 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.

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 (AT) or 2-9 (Δ8) 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 (Δ10), and mutants deleted of residues 2-23 (Δ22) or 2-39 (Δ38) 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, Δ70 and Δ22 are inserted into the membrane in a stable form and catalyze active lactose transport in a highly significant manner, whereas Δ38 is hardly detected in the membrane and exhibits no activity. Interestingly, a fusion protein consisting of Δ38 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 α-helix are not mandatory for either membrane insertion or transport activity.

The polytopic membrane protein lac permease harnesses energy from the electrochemical H⁺ gradient to transport β-galactosidases against a concentration gradient. Although high-resolution structural information is still lacking, the permease is thought to possess 12 membrane-spanning α-helical segments. Various experimental strategies, including site-directed mutagenesis, have been employed to probe the function of this membrane protein at the molecular level.