Aspartate Chemoreceptor Research Articles

Attractant signaling by an aspartate chemoreceptor dimer with a single cytoplasmic domain.

Signal transduction across cell membranes often involves interactions among identical receptor subunits, but the contribution of individual subunits is not well understood. The chemoreceptors of enteric bacteria mediate attractant responses by interrupting a phosphotransfer circuit initiated at receptor complexes with the protein kinase CheA. The aspartate receptor (Tar) is a homodimer, and oligomerized cytoplasmic domains stimulate CheA activity much more than monomers do in vitro. Intragenic complementation was used to show in Escherichia coli that heterodimers containing one full-length and one truncated Tar subunit mediated responses to aspartate in the presence of full-length Tar homodimers that could not bind aspartate. Thus, a Tar dimer containing only one cytoplasmic domain can initiate an attractant (inhibitory) signal, although it may not be able to stimulate kinase activity of CheA.

Purification and characterization of the periplasmic domain of the aspartate chemoreceptor.

In order to facilitate biochemical studies of cell-surface receptors, a plasmid allowing the expression of the periplasmic domain of the aspartate receptor from Salmonella typhimurium as a soluble periplasmic protein has been constructed. This 18-kDa protein is exported to the periplasm, where it may be extracted by mild osmotic lysis. This isolated domain behaves as a normal, soluble protein and has been purified to homogeneity by standard techniques. The purified periplasmic domain binds aspartate with a kD similar to that of the full-length receptor, and the binding occurs with negative cooperativity, i.e. the binding of one molecule of aspartate induces a conformational change that interferes with the binding of the second aspartate. Unlike the full-length receptor, the periplasmic domain undergoes a protein concentration- and aspartate-dependent monomer-dimer equilibrium. At low protein concentrations and in the absence of aspartate, the protein is monomeric. At higher protein concentrations or in the presence of saturating aspartate, the protein is dimeric. Two charge variants of the protein have been identified on native polyacrylamide gels. The more acidic form is blocked to Edman degradation, indicating that modification of the amino terminus of this protein can occur after cleavage of the signal peptide in the periplasm.

Ligand Binding to the Receptor Domain Regulates the Ratio of Kinase to Phosphatase Activities of the Signaling Domain of the Hybrid Escherichia coli Transmembrane Receptor, Taz1

Taz1 is a hybrid receptor, in which the periplasmic receptor domain of Tar, an aspartate chemoreceptor, is fused with the cytoplasmic signaling domain of EnvZ, an osmosensor. Taz1 is able to induce ompC-lacZ expression in response to aspartate added to the medium. We introduced amino acid substitution mutations in the highly conserved region of the signaling domain of Tar near the Tar-EnvZ junction. The same mutations in Tsr, a serine chemoreceptor, are known to lock the flagella rotation in either a clockwise (CW) or in a counter-clockwise (CCW) mode. It was found that a CW-biased mutation in Taz1 resulted in ompC-lacZ expression in the "off mode", or low ompC-lacZ expression in both the absence and presence of aspartate, while CCW-biased mutations caused ompC-lacZ expression in the "on mode", or constitutive expression regardless of aspartate. The OmpR kinase and phospho-OmpR phosphatase activities of the wild-type and mutant Taz proteins were also examined in response to aspartate. The phosphatase activity of the wild-type Taz1 was found to decrease in the presence of aspartate, while the OmpR kinase activity remained constant. This indicated that aspartate binding to the Taz1 receptor domain modulates the ratio of kinase to phosphatase activity of the signaling domain. An increased kinase to phosphatase ratio in the presence of aspartate resulted in higher levels of phospho-OmpR in the cell and therefore induced ompC-lacZ expression. In contrast to the wild-type Taz1 protein, the enzymatic activities of CW as well as CCW mutants did not change in response to aspartate, indicating that mutant Taz proteins are incapable of transducing the signal across the membrane as a result of a locked conformation of the signaling domain in either the on or off mode.

Cloning and characterization of the Salmonella typhimurium-specific chemoreceptor Tcp for taxis to citrate and from phenol.

Salmonella typhimurium shows an attractant response to citrate and a repellent response to phenol, and a chemoreceptor mediating these responses has been identified and named Tcp (taxis to citrate and away from phenol). Tcp is one of the methyl-accepting chemotaxis proteins that have a molecular mass of approximately 60 kDa estimated by SDS/PAGE, and its methylation level is increased by citrate and decreased by phenol. Tcp also mediates an attractant response to metal-citrate complexes. The complete nucleotide sequence of the tcp coding region has been determined. The deduced amino acid sequence of Tcp, consisting of 547-amino acid residues, is homologous with that of the aspartate chemoreceptor of S. typhimurium. Thus, Tcp is another member of the bacterial transmembrane chemoreceptor family. Because citrate is a good carbon source for S. typhimurium but is not a carbon source for the closely related species Escherichia coli and because citrate utilization is used as a key diagnostic character to distinguish these species, it is reasonable to assume that Tcp is specific to S. typhimurium.

Intermolecular complementation between two defective mutant signal-transducing receptors of Escherichia coli.

Taz1 is a hybrid signal-transducing membrane receptor between Tar, an aspartate chemoreceptor, and EnvZ, an osmosensor of Escherichia coli that is able to induce ompC expression by phosphorylating OmpR (a transcriptional activator) in response to aspartate. When the Taz1 His-277, the proposed autophosphorylation site in the cytoplasmic EnvZ domain, was replaced with a valine residue, the mutant Taz1 was unable to induce ompC expression. Similarly, when approximately two-thirds of the EnvZ domain was deleted, Taz1 was nonfunctional. However, when these two defective Taz1 proteins were coexpressed in a cell, ompC was constitutively expressed. Coinciding with this result, two mutant Taz1 molecules were able to intermolecularly complement each other to restore the OmpR kinase activity but not phosphatase activity in vitro. The identical result was also obtained with EnvZ. The present results suggest that the autophosphorylation of Taz1 and EnvZ is an intermolecular phosphorylation reaction, requiring formation of a dimer (or oligomer), and that ligand-dependent ompC expression requires not only kinase but also phosphatase activity.

Intrasubunit signal transduction by the aspartate chemoreceptor.

Receptors that transmit signals across cell membranes are typically composed of multiple subunits. To test whether subunit interactions are required for transmembrane signaling by the bacterial aspartate receptor, dimers were constructed with (i) two full-length subunits, (ii) one full-length subunit and one subunit lacking the cytoplasmic domain, or (iii) one full-length subunit and one subunit lacking both the cytoplasmic and the transmembrane domains. Methylation of the cytoplasmic domain of all three receptor constructs was stimulated by the binding of aspartate. These findings demonstrate that transmembrane signaling does not require interactions between cytoplasmic or transmembrane domains of adjacent subunits and suggest that signaling occurs via conformational changes transduced through a single subunit.

The amino terminus of the aspartate chemoreceptor is formylmethionine.

The amino terminus of the Salmonella typhimurium aspartate receptor has been identified as formylmethionine by mass spectral analysis of the amino-terminal tryptic peptide. Purification and analysis of the blocked amino-terminal peptide was facilitated by the use of a mutant aspartate receptor which has a cysteine residue at position 3. The sequence of this peptide confirms the translational start site predicted from the nucleotide sequence of the tar gene. Furthermore, in vivo labeling experiments reveal that the formyl group is present on chemotaxis receptors produced at wild-type levels in Escherichia coli, indicating that the presence of the formyl group is not a consequence of over-production of the receptor. The stability of the amino-terminal formyl group on the receptor may be a consequence of the membrane localization of the receptor and the dependence of this localization on the membrane transport machinery of the cell.

Transmembrane signaling by a chimera of the Escherichia coli aspartate receptor and the human insulin receptor.

Since many receptors apparently contain only one or two membrane-spanning segments, their transmembrane topology should be similar. This feature suggests that these receptors share common mechanisms of transmembrane signaling. To test the degree of conservation of signaling properties, a chimeric receptor containing the ligand-binding extracellular domain of the Escherichia coli aspartate chemoreceptor and the cytosolic portion of the human insulin receptor was constructed. This chimeric receptor is active as a tyrosine kinase, and aspartate stimulates its activity. Some interesting differences are noted in the target proteins phosphorylated by the chimera compared to the wild-type insulin receptor. These results indicate that features of the signaling mechanisms used by these diverse receptors are conserved, but that interesting changes in the protein properties are caused by differences in the neighboring domains.

Maltose chemoreceptor of Escherichia coli: interaction of maltose-binding protein and the tar signal transducer.

The maltose chemoreceptor in Escherichia coli consists of the periplasmic maltose-binding protein (MBP) and the Tar signal transducer, which is localized in the cytoplasmic membrane. We previously isolated strains containing malE mutations that cause specific defects in the chemotactic function of MBP. Four of these mutations have now been characterized by DNA sequence analysis. Two of them replace threonine at residue 53 of MBP with isoleucine (MBP-TI53), one replaces an aspartate at residue 55 with asparagine (MBP-DN55), and the fourth replaces threonine at residue 345 with isoleucine (MBP-TI345). The chemotactic defects of MBP-TI53 and MBP-DN55, but not of MBP-TI345, are suppressed by mutations in the tar gene. Of the tar mutations, the most effective suppressor (isolated independently three times) replaces Arg-73 of Tar with tryptophan. Two other tar mutations that disrupt the aspartate chemoreceptor function of Tar also suppress the maltose taxis defects associated with MBP-TI53 and MBP-DN55. One of these mutations introduces glutamine at residue 73 of Tar, the other replaces arginine at residue 69 of Tar with cysteine. These results suggest that regions of MBP that include residues 53 to 55 and residue 345 are important for the interaction with Tar. In turn, arginines at residues 69 and 73 of Tar must be involved in the recognition of maltose-bound MBP and/or in the production of the attractant signal generated by Tar in response to maltose-bound MBP.

Gamma-N-methylasparagine in phycobiliproteins. Occurrence, location, and biosynthesis.

The novel post-translationally modified residue gamma-N-methylasparagine, previously detected in the beta subunit of allophycocyanin (Klotz, A. V., Leary, J. A., and Glazer, A. N. (1986) J. Biol. Chem. 261, 15891-15894), has been found in the beta subunits of a variety of other phycobiliproteins. Representatives of C- and R-phycocyanins and B-, C-, and R-phycoerythrins all contain 1 eq of gamma-N-methylasparagine on their beta subunits as judged by the presence of methylamine in acid hydrolysates. Radiotracer experiments show that the methyl group is derived from the S-methyl of methionine, implicating S-adenosylmethionine as an intermediate methyl transfer agent. Isolation of peptides from C-phycocyanins, prepared from cells labeled by L-[methyl-14C]methionine, showed that the gamma-N-methylasparagine residue is at position beta-72, within a highly conserved region in phycobiliproteins. This location corresponds to that reported earlier for the position of gamma-N-methylasparagine in allophycocyanin and R-phycoerythrin. Phycobiliprotein alpha subunits contain insignificant amounts of the adduct. Methylamine is absent from the hydrolysates of the beta subunits or alpha beta monomers of phycobiliproteins from certain organisms. These latter data indicate that the gamma-N-methylasparagine residue is dispensable in some circumstances. The function of this modification remains to be established. gamma-N-methylasparagine was also absent from several other proteins including bovine histones, porcine myelin basic peptide, and the Salmonella typhimurium aspartate chemoreceptor, all known to undergo post-translational methylations.

Linking functional domains of the human insulin receptor with the bacterial aspartate receptor.

A hybrid receptor has been constructed that is composed of the extracellular domain of the human insulin receptor fused to the transmembrane and cytoplasmic domains of the bacterial aspartate chemoreceptor. This hybrid protein can be expressed in rodent (CHO) cells and displays several functional features comparable to wild-type insulin receptor. It is localized to the cell surface, binds insulin with high affinity, forms oligomers, and is recognized by conformation-specific monoclonal antibodies. Although most of the expressed protein accumulates as a 180-kDa proreceptor, some processed 135-kDa receptor can be detected on the cell surface by covalent cross-linking. Expression of the hybrid receptor inhibits the insulin-activated uptake of 2-deoxyglucose by CHO cells. Thus, this hybrid is partially functional and can be processed; however, it is incapable of native transmembrane signaling. The results indicate that the intact domains of different types of receptors can retain some of the native features in a hybrid molecule but specific requirements will need to be satisfied for transmembrane signaling.

Analysis of mutations in the transmembrane region of the aspartate chemoreceptor in Escherichia coli.

Site-specific mutagenesis was used to replace an alanine with a lysine residue and to create a deletion of seven amino acids into the first transmembrane region (TMI region) of the aspartate chemoreceptor in Escherichia coli. The mutations resulted in the loss of aspartate chemotaxis on tryptone motility plates. However, both mutant proteins were able to associate with the membrane and to bind aspartate. They were both refractory to methylation or to modification of the C-terminal region of the protein by the cheB gene product. These results suggested that the integrity of the TMI domain of the protein was required to maintain the function of the cytoplasmic portion of the receptor. The Lys-19 mutant retained the ability to generate a repellent response. Analysis of suppressor mutations of the Lys-19 mutation suggested that formation of an ion pair or specific changes in a 40 amino acid stretch in the cytoplasmic region of the protein (from amino acid 264 to amino acid 303) could suppress the effects of the Lys-19 mutation. The TMI region of the protein may be involved in transmembrane transmission of signals from the periplasmic portion of the cell to the cytoplasmic portion of the Tar protein.

Mutations in tar suppress defects in maltose chemotaxis caused by specific malE mutations.

Maltose-binding protein (MBP), which is encoded by the malE gene, is the maltose chemoreceptor of Escherichia coli, as well as an essential component of the maltose uptake system. Maltose-loaded MBP is thought to initiate a chemotactic response by binding to the tar gene product, the signal transducer Tar, which is also the aspartate chemoreceptor. To study the interaction of MBP with Tar, we selected 14 malE mutants which had specific defects in maltose taxis. Three of these mutants were fully active in maltose transport and produced MBP in normal amounts. The isoelectric points of the MBPs from these three mutants were identical to (malE461 and malE469) or only 0.1 pH unit more basic than (malE454) the isoelectric point of the wild-type protein (pH 5.0). Six of the mutations, including malE454, malE461, and malE469, were mapped in detail; they were located in two regions within malE. We also isolated second-site suppressor mutations in the tar gene that restored maltose taxis in combination with the closely linked malE454 and malE461 mutations but not with the malE469 mutation, which maps in a different part of the gene. This allele-specific suppression confirmed that MBP and Tar interact directly.

Proteolytic fragments identified with domains of the aspartate chemoreceptor.

Two proteolytic fragments generated during the preparation of the aspartate receptor from Salmonella typhimurium have been purified. These fragments are the products of a single cleavage by an endogenous protease after amino acid 259 in the sequence of the intact receptor. Proteolytic fragment 1 (PF1) represents amino acids 1-259 (Mr = 29,000); this unit retains the aspartate-binding function of the intact receptor. The second fragment (PF2) includes residues 260-552 (Mr = 31,000) and has the normal sites of reversible methylation for the receptor. Like the purified intact receptor, this fragment can be methylated in vitro, although at a much slower rate. Circular dichroic measurements suggest that both proteolytic fragments contain substantial alpha-helical structure, approximately 95 and 53% for PF1 and PF2, respectively. No beta-structure could be detected in either fragment. Molecular sieve chromatography in the presence of detergent suggests that PF1 occurs as a stable multimer of an order equivalent to that observed for the detergent-solubilized aspartate receptor, i.e. a tetramer (+/- 1). PF2 is found to have a multimeric form which is sensitive to the removal of detergent. It is proposed that these fragments represent structural and functional domains of the aspartate receptor.

Purification and characterization of the aspartate chemoreceptor.

The chemoreceptor for aspartate in Salmonella typhimurium was purified from an Escherichia coli strain containing a plasmid bearing the receptor's structural gene (tar). The receptor was solubilized from salt-washed membranes with the nonionic detergent octyl-beta-D-glucopyranoside and purified by a combination of ion exchange, molecular sieve and hydroxyapatite-agarose chromatography. The inclusion of glycerol and 1,10-phenanthroline in all buffers used prior to ion exchange chromatography prevented scission of the receptor by an endogenous proteolytic activity. The solubilized receptor was estimated to have a molecular weight of 248,000 from its behavior on Sephacryl S-300, suggesting that the receptor may be organized as a multimer containing 4 +/- 1 identical subunits. Circular dichroic measurements of the purified protein indicate that 78% of its residues are arranged in helical secondary structures.

Chemotaxis Toward Amino Acids in Escherichia coli

Escherichia coli cells are shown to be attracted to the l-amino acids alanine, asparagine, aspartate, cysteine, glutamate, glycine, methionine, serine, and threonine, but not to arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, phenylalanine, tryptophan, tyrosine, or valine. Bacteria grown in a proline-containing medium were, in addition, attracted to proline. Chemotaxis toward amino acids is shown to be mediated by at least two detection systems, the aspartate and serine chemoreceptors. The aspartate chemoreceptor was nonfunctional in the aspartate taxis mutant, which showed virtually no chemotaxis toward aspartate, glutamate, or methionine, and reduced taxis toward alanine, asparagine, cysteine, glycine, and serine. The serine chemoreceptor was nonfunctional in the serine taxis mutant, which was defective in taxis toward alanine, asparagine, cysteine, glycine, and serine, and which showed no chemotaxis toward threonine. Additional data concerning the specificities of the amino acid chemoreceptors with regard to amino acid analogues are also presented. Finally, two essentially nonoxidizable amino acid analogues, alpha-aminoisobutyrate and alpha-methylaspartate, are shown to be attractants for E. coli, demonstrating that extensive metabolism of attractants is not required for amino acid taxis.