A new method for the measurement of protein turnover.

A new technique for the determination of rate constants of protein degradation is described. By using the method, half-lives of total soluble protein of Lemna minor during growth on full culture medium and distilled water were measured. The method involves incubating Lemna on a growth medium containing 3H2O. After a short exposure (20 min) to 3H-labelled culture medium, 3H was found in soluble amino acids, especially aspartate, glutamate, glutamine and alanine. After transfer to a 3H-free medium for 30 min, 80% of the 3H originally present in soluble amino acids was lost. These results suggest that 3H enters and leaves amino acids at the alpha-carbon atom, a conclusion supported by the observed labelling of glutamates. The exchange of H and 3H on the alpha-carbon atom is catalysed by transaminases and the speed of this exchange ensures that when the 3H2O is removed, the 3H in free amino acids is rapidly lost, thereby eliminating problems connected with metabolic pools and recycling. After an exposure of 20 min to 3H-labelled medium all protein amino acids, except for arginine, were found to be radioactive. The loss of radioactivity from protein amino acids was used to measure protein degradation.     

Compartmentation of free amino acids for protein synthesis in rat liver

The concept that a general intracellular pool serves as the sole precursor of amino acids for protein biosynthesis has been vigorously debated in recent years. To help resolve this controversy, we followed the distribution of intraperitoneally administered [(3)H]valine in the tRNA and the extracellular and intracellular compartments of rat liver. The specific radioactivity of the valine released from isolated tRNA was 2-3 times higher than that of intracellular valine, suggesting that the intracellular pool cannot be the sole precursor of amino acids used for charging tRNA. In addition, the specific radioactivity of the tRNA was only half that of the extracellular valine. Therefore it is unlikely that the valyl-tRNA is charged exclusively with amino acids derived from the extracellular pool. A model is proposed which stipulates that both extracellular and intracellular amino acids contribute to a restricted compartment that funnels amino acids towards protein biosynthesis.     

A comparison of methods for the measurement of protein turnover in vivo.

Steady-state rates of turnover of two single proteins were measured in vivo by two independent methods. The fractional rate of synthesis of liver ornithine aminotransferase, measured by a continuous infusion of L-[2,6-3H]tyrosine, was 0.42 day-1, whereas in the same animals the fractional rate of degradation measured by loss of radioactivity from amino acids labelled via [14C]bicarbonate was 0.40 day-1. The agreement between methods confirms the reliability of each method for the study of hepatic protein turnover. In contrast, [14C]bicarbonate-labelled amino acids are extensively reutilized in muscle, and are therefore unsuitable for measuring rates of muscle protein breakdown.     

Stimulation of protein synthesis by both insulin and amino acids is unique to skeletal muscle in neonatal pigs

In neonatal pigs, the feeding-induced stimulation of protein synthesis in skeletal muscle, but not liver, can be reproduced by insulin infusion when essential amino acids and glucose are maintained at fasting levels. In the present study, 7- and 26-day-old pigs were studied during 1) fasting, 2) hyperinsulinemic-euglycemic-euaminoacidemic clamps, 3) euinsulinemic-euglycemic-hyperaminoacidemic clamps, and 4) hyperinsulinemic-euglycemic-hyperaminoacidemic clamps. Amino acids were clamped using a new amino acid mixture enriched in nonessential amino acids. Tissue protein synthesis was measured using a flooding dose of L-[4-(3)H]phenylalanine. In 7-day-old pigs, insulin infusion alone increased protein synthesis in various skeletal muscles (from +35 to +64%), with equivalent contribution of myofibrillar and sarcoplasmic proteins, as well as cardiac muscle (+50%), skin (+34%), and spleen (+26%). Amino acid infusion alone increased protein synthesis in skeletal muscles (from +28 to +50%), also with equivalent contribution of myofibrillar and sarcoplasmic proteins, as well as liver (+27%), pancreas (+28%), and kidney (+10%). An elevation of both insulin and amino acids did not have an additive effect. Similar qualitative results were obtained in 26-day-old pigs, but the magnitude of the stimulation of protein synthesis by insulin and/or amino acids was lower. The results suggest that, in the neonate, the stimulation of protein synthesis by feeding is mediated by either amino acids or insulin in most tissues; however, the feeding-induced stimulation of protein synthesis in skeletal muscle is uniquely regulated by both insulin and amino acids.     

The rate of protein degradation in developing brain. Methodological considerations.

Recently we reported that the rate of protein breakdown decreases during development. Breakdown rates were calculated from the rates of protein synthesis and the changes in brain protein content with age. A different study, measuring breakdown by monitoring the loss of label from brain protein after an H14CO3- pulse, came to the opposite conclusion: that the rate of breakdown is low in immature brain and increases during development. We have now investigated some of the factors (the distribution of label in protein and the potential for recycling) that might introduce errors into these measurements. The specific radioactivities of both protein-bound and free amino acids were determined in the brains of young rats several days after an intraperitoneal pulse of H14CO3-. For a number of amino acids the specific radioactivity of the free amino acid is high compared with that of the protein-bound amino acid, and therefore recycling could result in an underestimate of the degradation rate. Because glutamic acid had a relatively low specific-radioactivity ratio, [1-14C]glutamic acid was used in a pulse-labelling experiment to measure degradation. The rate so obtained, 0.6% . h-1, is twice the rate found with H14CO3- labelling (based on total protein-bound radioactivity). Insofar as recycling is a possible complication, 0.6% . h-1 may be a minimum value. Although somewhat higher degradation rates are found after labelling with an intracranial pulse, which was considered as a possible route to limit recycling, there are difficulties in interpreting these data.     

A novel UbcH10-binding protein facilitates the ubiquitinylation of cyclin B in vitro

UbcH10 is known to act as a ubiquitin-conjugating enzyme (E2) for anaphase-promoting complex/cyclosome. Since some E2s support different ubiquitin ligases (E3), it is possible that UbcH10 interacts with other proteins. We cloned a novel protein named H10BH by using a yeast two-hybrid screening method with UbcH10 as bait. The carboxyl terminus of H10BH showed a weak homology to the HECT (homologous to E6-AP carboxyl terminus) domain, which is conserved in one of the families of E3. H10BH bound UbcH10, and the amino acid sequence between 235 and 257 was necessary for this binding. H10BH showed a self-ubiquitinylation activity in a HECT-like sequence-dependent manner. The carboxyl terminal half (amino acids 188-389) showed stronger activity than the full-length H10BH. Furthermore, the carboxyl terminal half of H10BH was able to bind cyclin B and ubiquitinylate cyclin B in vitro. These results suggest that H10BH functions as an E3 using UbcH10 for its E2.     

Single column analysis of amino acids in protein hydrolysates utilizing the fluorescamine reaction

A system is described for the separation of the amino acids commonly found in protein hydrolysates at the picomole level using a single ion exchange column and for their quantitation by the fluorescamine (4-phenylspiro[furan-2 (3H),1′-phthalan]-3,3′-dione) reaction. Three sodium citrate buffers were required for the separation of the amino acids with an analysis time of approximately 3 hr. The amino acids in 1 μg of hydrolyzed bovine serum albumin were separated using a single ion exchange column and were detected in the effluent from the column by the fluorescamine assay. The results were compared with those obtained using a commercial amino acid analyzer and 150 μg of hydrolyzed bovine serum albumin. The chromatogram produced by the more sensitive analyzer utilizing the fluorescamine reaction to detect the amino acids compared favorably with the chromatogram produced by the commercial analyzer utilizing the ninhydrin reaction with the exception that the proline peak was missing. Proline and hydroxyproline fail to yield fluorescence on reaction with fluorescamine unless converted from imines to primary amines.     

A statistical analysis of random mutagenesis methods used for directed protein evolution

We have developed a statistical method named MAP (mutagenesis assistant program) to equip protein engineers with a tool to develop promising directed evolution strategies by comparing 19 mutagenesis methods. Instead of conventional transition/transversion bias indicators as benchmarks for comparison, we propose to use three indicators based on the subset of amino acid substitutions generated on the protein level: (1) protein structure indicator; (2) amino acid diversity indicator with a codon diversity coefficient; and (3) chemical diversity indicator. A MAP analysis for a single nucleotide substitution was performed for four genes: (1) heme domain of cytochrome P450 BM-3 from Bacillus megaterium (EC 1.14.14.1); (2) glucose oxidase from Aspergillus niger (EC 1.1.3.4); (3) arylesterase from Pseudomonas fluorescens (EC 3.1.1.2); and (4) alcohol dehydrogenase from Saccharomyces cerevisiae (EC 1.1.1.1). Based on the MAP analysis of these four genes, 19 mutagenesis methods have been evaluated and criteria for an ideal mutagenesis method have been proposed. The statistical analysis showed that existing gene mutagenesis methods are limited and highly biased. An average amino acid substitution per residue of only 3.15-7.4 can be achieved with current random mutagenesis methods. For the four investigated gene sequences, an average fraction of amino acid substitutions of 0.5-7% results in stop codons and 4.5-23.9% in glycine or proline residues. An average fraction of 16.2-44.2% of the amino acid substitutions are preserved, and 45.6% (epPCR method) are chemically different. The diversity remains low even when applying a non-biased method: an average of seven amino acid substitutions per residue, 2.9-4.7% stop codons, 11.1-16% glycine/proline residues, 21-25.8% preserved amino acids, and 55.5% are amino acids with chemically different side-chains. Statistical information for each mutagenesis method can further be used to investigate the mutational spectra in protein regions regarded as important for the property of interest.     

Two nonoverlapping domains on the Norwalk virus open reading frame 3 (ORF3) protein are involved in the formation of the phosphorylated 35K protein and in ORF3-capsid protein interactions

Expression of the Norwalk virus open reading frame 3 (ORF3) in Spodoptera frugiperda (Sf9) cells yields two major forms, the predicted 23,000-molecular-weight (23K) form and a larger 35K form. The 23K form is able to interact with the ORF2 capsid protein and be incorporated into virus-like particles. In this paper, we provide mass spectrometry evidence that both the 23K and 35K forms are composed only of the ORF3 protein. Two-dimensional gel electrophoresis and phosphatase treatment showed that the 35K form results solely from phosphorylation and that the 35K band is composed of several different phosphorylated forms with distinct isoelectric points. Furthermore, we analyzed deletion and point mutants of the ORF3 protein. Mutants that lacked the C-terminal 33 amino acids (ORF3(1-179), ORF3(1-152), and ORF3(1-107)) no longer produced the 35K form. An N-terminal truncation mutant (ORF3(51-212)) and a site-directed mutant (ORF3(T201V)) were capable of producing the larger form, which was converted to the smaller form by treatment with protein phosphatase. These data suggest that the region between amino acids 180 and 212 is phosphorylated, and mass spectrometry showed that amino acids Arg196 to Arg211 are not phosphorylated; thus, phosphorylation of the serine-threonine-rich region from Thr181 to Ser193 must be involved in the generation of the 35K form. Studies of the interaction between the ORF2 protein and full-length and mutated ORF3 proteins showed that the full-length ORF3 protein (ORF3(FL)), ORF3(1-179), ORF3(1-152), and ORF3(51-212) interacted with the ORF2 protein, while an ORF3(1-107) protein did not. These results indicate that the region of the ORF3 protein between amino acids 108 and 152 is responsible for interaction with the ORF2 protein.     

The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H(+)-ATPase.

Accumulating evidence suggests that 14-3-3 proteins are involved in the regulation of plant plasma membrane H(+)-ATPase activity. However, it is not known whether the 14-3-3 protein interacts directly or indirectly with the H(+)-ATPase. In this study, detergent-solubilized plasma membrane H(+)-ATPase isolated from fusicoccin-treated maize shoots was copurified with the 14-3-3 protein (as determined by protein gel blotting), and the H(+)-ATPase was recovered in an activated state. In the absence of fusicoccin treatment, H(+)-ATPase and the 14-3-3 protein were well separated, and the H(+)-ATPase was recovered in a nonactivated form. Trypsin treatment removed the 10-kD C-terminal region from the H(+)-ATPase as well as the 14-3-3 protein. Using the yeast two-hybrid system, we could show a direct interaction between Arabidopsis 14-3-3 GF14-phi and the last 98 C-terminal amino acids of the Arabidopsis AHA2 plasma membrane H(+)-ATPase. We propose that the 14-3-3 protein is a natural ligand of the plasma membrane H(+)-ATPase, regulating proton pumping by displacing the C-terminal autoinhibitory domain of the H(+)-ATPase.     

Determination of protein replacement rates by deuterated water: validation of underlying assumptions

H(2)O administration has recently been proposed as a simple and convenient method to measure protein synthesis rates. (2)H(2)O administration results in deuterium labeling of free amino acids such as alanine, and incorporation into proteins of labeled alanine can then be used to measure protein synthesis rates. We examined first whether during (2)H(2)O administration plasma free alanine enrichment is a correct estimate of the enrichment in the tissue amino acid pools used for protein synthesis. We found that, after (2)H(2)O administration, deuterium labeling in plasma free alanine equilibrated rapidly with body water, and stable enrichment values were obtained within 20 min. Importantly, oral administration of (2)H(2)O induced no difference of labeling between portal and peripheral circulation except for the initial 10 min after a loading dose. The kinetics of free alanine labeling were comparable in various tissues (liver, skeletal muscle, heart) and in plasma with identical plateau values. We show next that increased glycolytic rate or absorption of unlabeled amino acids from ingested meals do not modify alanine labeling. Calculated synthesis rates of mixed proteins were much higher (20- to 70-fold) in plasma and liver than in muscle and heart. Last, comparable replacement rates of apoB100-VLDL were obtained in humans by using the kinetics of incorporation into apoB100 of infused labeled leucine or of alanine labeled by (2)H(2)O administration. All of these results support (2)H(2)O as a safe, reliable, useful, and convenient tracer for studies of protein synthesis, including proteins with slow turnover rate.     

An oral essential amino acid-carbohydrate supplement enhances muscle protein anabolism after resistance exercise.

This study was designed to determine the response of muscle protein to the bolus ingestion of a drink containing essential amino acids and carbohydrate after resistance exercise. Six subjects (3 men, 3 women) randomly consumed a treatment drink (6 g essential amino acids, 35 g sucrose) or a flavored placebo drink 1 h or 3 h after a bout of resistance exercise on two separate occasions. We used a three-compartment model for determination of leg muscle protein kinetics. The model involves the infusion of ring-(2)H(5)-phenylalanine, femoral arterial and venous blood sampling, and muscle biopsies. Phenylalanine net balance and muscle protein synthesis were significantly increased above the predrink and corresponding placebo value (P < 0.05) when the drink was taken 1 or 3 h after exercise but not when the placebo was ingested at 1 or 3 h. The response to the amino acid-carbohydrate drink produced similar anabolic responses at 1 and 3 h. Muscle protein breakdown did not change in response to the drink. We conclude that essential amino acids with carbohydrates stimulate muscle protein anabolism by increasing muscle protein synthesis when ingested 1 or 3 h after resistance exercise.     

The substrate specificity of adenosine 3'':5''-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle.

The known amino acid sequences at the two sites on phosphorylase kinase that are phosphorylated by cyclic AMP-dependent protein kinase were extended. The sequences of 42 amino acids around the phosphorylation site on the alpha-subunit and of 14 amino acids around the phosphorylation site on the beta-subunit were shown to be: alpha-subunit Phe-Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser-Glx-Pro-Asx-Gly-Gly-His-Ser-Leu-Gly-Ala-Asp-Leu-Met-Ser-Pro-Ser-Phe-Leu-Ser-Pro-Gly-Thr-Ser-Val-Phe(Ser,Pro,Gly)His-Thr-Ser-Lys; beta-subunit, Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-VALIle-Tyr-Glu-Pro-Leu-Lys. The sites on histone H2B which are phosphorylated by cyclic AMP-dependent protein kinase in vitro were identified as serine-36 and serine-32. The amino acid sequence in this region is: Lys-Lys-Arg-Lys-Arg-Ser32(P)-Arg-Lys-Glu-Ser36(P)-Tyr-Ser-Val-Tyr-Val- [Iwai, K., Ishikawa, K. & Hayashi, H. (1970) Nature (London) 226, 1056-1058]. Serine-36 was phosphorylated at 50% of the rate at which the beta-subunit of phosphorylase kinase was phosphorylated, and it was phosphorylated 6-7-fold more rapidly than was serine-32. The amino acid sequences when compared with those at the phosphorylation sites of other physiological substrates suggest that the presence of two adjacent basic amino acids on the N-terminal side of the susceptible serine residue may be critical for specific substrate recognition in vivo.     

Structural analysis of human complement protein H: homology with C4b binding protein, beta 2-glycoprotein I, and the Ba fragment of B2

We report here a partial primary structure for human complement protein H. Tryptic peptides comprising 27% of the H molecule were isolated by conventional techniques and were sequenced (333 amino acid residues). Several mixed-sequence oligonucleotide probes were constructed, based on the peptide sequence data, and were used to screen a human liver cDNA library. The largest recombinant plasmid (pH1050), which hybridized with two probes, was further characterized. The cDNA insert of this plasmid contained coding sequence (672 bp) for 224 amino acids of H. The 3' end of this clone had a polyadenylated tail preceded by a polyadenylation recognition site (ATTAAA) and a 3'-untranslated region (229 bp). Four regions of internal homology, each about 60 amino acids in length, were observed in the derived protein sequence from this cDNA clone, and a further seven from the tryptic peptide sequences. The consensus sequence for each of the repetitive units of H was four cysteines, two prolines, three glycines, one tryptophan, and two tyrosines/phenylalanines. Based on the mole percent values for each of these amino acids, it is likely that H is composed of about 20 repetitive units of this nature. Furthermore, the repetitive unit of H shows pronounced homology with the Ba fragment of B, the C4b binding protein, and beta 2-glycoprotein I. Therefore, it seems that at least portions of these proteins have evolved from a common ancestral DNA element.     

Problems associated with the labelling of RNA with radioactive precursors in vivo (author's transl)]

The radioactivity of RNA, DNA and proteins in the liver, muscles and cerebrum of 30-day-old rats after labelling with [3H]uridine, [14C]uridine, [3H]cytidine or [3H]orotic acid was measured. It was found that after administration of [3H]uridine, the proteins were 5 - 10 times more radioactive than the RNA. After administration of [14C]uridine, the proteins were 1 - 2 times more heavily labelled than the RNA. Hydrolysis of the proteins followed by chromatography of the amino acids revealed that the protein labelling was mostly due to [3H]glutamate. In the liver, [3H]orotic acid produced very specific labelling of the RNA. The radioactivity of the proteins is very slight. However, the specific labelling of the RNA in the muscles and cerebrum is not so pronounced with this precursor. [3H]Cytidine is an ideal precursor for RNA. The labelling of protein in all three organs examined is very slight, and furthermore, the specific activity of the RNA is 10 - 20 times higher than after labelling with uridine. We were also able to show that after labelling with radioactive uridine, the method of isolation of RNA by alkaline hydrolysis gives incorrect results, because [3H]amino acids interfere with the measurement of the specific activity of the RNA. The heavy labelling of proteins by [3H]-uridine must also be taken into account in histoautoradiography, because our experiments showed that in liver, the proteins in the cell nucleus are 3 times as radioactive as the nucleic acids. The particulate components of the cytoplasm are even 20 times more radioactive than the nucleic acids.     

Aminoacyl-tRNA enrichment after a flood of labeled phenylalanine: insulin effect on muscle protein synthesis

Muscle protein synthesis in dogs measured by flooding with L-[(2)H(5)]phenylalanine (70 mg/kg) was significantly stimulated by infusion of insulin with amino acids. The stimulation of muscle protein synthesis was similar when calculated from the enrichment of phenylalanyl-tRNA (61 +/- 10%, P < 0.001), plasma phenylalanine (61 +/- 10%, P < 0.001), or tissue fluid phenylalanine (54 +/- 10%, P < 0.001). The time course for changes in enrichment of L-[(2)H(5)]phenylalanine throughout the flooding period was determined for plasma, tissue fluid, and phenylalanyl-tRNA in the basal state and during the infusion of insulin with amino acids. Enrichments of plasma free phenylalanine and phenylalanyl-tRNA were equalized between 20 and 45 min, although the enrichment of phenylalanyl-tRNA was lower at early time points. Rates of muscle protein synthesis obtained with the flooding method and calculated from plasma phenylalanine enrichment were comparable to those calculated from phenylalanyl-tRNA and also to those obtained previously with a continuous infusion of phenylalanine with phenylalanyl-tRNA as precursor. This study confirms that, with a bolus injection of labeled phenylalanine, the enrichment of aminoacyl-tRNA, the true precursor pool for protein synthesis, can be assessed from more readily sampled plasma phenylalanine.     

Carbodiimide-binding protein of H+-translocating ATPase and inhibition of H+ conduction by dicyclohexylcarbodiimide.

H+-Translocating ATPase, which catalyzes ATP synthesis in biomembranes, is composed of a head piece (F1) and a membrane moiety (F0). Using highly-purified F0 from a thermophilic bacterium PS3 (TF0), the following results were obtained. 1. Inhibition by N,N'-dicyclohexylcarbodiimide (DCCD) of H+ conduction through TF0 followed pseudo-first-order kinetics. The second-order rate constant for inhibitor-enzyme interaction was 5 times 10(3) M(-1)-min(-1). 2. H+ conductivity blocked by DCCD was proportional to the amount of DCCD incorporated in the band 8 protein of TF0. When only one-third of the band 8 protein was labeled with DCCD, TF0 hardly transported any H+. 3. By extracting TF0 with chloroform-methanol, the band 8 protein was obtained as a proteolipid. Polyacrylamide gel electrophoresis with dodecyl sulfate and urea showed that the molecular weight was about 6,000. 4. The amino acid composition of band 8 protein indicated that this protein contained an extremely high percentage of hydrophobic amino acids (0.29 in polarity) and was devoid of histidine, tryptophan, cysteine, and lysine. Its minimum molecular weight was 6,500. 5. The role of band 8 protein (DCCD-binding protein) in H+ conduction through TF0 is discussed on the basis of these results.     

Linker mutagenesis of a bacterial fatty acid transport protein. Identification of domains with functional importance

The product of the fadL gene (FadL) of Escherichia coli is a multifunctional integral outer-membrane protein required for the specific binding and transport of exogenous long-chain fatty acids [C12-C18]. FadL also serves as a receptor for the bacteriophage T2. In order to define regions of functional importance within FadL, the fadL gene has been mutagenized by the insertion of single-stranded hexameric linkers into the unique SalI restriction site that lies towards the 3' end of the gene and into four HpaII restriction sites distributed throughout the coding region. The five insertion mutants were classified into three groups based on their specific growth rates (alpha) in minimal media containing the long-chain fatty acid oleate (C18:1) as a sole carbon and energy source: Oleslow, alpha = 0.035-0.045; Ole +/-, alpha = 0.020-0.035; and Ole-, alpha less than or equal to 0.005 (wild-type, alpha = 0.07-0.10). The hexameric insertion at the SalI site (fadL allele termed S1; insertion after amino acid 410) conferred an Oleslow phenotype and resulted in a reduction of long-chain fatty acid transport (36% the wild-type level). This insertion mutant, however, bound oleic acid at wild-type levels and was fully functional as a receptor for the bacteriophage T2. The modified FadL-S1 protein did not have the heat-modifiable property characteristic of wild-type FadL. Insertions in the four HpaII sites (fadL alleles termed H1, H2, H3, and H5; after amino acids 41, 81, 238, and 389, respectively) resulted in all three classes of mutants. The fadL insertion mutant H5 was defective for long-chain fatty acid transport but bound oleic acid at significant levels. Together with the S1 allele, these data suggest that the carboxyl terminus of FadL is crucial for long-chain fatty acid transport. The insertion mutants H1 and H2 were defective for both oleic acid binding and transport suggesting that the amino terminus of FadL is important for long-chain fatty acid binding and transport. The fadL linker mutant H3 was defective in oleic acid binding yet had significant levels of oleic acid transport. These studies delineated for the first time different regions of the fadL gene that encode domains of FadL implicated in the binding and transport of long-chain fatty acids.     

BasT, a membrane-bound transducer protein for amino acid detection in Halobacterium salinarum

Halophilic archaea, such as eubacteria, use methyl-accepting chemotaxis proteins (MCPs) to sense their environment. We show here that BasT is a halobacterial transducer protein (Htp) responsible for chemotaxis towards five attractant amino acids. The C-terminus of the protein exhibits the highly conserved regions that are diagnostic for MCPs: the signalling domain for communication with the histidine kinase and the methylation sites that interact with the methylation/demethylation enzymes for adaptation. Hydropathy analysis predicts an enterobacterial-type transducer protein topology for BasT, with an extracellular putative ligand-binding domain flanked by two transmembrane helices and a cytoplasmic domain. BasT-inactivated mutant cells are missing a membrane protein radiolabelled with L-[methyl-3H]-methionine in wild-type cells, confirming that BasT is methylatable and membrane bound. Behavioural analysis of the basT mutant cells by capillary and chemical-in-plug assays demonstrates complete loss of chemotactic responses towards five (leucine, isoleucine, valine, methionine and cysteine) of the six attractant amino acids for Halobacterium salinarum, whereas they still respond to arginine. The volatile methyl group production assays also corroborate these findings and confirm that BasT signalling induces methyl group turnover. Our data identify BasT as the chemotaxis transducer protein for the branched chain amino acids leucine, isoleucine and valine as well as for methionine and cysteine. Thus, BasT and the arginine sensor Car cover the entire spectrum of chemotactic responses towards attractant amino acids in H. salinarum.