Topological analysis of the peripheral benzodiazepine receptor in yeast mitochondrial membranes supports a five-transmembrane structure.

The peripheral benzodiazepine receptor, implicated in the transport of cholesterol from the outer to the inner mitochondrial membrane, is predicted by hydropathy analysis to feature five membrane-spanning domains, with the amino terminus within the mitochondrial periplasm and the carboxyl terminus in the external cytoplasm. We have tested these structural predictions directly by immunodetection of c-Myc-tagged peripheral benzodiazepine receptor on intact yeast mitochondria and by specific labeling in yeast membranes of cysteine residues introduced by site-directed mutagenesis. The combined results support the model originally proposed with some minor but important modifications. The theoretical model predicted relatively short alpha-helical domains, only long enough to span a phospholipid monolayer, whereas the results presented here would support a model with extended alpha-helices sufficiently long to span an entire membrane bilayer, with concomitant shorter loop and tail regions.

The Mr 18,000 subunit of the peripheral-type benzodiazepine receptor exhibits both benzodiazepine and isoquinoline carboxamide binding sites in the absence of the voltage-dependent anion channel or of the adenine nucleotide carrier.

The peripheral type benzodiazepine receptor (PBR) binds benzodiazepines such as RO5-4864 and isoquinoline carboxamide derivatives such as PK11195. This receptor includes an Mr 18,000 isoquinoline-binding subunit predominantly located in mitochondrial mem- branes. This protein has been found to copurify with two other mitochondrial proteins, namely the outer membrane voltage-dependent anion channel (VDAC), also known as mitochondrial porin, and the inner membrane adenine nucleotide carrier. In vitro reconstitution experiments suggested that the PBR was a multimeric complex in which the isoquinoline binding site was on the Mr 18,000 subunit, denoted pk18, whereas the benzodiazepine binding site required the association of this subunit with VDAC to be expressed. Untransformed cells of the yeast Saccharomyces cerevisiae are devoid of specific binding sites for isoquinolines and benzodiazepines, whereas yeast cells transformed with a pk18-expressing vector exhibit RO5-4864 and PK11195 binding sites that are pharmacologically identical to those of the PBR. To clarify the role of VDAC and of the adenine nucleotide carrier, if any, in the constitution of the benzodiazepine binding site, yeast host strains were constructed in which the corresponding genes had been knocked out. Mitochondria prepared from pk18-producing cells devoid of either VDAC or adenine nucleotide carrier exhibit both benzodiazepine and isoquinoline carboxamide binding sites with little or no change in the Kd values as compared with the wild-type background. These results rule out the contention that VDAC is indispensable for establishing the benzodiazepine binding site and are in agreement with the hypothesis that the Mr 18,000 subunit carries both the isoquinoline carboxamide and benzodiazepine binding domains.

Isolation and characterization of a priB mutant of Escherichia coli influencing plasmid copy number of delta rop ColE1-type plasmids.

The lethality induced by the overproduction in Escherichia coli of a heterologous protein was used to select bacterial mutants. In one of these, the mutation responsible was mapped to priB. We describe the isolation of this mutant, the sequencing of the mutated gene, and its in vivo effect on plasmid replication.

Combined effects of the signal sequence and the major chaperone proteins on the export of human cytokines in Escherichia coli.

We have studied the export of two human proteins in the course of their production in Escherichia coli. The coding sequences of the granulocyte-macrophage colony-stimulating factor and of interleukin 13 were fused to those of two synthetic signal sequences to direct the human proteins to the bacterial periplasm. We found that the total amount of protein varies with the signal peptide-cytokine combination, as does the fraction of it that is soluble in a periplasmic extract. The possibility that the major chaperone proteins such as SecB and the GroEL-GroES and DnaK-DnaJ pairs are limiting factors for the export was tested by overexpressing one or the other of these chaperones concomitantly with the heterologous protein. The GroEL-GroES chaperone pair had no effect on protein production. Overproduction of SecB or DnaK plus DnaJ resulted in a marked increase of the quantity of human proteins in the periplasmic fraction, but this increase depends on the signal peptide-heterologous protein-chaperone association involved.

High-level production of a human membrane protein in yeast: the peripheral-type benzodiazepine receptor.

The human peripheral-type benzodiazepine receptor (PBR) has been produced in Saccharomyces cerevisiae where it retains its pharmacological properties [Riond et al., Eur. J. Pharmacol. 208 (1991) 307-312]. As the rate of production was low, we analysed the mRNA level, the effect of variation of the 5' sequence and the production in mitochondria. Translation rather than transcription or targeting was found to be the main limiting factor. We were able to produce a chimeric PBR, with an N-terminal extension, to a very high level in the yeast mitochondrial membrane.

Site-directed mutagenesis of the peripheral benzodiazepine receptor: identification of amino acids implicated in the binding site of Ro5-4864.

The peripheral benzodiazepine receptor (PBR) is an 18-kDa protein present in the outer mitochondrial membrane. The human PBR can be labeled with the benzodiazepine Ro5-4864 and with the isoquinoline carboxamide PK11195. The two ligands compete with each other in binding experiments, with previous results suggesting overlapping but not identical binding sites. To define the regions of the receptor interacting with PK11195 and Ro5-4864 and to address the question of the topology of the molecule in the membrane, we generated mutant human PBRs with amino- and carboxyl-terminal deletions and with point mutations in potentially accessible cytoplasmic regions. The mutant genes were expressed in yeast and analyzed in binding experiments using radiolabeled PK11195 and Ro5-4864. The results showed that, whereas deletions in the amino-terminal sequence had marked consequences for the binding affinity of both ligands, the final 13 amino acids at the carboxyl terminus could be deleted with no effect on the binding of either Ro5-4864 or PK11195. The site-directed mutagenesis experiments pinpointed four amino acids as participating in the binding site of Ro5-4864. Three of these, Glu-29, Arg-32, and Lys-39, which are located in the first putative cytoplasmic loop, are conserved in human, bovine, rat, and mouse PBRs. The remaining residue, Val-154, which is found at the interface between the putative fifth transmembrane region and the cytoplasm, is present in the human, rat, and mouse sequences but is replaced by methionine in the bovine sequence. The exchange of Met-154 for valine in the bovine PBR introduced a binding site for Ro5-4864, which is absent in the native PBR. These four amino acids played a minor role, if any, in the binding site of PK11195. We also showed that the histidines previously suggested to be part of the binding site of PK11195 are not directly involved in the interaction of the human receptor with either PK11195 or Ro5-4864.

Molecular basis for the different binding properties of benzodiazepines to human and bovine peripheral-type benzodiazepine receptors.

The 18 kDa peripheral benzodiazepine receptor (PBR) can be labelled by benzodiazepines, such as Ro5-4864, and isoquinoline carboxamides such as PK11195. These two compounds are reversible competitive inhibitors of each other. However, while the binding affinity of Ro5-4864 varies enormously across species, PK11195 always displays high affinity, suggesting that their binding domains are overlapping but not identical. We report here that recombinant human and bovine PBR produced in yeast, a microorganism devoid of endogenous PBR, can be labelled with [3H]PK11195, but only the human receptor can be labelled with [3H]Ro5-4864. Furthermore, we identified, through the binding analysis of human-bovine chimaeric receptors, a region near the C-terminal end of the PBR, with only five non-conserved amino acids between human and bovine sequences, as responsible for the difference in high affinity binding of Ro5-4864 to the two receptors.

Human recombinant interleukin-1 beta isolated from Escherichia coli by simple osmotic shock.

A synthetic gene coding for the C-terminal 153 amino acids of the human interleukin-1 beta (IL-1 beta) was used to produce large quantities of recombinant IL-1 beta in Escherichia coli. The expression of the synthetic gene was under the control of an inducible promoter. The recombinant protein was released from the cells by an osmotic shock. This procedure did not lyse the cells. The IL-1 beta that represented 90% of the total extracted protein was purified to homogeneity by a single chromatographic step. Sequence analysis revealed a heterogeneous N-terminal sequence resulting from the cleavage of the N-terminal methionine in 50% of the molecules and of both the N-terminal methionine and alanine in the other 50%. This recombinant IL-1 beta had a specific activity of 1.3 x 10(8) international units per mg.

Transposable elements for efficient manipulation of a wide range of gram-negative bacteria: promoter probes and vectors for foreign genes.

We describe here the construction and use of a series of modified transposons based on the insertion sequence IS1. Like their parent, omegon-Km [Fellay et al., Gene 76 (1989) 215-226], these elements permit efficient insertional mutagenesis of a variety of Gram-negative bacteria. The presence of a functional pBR322 origin of replication within the transposable element facilitates subsequent cloning of the mutated gene. The omegon-Km system was previously shown to function in Pseudomonas putida, Rhizobium leguminosarum and Paracoccus denitrificans. The results we present here demonstrate that its use can be extended to Xanthomonas campestris, a plant pathogen, and to the microaeroduric Zymomonas mobilis. Derivative transposons carrying unique restriction sites for ScaI, NdeI, XbaI and XhoI have been constructed, allowing the cloning and introduction of foreign genes. We have also constructed two derivatives which can be used to generate operon fusions upon insertion and are thus useful for isolating and characterising indigenous promoters. One carries a promoterless chloramphenicol acetyl-transferase (CAT)-encoding gene (cat) and the second, the entire promoterless Escherichia coli lac operon. We demonstrate the utility of the cat promoter probe in X. campestris to target conditional promoters inducible by high salt or subject to repression by glucose.

Expression vector promoting the synthesis and export of the human growth-hormone-releasing factor in Escherichia coli.

We have studied the synthesis, processing and export of human growth-hormone-releasing factor (hGRF) in Escherichia coli transformed with a plasmid constructed for the expression of hGRF as a hybrid protein. A DNA fragment containing the entire sequence of phosphate-binding protein gene (phoS) is fused to a modified hGRF-coding sequence (phoS-mhGRF). The hybrid protein, PhoS-mhGRF, was recovered in the supernatant fluid after spheroplasting treatment indicating correct export to the periplasmic space. Pulse-chase experiments demonstrated that the hybrid protein was similarly processed as the PhoS precursor.