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 ␣-helical domains, only long enough to span a phospholipid monolayer, whereas the results presented here would support a model with extended ␣-helices sufficiently long to span an entire membrane bilayer, with concomitant shorter loop and tail regions.The peripheral (or mitochondrial) benzodiazepine receptor (PBR) 1 possesses a benzodiazepine binding site that is clearly distinct from the modulator site of the neurotransmitter ␥-aminobutyric acid receptor. PBR is present in most, if not all, tissues and is particularly abundant in the outer membrane of mitochondria. PBR has been suggested to be required for the transport of cholesterol from the outer to the inner mitochondrial membrane where steroid biosynthesis takes place (1). The receptor also appears to play a key role in modulating mitochondrial electrophysiology, which suggests its implication in the side effects of benzodiazepine pharmacology (for recent review, see Ref. 2). An outer membrane sensory protein of the proteobacterium Rhodobacter sphaeroides has recently been shown (3) to have a close structural and functional relationship with the PBR, supporting the hypothesis that mammalian mitochondria are of photosynthetic bacterial origin. The identification of an 18-kDa protein in human tissues (4) followed by the isolation of the corresponding cDNA (5) allowed us to produce recombinant human PBR in Saccharyomyces cerevisiae (6, 7), an organism normally devoid of binding sites for PBR ligands, thus opening up new avenues for the study of PBR structure-activity relationships.Hydrophobicity analysis of the amino acid sequences of the rat (8) (rPBR), murine (9) (mPBR), bovine (10) (bPBR), and human (5) (hPBR), together with the positive inside rule (11) led to a two-dimensional membrane topological model comprising an intramitochondrial short amino-terminal region and five putative amphipathic ␣-helices linked by hydrophilic loops leading to an extramitochondrial carboxyl-terminal tail (12). A similar pentahelix topology has been found at 2.2-Å resolution for a crystallized apolipoprotein (13) and from gene fusion studies of the cytochrome c terminal oxidase complex of Escherichia coli (14). A three-dimensional model for PBR was proposed and studied using molecular dynamics simulations (12). It was concluded from this model that t...

The 18 kDa peripheral benzodiazepine receptor (PBR) can be labelled by benzodiazepines, such as Ro54864, 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 ['H]PKll195, but only the human receptor can be labelled with [3H]Ro54864. 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.

The peripheral type benzodiazepine receptor (PBR) binds benzodiazepines such as RO5-4864 and isoquinoline carboxamide derivatives such as PK11195. This receptor includes an M r 18,000 isoquinoline-binding subunit predominantly located in mitochondrial membranes. 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 M r 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 K d 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 M r 18,000 subunit carries both the isoquinoline carboxamide and benzodiazepine binding domains.

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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.The product of priB, initially called the n protein, was identified biochemically as one of the proteins necessary for the assembly on the DNA of the primosomal protein complex (1,14,16,19). The proposed role of this element in DNA replication is to eventually provide an entry point for the DNA polymerase III holoenzyme. The assembly of the primosome requires six proteins, PriA, PriB, PriC, DnaB, DnaC, and DnaT, the functions of which have mostly been explored by in vitro experiments (2, 13, 16). PriA initiates primosome assembly by binding to a specific site on the DNA, the pas sequence (2,16,20). The ensuing structure is recognized by proteins PriB and PriC, to generate the preprimosome complex. However, no direct evidence for an in vivo role of PriB in replication had been obtained until now, probably because no priB mutant had yet been described. The results described below on the effect on plasmid copy number of a priB mutant that we have isolated suggests that PriB may indeed be involved in replication of ColE1-related plasmids in vivo.Selection of hGM-CSF-tolerant mutants. In plasmid pEMR727 (3, 4), the gene coding for the human granulocytemacrophage colony-stimulating factor (hGM-CSF) lies downstream of the tac promoter, so that in strain JS219 (6), its synthesis is efficiently repressed by the LacI protein encoded by both the plasmid and the chromosome. Addition of isopropyl-␤-D-thiogalactopyranoside (IPTG) inactivates the repressor to allow transcription to proceed from the tac promoter. For the bacterium, the result of inducing hGM-CSF expression is severe growth inhibition (IPTG s phenotype): the pEMR727-containing strain forms colonies on medium supplemented with 1 mM IPTG at a frequency of only 3.5 ϫ 10 Ϫ6 (IPTG r phenotype). Independent cultures of strain JS219 were treated with nitrosoguanidine (17), transformed with plasmid pEMR727, and plated on Luria-Bertani (LB) medium (17) supplemented with 1 mM IPTG and ampicillin. Of 40 IPTG r clones, only 1 displayed an increase, by a factor of 2, in the level of recombinant protein recovered in the soluble fraction of the periplasm (data not shown). We focused on the physiological and genetical characterization of this mutant, which turned out to be affected primarily in the regulation of plasmid copy number.Genetic mapping to the priB gene of the mutation of strain OFB3085. The original mutant strain was first cured of plasmid pEMR727, to give strain OFB3085. Insertions of mini-Tn10 genetically linked to the mutation in OFB3085 were then selected (11, 17). Two, Tet r -7 and Tet r -26, were retained because bacteriophage P1 cotransduction data suggested they were derived from independent insertions events. To determine more precisely the position...