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, 94 (10), 5101-6

A Mammalian Mitochondrial Drug Receptor Functions as a Bacterial "Oxygen" Sensor

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A Mammalian Mitochondrial Drug Receptor Functions as a Bacterial "Oxygen" Sensor

A A Yeliseev et al. Proc Natl Acad Sci U S A.

Abstract

The rat mitochondrial outer membrane-localized benzodiazepine receptor (MBR) was expressed in wild-type and TspO- (tryptophan-rich sensory protein) strains of the facultative photoheterotroph, Rhodobacter sphaeroides 2.4.1, and was shown to retain its structure within the bacterial outer membrane as assayed by its binding properties with a variety of MBR ligands. Functionally, it was able to substitute for TspO by negatively regulating the expression of photosynthesis genes in response to oxygen. This effect was reversed pharmacologically with the MBR ligand PK11195. These results suggest a close evolutionary and functional relationship between the bacterial TspO and the MBR. This relationship provides further support for the origin of the mammalian mitochondrion from a "photosynthetic" precursor. Finally, these findings provide novel insights into the physiological role that has been obscure for the MBR in situ.

Figures

Figure 1
Figure 1
Alignment of R. sphaeroides 2.4.1 TspO (RsTspO) (2), Rhodobacter capsulatus CrtK (RcCrtK) (1), pk18 from rat (Rpk18) (3), mouse (Mpk18) (4), human (Hpk18) (5), bovine (Bpk18) (6), and hypothetical protein (ScTspO) from Synechocystis sp. strain PCC6803 (7). The alignments were generated by the seqvu computer program and by sight. Residues that are identical in five or more sequences are framed; residues conservatively changed relative to the RsTspO are shaded; dashes indicate gaps to maximize alignments. Numbers indicate the amino acid positions of the aligned sequences.
Figure 2
Figure 2
Western blot analysis of rat pk18 expressed in R. sphaeroides. The proteins (8.5 μg of protein per lane) were separated by SDS/Tricine electrophoresis, electroblotted onto nitrocellulose membrane, and probed with antibodies raised against rat pk18. Lane 1, urea-treated rat adrenal mitochondria; lanes 2–5, outer membrane preparations of TSPO1 (pAY13), 2.4.1(pAY13), TSPO1 (pRK415), and 2.4.1 (pRK415), respectively.
Figure 3
Figure 3
Relative growth rates of R. sphaeroides strains under photosynthetic, 10 W/m2 (A), and anaerobic dark/dimethyl sulfoxide (B) conditions. (○), R. sphaeroides 2.4.1; (•), 2.4.1 (pAY13); (□), TSPO1; (▪), TSPO1 (pAY13). Growth conditions were as described (1).
Figure 4
Figure 4
Analysis of the transcriptional effect of pk18 expressed in R. sphaeroides. Cells were grown to an OD660 of 0.2–0.3 and collected by centrifugation. Aerobic and semiaerobic growth was maintained by sparging with a gas mixture of 20% O2/77% N2/3% CO2 or 2% O2/95% N2/3% CO2, respectively. Photosynthetic cells were grown in front of the light at 10 W/m2 (for crtI::lacZ assay) or 100 W/m2 (for puc::lacZ assay) by sparging with a gas mixture of 97% N2/3% CO2, and β-Galactosidase activity was determined as described. Data represent the mean value ± SD of three independent experiments.
Figure 5
Figure 5
Scatchard analysis of [3H]PK11195 binding and [3H]PK14105 photolabeling in TSPO1 (pAY13) membranes. A representative Scatchard plot is shown where specific binding of [3H]PK11195 was calculated as total binding (in the absence of competitor) minus nonspecific binding (determined in the presence of 10 μM PK11195). Radioligand concentrations were in the range of 0.8–185 nM; however, the points shown here extend only to 15 nM to highlight the contribution from the high affinity sites (Kd = 4 nM) detected with expression of rat pk18, as represented by the solid line. Each point represents the average of triplicate determinations at each concentration. The dashed line indicates low affinity, high density sites that are not dependent upon rat pk18 expression. Inset shows autoradiography of membrane proteins photolabeled with 100 nM [3H]PK14105 in the absence (T) or presence (N) of 10 μM PK11195. The arrow points to the specifically photolabeled 18-kDa protein.
Figure 6
Figure 6
Effect of PK11195 on puc::lacZ expression. TSPO1 (filled columns) and TSPO1 (pAY13) (hatched columns) were grown in the presence (+) or absence (−) of 10 μM PK11195. Growth conditions and β-Galactosidase assay are the same as in Fig. 4. Data are the mean value ± SD of at least three independent assays.

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