FIGURE 6.

Simultaneous membrane interaction favored hetero-oligomerization over formation of translocator homo-oligomers. A, formation of the PopD homo-oligomer is not reversed by PopB addition to membranes. Left bar, PopD^BpyFL was incubated with membranes and the final self-quenched emission determined (PopD only). Center bar, PopD^BpyFL was mixed in 6 m urea buffer with a 10-fold excess of PopB^WT and the mixture was added to membranes. Excess PopB favored the incorporation of PopD into hetero-oligomers, therefore it de-quenches the emission of the BpyFl probe (see also Fig. 3B). Right bar, PopD^BpyFL was incubated with membranes to allow homo-oligomer formation and a 10-fold excess of PopB was subsequently added. No emission de-quenching was observed in this case, indicating that the simultaneous interaction with membranes of both translocators is necessary to form hetero-oligomers. A constant amount of PopD^BpyFL (83% labeled with BpyFL) was present in every sample at a final concentration of 10 nm. The protein to lipid ratio was 1/6000 in all samples. Error bars indicate the range between two measurements. B, PopD size distribution shifts from 6 to 8 subunits per complex when membrane incubation is done in the presence of PopB. PopD^BpyFL was incubated with membranes in the presence of 10-fold excess PopB whereas maintaining a constant protein:lipid ratio (black bars). Size distributions were obtained using single-molecule photobleaching and compared with the distribution obtained when PopD^BpyFL was incubated alone with membranes (Fig. 4C, reproduced in gray bars). Lines indicate Gaussian fits to the histogram data. Oligomers are represented as coded in Fig. 3.