Deciphering the Subunit Stoichiometry and Structural Assembly of Bacterial ABC Transporter

The ATP-binding-cassette (ABC) transporters are protein nanomachines associated with membranes and common to all living cells. ABC transporters utilize the energy of ATP hydrolysis to transport a variety of solutes across the membrane. Wzm (the transmembrane component) and Wzt (the nucleotide binding component) proteins constitute an ABC transporter that translocates the endotoxic polysaccharides through the inner membrane of the pathogenic bacterium Pseudomonas aeruginosa. To gain insights into the subunit stoichiometry and structural assembly of this ABC transporter, we reconstituted the ABC transporter in CHO cell lines. We expressed Wzm and Wzt proteins fused with green fluorescent protein (GFP2) or its variant, yellow fluorescent protein (YFP). The three-dimensional cellular localization of the expressed ABC components were constructed by stacking images of different sections of the cells utilizing a newly developed and spectrally-resolved two-photon microscope. We next used Fluorescence Resonance Energy Transfer (FRET) to obtain a quantitative understanding of the interaction between the ABC components by calculating the apparent FRET efficiencies for single pixels. When Wzm-GFP2 and Wzm-YFP were co-expressed, our FRET analysis indicated that Wzm self-associates within the cell membrane as an oligomer. This correlates with our biochemical characterization of membrane-extracted Wzm. When Wzt-GFP2 and Wzm-YFP were co-expressed, our FRET analysis indicated that Wzm and Wzt interact at the inner membrane surface. This correlates with our in vitro studies that show Wzm and Wzt interact with each other. The distribution of FRET efficiencies were compared when Wzt-GFP2 and Wzm-YFP or Wzt-YFP and Wzm-GFP2 were co-expressed to study the stoichiometry of Wzm and Wzt in the ABC transporter. The combination of FRET analysis and biochemical approaches will lead to a comprehensive investigation of the structural assembly and subunit composition of this ABC transporter and ultimately other transporters.