Membrane topology of the Rickettsia prowazekii ATP/ADP translocase revealed by novel dual pho-lac reporters

Here, we report the construction and characterization of dual reporters, co nsisting of both an Escherichia coli alkaline phosphatase (AP) gene and an alpha-fragment of the beta-galactosidase (BG) gene, for studying membrane p rotein topology by the gene fusion approach. Each of the reporters, when fu sed to periplasmic domains of polytopic proteins, produces fusions with hig h AP activity and, when fused to cytoplasmic domains, produces fusions with high BG activity in E. coli strains capable of ct-complementation. The dua l nature of these reporters simplifies interpretation of data obtained with poorly expressed fusions and allows one to evaluate the reliability of top ological data. Deleterious effects resulting from the cell's attempt to exp ort the full-length BG are eliminated in this approach. We describe dual in dicator plates that allow for discrimination between colonies bearing cytop lasmic fusions, periplasmic fusions, and no fusions. We have generated a se t of fusions to the topologically well-studied lactose permease of E. coli and demonstrated that topological information generated by these new report ers is in good agreement with the existing model. We used this new methodol ogy for the determination of membrane topology of the Rickettsia prowazekii ATP/ADP translocase (Tlc). Our results were in agreement with the proposed in silico topological model in which Tie traverses the cytoplasmic membran e of E. coli 12 times with its N and C termini facing the cytoplasm. (C) 19 99 Academic Press.