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.