The lactose permease of Escherichia coli has 12 transmembrane hydropho
bic domains in probable and-helical conformation connected by hydrophi
lic loops. Previous studies [Consler, T. G., Persson, B., et al. (1993
) Proc. Natl. Acad. Sci. U.S.A. 90, 6934-6938] demonstrate that a pept
ide fragment (the XB domain) containing a factor Xa protease site imme
diately upstream of a biotin acceptor domain can be engineered into th
e permease, thereby allowing rapid purification to a high state of pur
it:y. Here we describe the use of the XB domain to probe topology and
insertion. Cells expressing permease with the XB domain at the N termi
nus, at the C terminus, or in loop 6 or 10 on the cytoplasmic face of
the membrane catalyze active transport, although only the chimeras wit
h the XB domain at the C terminus or in loop 6 are biotinylated. In co
ntrast, chimeras with the XB domain in periplasmic loop 3 or 7 are ina
ctive, but strikingly, both constructs are biotinylated. Furthermore,
the XB domain in all the constructs, particularly in the loop 3 and lo
op 7 chimeras, is accessible from the cytoplasmic face of the membrane
, as evidenced by factor Xa proteolysis or avidin binding studies with
spheroplasts and disrupted membrane preparations. Finally, alkaline p
hosphatase fusions one loop downstream from each periplasmic XB domain
exhibit high phosphatase activity. Thus, the presence of the XB domai
n in a periplasmic loop apparently blocks translocation of a discrete
segment of the permease consisting of the loop and the two adjoining h
elices without altering insertion of the remainder of the protein. The
results provide a strong indication that XB domain insertion cannot b
e used to study the topology of polytopic membrane proteins. On the ot
her hand, the approach yields unique and important information regardi
ng insertion, and it seems likely that certain regions of lactose perm
ease may be inserted as helical hairpins. The findings are discussed i
n the context of other observations indicating that different regions
of the permease may be inserted by different mechanisms.