A. Zelazny et E. Bibi, BIOGENESIS AND TOPOLOGY OF INTEGRAL MEMBRANE-PROTEINS - CHARACTERIZATION OF LACTOSE PERMEASE-CHLORAMPHENICOL ACETYLTRANSFERASE HYBRIDS, Biochemistry, 35(33), 1996, pp. 10872-10878
Use of beta-lactamase in gene fusions to study membrane protein topolo
gy permits exploitation of its biological activity to select for posit
ive (external) hybrids on ampicillin agar plates. When the enzyme is a
ttached to cytoplasmic loops of a membrane protein, it is not secreted
and is therefore unable to confer ampicillin resistance. In this stud
y, we examine the use of the cytoplasmic enzyme chloramphenicol acetyl
transferase (Cat) as a complement to the use of periplasmic beta-lacta
mase, in gene fusion studies. This enzyme is responsible for chloramph
enicol resistance in Escherichia coil. We show that Cat confers substa
ntial antibiotic resistance when fused to cytoplasmic loops of lactose
permease. As expected, periplasmically exposed Cat is enzymatically a
ctive in vitro but unable to confer significant chloramphenicol resist
ance, presumably because of the absence of acetylcoenzyme A in the per
iplasm. Therefore, Cat may serve as a topogenic sensor in gene fusion
studies. The new Cat fusion approach is discussed with regard to its p
otential use for selecting E. coli mutants which are defective in the
assembly of membrane proteins.