BIOGENESIS AND TOPOLOGY OF INTEGRAL MEMBRANE-PROTEINS - CHARACTERIZATION OF LACTOSE PERMEASE-CHLORAMPHENICOL ACETYLTRANSFERASE HYBRIDS

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.