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 topology permits exploitation of its biological activity to select for positive (external) hybrids on ampicillin agar plates. When the enzyme is attached to cytoplasmic loops of a membrane protein, it is not secreted and is therefore unable to confer ampicillin resistance. In this study, we examine the use of the cytoplasmic enzyme chloramphenicol acetyltransferase (Cat) as a complement to the use of periplasmic beta-lactamase, in gene fusion studies. This enzyme is responsible for chloramphenicol resistance in Escherichia coli. We show that Cat confers substantial antibiotic resistance when fused to cytoplasmic loops of lactose permease. As expected, periplasmically exposed Cat is enzymatically active in vitro but unable to confer significant chloramphenicol resistance, presumably because of the absence of acetylcoenzyme A in the periplasm. Therefore, Cat may serve as a topogenic sensor in gene fusion studies. The new Cat fusion approach is discussed with regard to its potential use for selecting E. coli mutants which are defective in the assembly of membrane proteins.
Zelazny
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A
Department of Biochemistry, Weizmann Institute of Science, Rehovot, Israel.
Bibi
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E
D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D027981:Symporters; C021499:lactose permease; D015500:Chloramphenicol O-Acetyltransferase