Membrane topology of penicillin‐binding protein 3 of Escherichia coli

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Abstract
The .beta.-lactamase fusion vector, pJBS633, has been used to analyse the organizaton of penicillin-binding protein 3 (PBP3) in the cytoplasmic membrane of EEscherichia coli. The fusion junctions in 84 in-frame fusions of the coding region of mature TEM .beta.-lactamase to random positions within the PBP3 gene were determined. Fusions of .beta.-lactamase to 61 different positions in PBP3 were obtained. Fusions to positions within the first 31 residues of PBP3 resulted in enzymatically active fusion proteins which could not protect single cells of E. coli from killing by ampicillin, indicating that the .beta.-lactamase moieties of these fusion proteins were not translocated to the periplasm. However, all fusions that contained .gtoreq. 36 residues of PBP3 provided single cells of E. coli with substantial levels of resistance to ampicillin, indicating that the .beta.-lactamase moieties of these fusion poteins were translocated to the periplasm. PBP3 therefore appeared to have a simple membrane topology with residues 36 to the carboxy-terminus exposed on the periplasmic side of the cytoplasmic membrane. This topology was confirmed by showing that PBP3 was protected from proteolytic digestion at the cytoplasmic side of the inner membrane but was completely digested by proteolytic attack from the periplasmic side. PBP3 was only inserted in the cytoplasmic membrane at its amino terminus since replacement of its putative lipoprotein signal peptide with a normal signal peptide resulted in a water-soluble, periplasmic form of the enzyme. The periplasmic form of PBP3 retained its penicillin-binding activity and appeared to be truly water-soluble since it fractionated, in the absence of detergents, with the expected molecular weight in Sephadex G-100 was not retarded by hydrophobic interaction chromatography on Phenyl-Superose.