The murG gene ofEscherichia coli encodes the N-acetylglucosaminyltransferase responsible for the final step in the formation of the lipid-linked disaccharide-pentapeptide subunit of peptidoglycan. Using trypsin to probe maxicell spheroplasts, we show that this enzyme is peripherally associated with the inner face of the cytoplasmic membrane. Therefore, the peptidoglycan subunit is completely assembled before it traverses the cytoplasmic membrane.The final steps of Escherichia coli peptidoglycan subunit assembly occur on a lipid carrier molecule located in the cytoplasmic membrane (8, 18). First, phospho-MurNAc-might also be used in this reaction (18). Next, GlcNAc (Nacetylglucosamine) is transferred from UDP to the lipid-linked MurNAc-pentapeptide (or MurNAc-tripeptide). The MurNAc-(pentapeptide/tripeptide)-GlcNAc subunit thus formed is then polymerized into peptidoglycan in the periplasm (2). These lipid-linked subunit assembly reactions were previously shown to be associated both with the inner membrane and with a membrane fraction which probably corresponds to zones of adhesion between the inner and outer membranes (7).Although the biochemical details of these reactions have been known for some time, the precise point at which cytoplasmic synthesis becomes periplasmic synthesis was not known. Recently, the GlcNAc transferase was identified as the product of the murG gene of E. coli (6,12). In that work (12), Mengin-Lecreulx et al. also showed that the MurG protein was membrane associated. Here we show that the GlcNAc transferase is associated with the cytoplasmic face of the inner membrane and that therefore the entire peptidoglycan subunit is assembled before being transported across the cytoplasmic membrane.To assess the topology of the GlcNAc transferase with respect to the cytoplasmic membrane of E. coli, we used trypsin to probe maxicell spheroplasts. Sodium dodecyl sulfate (SDS) sample buffer was added to each sample, the samples were boiled and run on SDS-10% polyacrylamide gels. The gels were treated with Amplify (Amersham), and exposure was carried out for 1 to 2 days at -70'C. Proteins were identified by comparison with strains not containing the indicated plasmids and by comparison with 14C-methylated molecular weight markers (Amersham) (data not shown).Using this approach, we found that the GlcNAc transferase was insensitive to trypsin unless trypsin gained access to the cytoplasm. Access could be gained either by permeabilizing the membrane with Triton X-100 or by the use of very high concentrations (.1 mg/ml) of trypsin in the absence of the detergent. Preliminary experiments showed that permeabilization of the cytoplasmic membrane by the addition of 1% Triton X-100 to maxicell spheroplasts resulted in increased sensitivity of the MurG protein to tryptic digestion (data not shown).To demonstrate that the increased sensitivity of the GlcNAc transferase to trypsin in the presence of Triton X-100 was a consequence of the disruption of the membrane rather than denaturation of the transferase, we per...