A novel labeling approach supports the five-transmembrane model of subunita of the Escherichia coli ATP synthase

Cysteine mutagenesis and surface labeling has been used to define more prec isely the transmembrane spans of subunit a of the Escherichia coli ATP synt hase, Regions of subunit a that are exposed to the periplasmic space have b een identified by a new procedure, in which cells are incubated with polymy xin B nonapeptide (PMBN), an antibiotic derivative that partially permeabil izes the outer membrane of E. coli, along with a sulfhydryl reagent, 3-(N-m aleimidylpropionyl) biocytin (MPB). This procedure permits reaction of sulf hydryl groups in the periplasmic space with MPB, but residues in the cytopl asm are not labeled, Using this procedure, residues 8, 27, 37, 127, 131, 23 0, 231, and 232 were labeled and so are thought to be exposed in the peripl asm, Using inside-out membrane vesicles, residues near the end of transmemb rane spans 1, 64, 67, 68, 69, and 70 and residues near the end of transmemb rane spans 5, 260, 263, and 265 were labeled. Residues 62 and 257 were not labeled. None of these residues were labeled in PMBN-permeabilized cells. T hese results provide a more detailed view of the transmembrane spans of sub unit a and also provide a simple and reliable technique for detection of pe riplasmic regions of inner membrane proteins in E, coli,