A Novel Labeling Approach Supports the Five-transmembrane Model of Subunit a of the Escherichia coli ATP Synthase

Julie C Long,Steven B Vik,Takaaki Wada,Di Zhang

doi:10.1074/jbc.274.24.17353

Julie C Long, Steven B Vik + Show 2 more

Open Access

https://doi.org/10.1074/jbc.274.24.17353

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Abstract

Cysteine mutagenesis and surface labeling has been used to define more precisely the transmembrane spans of subunit a of the Escherichia coli ATP synthase. Regions of subunit a that are exposed to the periplasmic space have been identified by a new procedure, in which cells are incubated with polymyxin B nonapeptide (PMBN), an antibiotic derivative that partially permeabilizes the outer membrane of E. coli, along with a sulfhydryl reagent, 3-(N-maleimidylpropionyl) biocytin (MPB). This procedure permits reaction of sulfhydryl groups in the periplasmic space with MPB, but residues in the cytoplasm are not labeled. Using this procedure, residues 8, 27, 37, 127, 131, 230, 231, and 232 were labeled and so are thought to be exposed in the periplasm. Using inside-out membrane vesicles, residues near the end of transmembrane spans 1, 64, 67, 68, 69, and 70 and residues near the end of transmembrane 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. These results provide a more detailed view of the transmembrane spans of subunit a and also provide a simple and reliable technique for detection of periplasmic regions of inner membrane proteins in E. coli.

Highlights

The mechanism by which an electrochemical proton gradient across the membrane drives ATP synthesis is thought to involve a rotary mechanism
Periplasmic exposure of the cysteine residues was detected by labeling with a new procedure in which cells were partially permeabilized by incubating with 50 ␮M polymyxin B nonapeptide in the presence of the sulfhydryl reagent MPB
Cytoplasmic residues were identified as those that can be labeled in inside-out membrane vesicles but not in polymyxin B nonapeptide (PMBN)-permeabilized cells

Summary

Introduction

The mechanism by which an electrochemical proton gradient across the membrane drives ATP synthesis is thought to involve a rotary mechanism. Studies using the labeling of cysteine substitutions [18, 19] concluded five-transmembrane spans, with a periplasmic N terminus.

Results

Conclusion