Global Topology Analysis of the <i>Escherichia coli</i> Inner Membrane Proteome

TLDR

The protein complement of cellular membranes is notoriously resistant to standard proteomic analysis, leaving membrane proteomes ill‑defined. This study reports a global topology analysis of the Escherichia coli inner membrane proteome. By C‑terminal tagging with alkaline phosphatase and green fluorescent protein, the authors determined the periplasmic or cytoplasmic location of the C termini for 601 inner membrane proteins. Constraining a topology prediction algorithm with this data yielded high‑quality topology models for the 601 proteins and showed that 397 GFP fusions can be overexpressed in sufficient quantities for biochemical and structural work, providing a firm foundation for future functional studies.

Abstract

The protein complement of cellular membranes is notoriously resistant to standard proteomic analysis and structural studies. As a result, membrane proteomes remain ill-defined. Here, we report a global topology analysis of the Escherichia coli inner membrane proteome. Using C-terminal tagging with the alkaline phosphatase and green fluorescent protein, we established the periplasmic or cytoplasmic locations of the C termini for 601 inner membrane proteins. By constraining a topology prediction algorithm with this data, we derived high-quality topology models for the 601 proteins, providing a firm foundation for future functional studies of this and other membrane proteomes. We also estimated the overexpression potential for 397 green fluorescent protein fusions; the results suggest that a large fraction of all inner membrane proteins can be produced in sufficient quantities for biochemical and structural work.