The Escherichia coli Peripheral Inner Membrane Proteome

Malvina Papanastasiou,Georgia Orfanoudaki,Marina Koukaki,Nikos Kountourakis,Marios Frantzeskos Sardis,Michalis Aivaliotis,Spyridoula Karamanou,Anastassios Economou

doi:10.1074/mcp.m112.024711

Abstract

Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ∼19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ∼25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with biological membrane surfaces that we are only beginning to decipher.

Highlights

From the ‡Institute of Molecular Biology and BiotechnologyFORTH, Iraklio, Crete 711110, Greece; §Department of Biology-University of Crete, P.O
A total of 138 K-12 proteins were annotated as PIM in EchoLOCATION and Uniprot, but this number was subsequently reduced to 123 after manual validation
Our analysis relied on experimental proteomics data, as current in silico approaches cannot predict the cell topology of cytoplasmic proteins that associate with membranes

Summary

Introduction

From the ‡Institute of Molecular Biology and BiotechnologyFORTH, Iraklio, Crete 711110, Greece; §Department of Biology-University of Crete, P.O. Peripheral inner membrane proteins on the cytoplasmic side constitute a sub-proteome of central importance because of their interaction with the cytoplasmic proteome, the nucleoid, and most of the cell’s metabolism. Thanks to their soluble character and the nature of their interactions with the membrane (mostly electrostatic and moderate hydrophobic interactions [7]), peripheral inner membrane proteins can be extracted using high salt concentrations, extreme pH levels, or chaotropes without disrupting the lipid bilayer (8 –11). Of 1133 predicted integral inner membrane proteins, only half were experimentally identified through proteomics approaches [14]. These figures are constantly being re-evaluated, but most protein identifications appear robust. We demonstrate that a significant, previously unsuspected percentage of the expressed polypeptides constitute the PIM proteome

Methods

Results

Conclusion