A Derivative of Lipid A Is Involved in Signal Recognition Particle/SecYEG-dependent and -independent Membrane Integrations

Ken-Ichi Nishiyama,Ayao Ikegami,Michael Moser,Emile Schiltz,Hajime Tokuda,Matthias Müller

doi:10.1074/jbc.m608228200

Ken-Ichi Nishiyama, Ayao Ikegami + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m608228200

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Abstract

A cell-free system was developed that allows the correct integration of single and multispanning membrane proteins of Escherichia coli into proteoliposomes. We found that physiological levels of diacylglycerol were required to prevent spontaneous integration into liposomes even of the polytopic mannitol permease. Using diacylglycerol-containing proteoliposomes, we identified a novel integration-stimulating factor. Integration of mannitol permease was dependent on both the SecYEG translocon and this factor and was mediated by signal recognition particle and signal recognition particle receptor. Integration of M13 procoat, which is independent of both signal recognition particle/signal recognition particle receptor and SecYEG, was also promoted by this factor. Furthermore, the factor stimulated the post-translational translocation of presecretory proteins, suggesting that it also mediates integration of a signal sequence. This factor was found to be a lipid A-derived membrane component possessing a peptide moiety.

Highlights

In contrast to the original idea of a spontaneous process, the integration of those small signal recognition particle (SRP)- and Sec-independent membrane proteins was recently found to depend on the inner membrane YidC [13,14,15], which is homologous to the mitochondrial Oxa1p and the chloroplast Alb3 protein
By using mannitol permease (MtlA), we have reconstituted for the first time the biogenesis of a polytopic membrane protein in a liposome-based system that is not perturbed by spontaneous integration events
Spontaneous Protein Integration into Phospholipids Is Prevented by DAG—When MtlA is synthesized in vitro in the presence of native inverted, inner membrane vesicles (INVs), proteinase K (PK) digestion leaves behind a 30-kDa MPF

Summary

Introduction

In contrast to the original idea of a spontaneous process, the integration of those small SRP- and Sec-independent membrane proteins was recently found to depend on the inner membrane YidC [13,14,15], which is homologous to the mitochondrial Oxa1p and the chloroplast Alb protein. By using MtlA, we have reconstituted for the first time the biogenesis of a polytopic membrane protein in a liposome-based system that is not perturbed by spontaneous integration events. In this way we have identified a minimal integration machinery consisting of SecYEG and a novel integration-stimulating factor that was extracted from the inner membrane of E. coli. This factor, which stimulated the integration of the M13 procoat protein in the absence of any other protein if spontaneous integration was prevented, turned out to be a lipid A-derived compound

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