Faculty, Staff and Student Publications

Publication Date

1-1-2024

Journal

Methods in Molecular Biology

Abstract

The complex double-membrane organization of the envelope in Gram-negative bacteria places unique biosynthetic and topological constraints that can affect translocation of lipids and proteins synthesized on cytoplasm facing leaflet of cytoplasmic (inner) membrane (IM), across IM and between IM and outer membrane (OM). Uniformly oriented inside-out (ISO) vesicles became functional requisite for many biochemical reconstitution functional assays, vectorial proteomics, and vectorial lipidomics. Due to these demands, it is necessary to develop simple and reliable approaches for preparation of uniformly oriented IM membrane vesicles and validation of their sidedness. The uniformly ISO oriented membrane vesicles which have the cytoplasmic face of the membrane on the outside and the periplasmic side facing the sealed lumen can be obtained following intact cell disruption by a single passage through a French pressure cell (French press) at desired total pressure. Although high-pressure lysis leads to the formation of mostly inverted membrane vesicles (designated and abbreviated usually as ISO vesicles, everted or inverted membrane vesicles (IMVs)), inconclusive results are quite common. This uncertainty is due mainly by applying a different pressures, using either intact cells or spheroplasts and presence or absence of sucrose during rupture procedure. Many E. coli envelope fractionation techniques result in heterogeneity among isolated IM membrane vesicles. In part, this is due to difficulties in simple validation of sidedness of oriented membrane preparations of unknown sidedness. The sidedness of various preparations of membrane vesicles can be inferred from the orientation of residing uniformly oriented transmembrane protein. We outline the method in which the orientation of membrane vesicles can be verified by mapping of uniform or mixed topologies of essential protein E. coli protein leader peptidase (LepB) by advanced SCAM™. Although the protocol discussed in this chapter has been developed using Escherichia coli and Yersinia pseudotuberculosis, it can be directly adapted to other Gram-negative bacteria including pathogens.

Keywords

Escherichia coli, Cell Membrane, Membranes, Cytoplasmic Vesicles, Gram-Negative Bacteria, Membrane, ISO, inside-out vesicles, IMV, inverted membrane vesicles, SCAM™

DOI

10.1007/978-1-0716-3445-5_10

PMID

37930527

PMCID

PMC10724710

PubMedCentral® Posted Date

1-1-2025

PubMedCentral® Full Text Version

Author MSS

Published Open-Access

yes

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