Dataset Information

Periplasmic orientation of nascent lipid A in the inner membrane of an Escherichia coli LptA mutant.

ABSTRACT: The core-lipid A domain of Escherichia coli lipopolysaccharide (LPS) is synthesized on the inner surface of the inner membrane (IM) and flipped to its outer surface by the ABC transporter MsbA. Recent studies with deletion mutants implicate the periplasmic protein LptA, the cytosolic protein LptB, and the IM proteins LptC, LptF, and LptG in the subsequent transport of nascent LPS to the outer membrane (OM), where the LptD/LptE complex flips LPS to the outer surface. We have isolated a temperature-sensitive mutant (MB1) harboring the S22C and Q111P substitutions in LptA. MB1 stops growing after 30 min at 42 degrees C. (32)P(i) and [(35)S]methionine labeling show that export of newly synthesized phospholipids and proteins is not severely impaired, but export of LPS is defective. Using the lipid A 1-phosphatase LpxE as a periplasmic IM marker and the lipid A 3-O-deacylase PagL as an OM marker, we show that core-lipid A reaches the periplasmic side of the IM at 42 degrees C in MB1 but not the outer surface of the OM. Electron microscopy of MB1 reveals dense periplasmic material and a smooth OM at 42 degrees C, consistent with a role for LptA in shuttling LPS across the periplasm.

SUBMITTER: Ma B

PROVIDER: S-EPMC2544538 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Periplasmic orientation of nascent lipid A in the inner membrane of an Escherichia coli LptA mutant.

Ma Bing B Reynolds C Michael CM Raetz Christian R H CR

Proceedings of the National Academy of Sciences of the United States of America 20080903 37

The core-lipid A domain of Escherichia coli lipopolysaccharide (LPS) is synthesized on the inner surface of the inner membrane (IM) and flipped to its outer surface by the ABC transporter MsbA. Recent studies with deletion mutants implicate the periplasmic protein LptA, the cytosolic protein LptB, and the IM proteins LptC, LptF, and LptG in the subsequent transport of nascent LPS to the outer membrane (OM), where the LptD/LptE complex flips LPS to the outer surface. We have isolated a temperatur ...[more]

PMID: 18768814

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Project description: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.

Dataset Information

Periplasmic orientation of nascent lipid A in the inner membrane of an Escherichia coli LptA mutant.

Publications

Periplasmic orientation of nascent lipid A in the inner membrane of an Escherichia coli LptA mutant.

Similar Datasets

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