Analysis of Transmembrane β-Barrel Proteins by Native and Semi-native Polyacrylamide Gel Electrophoresis.

Membrane-embedded β-barrel proteins are important regulators of the outer membrane permeability barrier of Gram-negative bacteria. β-barrels are highly structured domains formed by a series of antiparallel β-strands. Each β-strand is locked in position by hydrogen bonds between its polypeptide backbone and those of the two neighbouring strands in the barrel structure.

In Vivo Site-Directed and Time-Resolved Photocrosslinking of Envelope Proteins.

In vivo site-directed photocrosslinking provides a means to probe the vicinity of proteins in their native cellular environment. Because this method relies on the incorporation of unnatural amino acid analogs that are similar in size to natural amino acids, crosslink products are indicative of direct protein-protein interactions. Here, we present the use of this approach to monitor both transient and stable interactions of two proteins of the envelope of Escherichia coli.

LptM promotes oxidative maturation of the lipopolysaccharide translocon by substrate binding mimicry.

Insertion of lipopolysaccharide (LPS) into the bacterial outer membrane (OM) is mediated by a druggable OM translocon consisting of a β-barrel membrane protein, LptD, and a lipoprotein, LptE. The β-barrel assembly machinery (BAM) assembles LptD together with LptE at the OM. In the enterobacterium Escherichia coli, formation of two native disulfide bonds in LptD controls translocon activation.

Lipoprotein DolP supports proper folding of BamA in the bacterial outer membrane promoting fitness upon envelope stress.

In Proteobacteria, integral outer membrane proteins (OMPs) are crucial for the maintenance of the envelope permeability barrier to some antibiotics and detergents. In Enterobacteria, envelope stress caused by unfolded OMPs activates the sigmaE (σ) transcriptional response. σ upregulates OMP biogenesis factors, including the β-barrel assembly machinery (BAM) that catalyses OMP folding.

Transmembrane Coordination of Preprotein Recognition and Motor Coupling by the Mitochondrial Presequence Receptor Tim50.

Mitochondrial preproteins contain amino-terminal presequences directing them to the presequence translocase of the mitochondrial inner membrane (TIM23 complex). Depending on additional downstream import signals, TIM23 either inserts preproteins into the inner membrane or translocates them into the matrix. Matrix import requires the coupling of the presequence translocase-associated motor (PAM) to TIM23.

The Mgr2 subunit of the TIM23 complex regulates membrane insertion of marginal stop-transfer signals in the mitochondrial inner membrane.

The TIM23 complex mediates membrane insertion of presequence-containing mitochondrial proteins via a stop-transfer mechanism. Stop-transfer signals consist of hydrophobic transmembrane segments and flanking charges. Mgr2 functions as a lateral gatekeeper of the TIM23 complex.

Mitochondrial presequence import: Multiple regulatory knobs fine-tune mitochondrial biogenesis and homeostasis.

Mitochondria are pivotal organelles for cellular signaling and metabolism, and their dysfunction leads to severe cellular stress. About 60-70% of the mitochondrial proteome consists of preproteins synthesized in the cytosol with an amino-terminal cleavable presequence targeting signal. The TIM23 complex transports presequence signals towards the mitochondrial matrix.

Bacterial machineries for the assembly of membrane-embedded β-barrel proteins.

The outer membrane (OM) of Gram-negative bacteria is an essential organelle that protects cells from external aggressions and mediates the secretion of virulence factors. Efficient assembly of integral OM β-barrel proteins (OMPs) is crucial for the correct functioning of the OM. Biogenesis of OMPs occurs in a stepwise manner that is finalized by the β-barrel assembly machinery (BAM complex).

Job contenders: roles of the β-barrel assembly machinery and the translocation and assembly module in autotransporter secretion.

In Gram-negative bacteria, autotransporters secrete effector protein domains that are linked to virulence. Although they were once thought to be simple and autonomous secretion machines, mounting evidence reveals that multiple factors of the bacterial envelope are necessary for autotransporter assembly. Secretion across the outer membrane of their soluble effector "passenger domain" is promoted by the assembly of an outer membrane-spanning "β-barrel domain".

Site-Directed and Time-Resolved Photocrosslinking in Cells Metabolically Labeled with Radioisotopes.

To efficiently transport proteins into and across cellular membranes, specialized transport machineries engage in recognition events with different domains of their client proteins, forming sequential intermediate complexes. The short-lived nature of these interactions poses a big challenge in the identification of the key factors involved in transport reactions and their mechanism of action. Site-directed photocrosslinking is a powerful method for the detection and accurate mapping of interacting protein domains.

Analysis of Mitochondrial Membrane Protein Complexes by Electron Cryo-tomography.

The visualization of membrane protein complexes in their natural membrane environment is a major goal in an emerging area of research termed structural cell biology. Such approaches provide important information on the spatial distribution of protein complexes in their resident cellular membrane systems and on the structural organization of multi-subunit membrane protein assemblies. We have developed a method to specifically label active membrane protein complexes in their native membrane environment with electron-dense nanoparticles coupled to an activating ligand, in order to visualize them by electron cryo-tomography.

Two distinct membrane potential-dependent steps drive mitochondrial matrix protein translocation.

Two driving forces energize precursor translocation across the inner mitochondrial membrane. Although the membrane potential (Δψ) is considered to drive translocation of positively charged presequences through the TIM23 complex (presequence translocase), the activity of the Hsp70-powered import motor is crucial for the translocation of the mature protein portion into the matrix. In this study, we show that mitochondrial matrix proteins display surprisingly different dependencies on the Δψ.

Protein Import by the Mitochondrial Presequence Translocase in the Absence of a Membrane Potential.

The highly organized mitochondrial inner membrane harbors enzymes that produce the bulk of cellular ATP via oxidative phosphorylation. The majority of inner membrane protein precursors are synthesized in the cytosol. Precursors with a cleavable presequence are imported by the presequence translocase (TIM23 complex), while other precursors containing internal targeting signals are imported by the carrier translocase (TIM22 complex).

Mgr2 functions as lateral gatekeeper for preprotein sorting in the mitochondrial inner membrane.

The majority of preproteins destined for mitochondria carry N-terminal presequences. The presequence translocase of the inner mitochondrial membrane (TIM23 complex) plays a central role in protein sorting. Preproteins are either translocated through the TIM23 complex into the matrix or are laterally released into the inner membrane.

Visualizing active membrane protein complexes by electron cryotomography.

Unravelling the structural organization of membrane protein machines in their active state and native lipid environment is a major challenge in modern cell biology research. Here we develop the STAMP (Specifically TArgeted Membrane nanoParticle) technique as a strategy to localize protein complexes in situ by electron cryotomography (cryo-ET). STAMP selects active membrane protein complexes and marks them with quantum dots.

The presequence pathway is involved in protein sorting to the mitochondrial outer membrane.

The mitochondrial outer membrane contains integral α-helical and β-barrel proteins that are imported from the cytosol. The machineries importing β-barrel proteins have been identified, however, different views exist on the import of α-helical proteins. It has been reported that the biogenesis of Om45, the most abundant signal-anchored protein, does not depend on proteinaceous components, but involves direct insertion into the outer membrane.

Monitoring the assembly of a secreted bacterial virulence factor using site-specific crosslinking.

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest.

Mitochondrial inner membrane protease promotes assembly of presequence translocase by removing a carboxy-terminal targeting sequence.

The presequence translocase of the inner mitochondrial membrane (TIM23 complex) is essential for importing cleavable preproteins into mitochondria. The preproteins contain amino-terminal targeting sequences that are removed by the mitochondrial processing peptidase (MPP). Some preproteins carry bipartite presequences that are cleaved twice, by MPP and the inner membrane protease (IMP).

Mechanistic link between β barrel assembly and the initiation of autotransporter secretion.

Autotransporters are bacterial virulence factors that contain an N-terminal extracellular ("passenger") domain and a C-terminal β barrel ("β") domain that anchors the protein to the outer membrane. The β domain is required for passenger domain secretion, but its exact role in autotransporter biogenesis is unclear. Here we describe insights into the function of the β domain that emerged from an analysis of mutations in the Escherichia coli O157:H7 autotransporter EspP.

Mgr2 promotes coupling of the mitochondrial presequence translocase to partner complexes.

Many mitochondrial proteins are synthesized with N-terminal presequences in the cytosol. The presequence translocase of the inner mitochondrial membrane (TIM23) translocates preproteins into and across the membrane and associates with the matrix-localized import motor. The TIM23 complex consists of three core components and Tim21, which interacts with the translocase of the outer membrane (TOM) and the respiratory chain.

Sequential and spatially restricted interactions of assembly factors with an autotransporter beta domain.

Autotransporters are bacterial virulence factors that consist of an N-terminal extracellular ("passenger") domain and a C-terminal β barrel domain ("β domain") that resides in the outer membrane. Here we used an in vivo site-specific photocrosslinking approach to gain insight into the mechanism by which the β domain is integrated into the outer membrane and the relationship between β domain assembly and passenger domain secretion. We found that periplasmic chaperones and specific components of the β barrel assembly machinery (Bam) complex interact with the β domain of the Escherichia coli O157:H7 autotransporter extracellular serine protease P (EspP) in a temporally and spatially regulated fashion.

Secretion of a bacterial virulence factor is driven by the folding of a C-terminal segment.

Autotransporters are bacterial virulence factors consisting of an N-terminal "passenger domain" that is secreted in a C- to-N-terminal direction and a C-terminal "β domain" that resides in the outer membrane (OM). Although passenger domain secretion does not appear to use ATP, the energy source for this reaction is unknown. Here, we show that efficient secretion of the passenger domain of the Escherichia coli O157:H7 autotransporter EspP requires the stable folding of a C-terminal ≈17-kDa passenger domain segment.

Interaction of an autotransporter passenger domain with BamA during its translocation across the bacterial outer membrane.

Autotransporters are a superfamily of virulence factors produced by Gram-negative bacteria consisting of a large N-terminal extracellular domain ("passenger domain") and a C-terminal beta barrel domain ("beta domain"). The mechanism by which the passenger domain is translocated across the outer membrane (OM) is unknown. Here we show that the insertion of a small linker into the passenger domain of the Escherichia coli O157:H7 autotransporter EspP effectively creates a translocation intermediate by transiently stalling translocation near the site of the insertion.

OxyR tightly regulates catalase expression in Neisseria meningitidis through both repression and activation mechanisms.

Mechanisms for coping with oxidative stress (OS) are crucial for the survival of pathogenic Neisseria spp. in the human host. In this study we investigate the mechanism by which OxyR finely regulates the catalase gene (kat) in Neisseria meningitidis.