Assembly Of Beta-barrel Proteins Research Articles

The Assembly of Beta-Barrel Proteins into Bacterial Outer Membranes

In order to cause disease, bacterial pathogens must (i) avoid immune detection, (ii) inhibit immune responses, and (iii) induce pathology favoring bacterial replication. In gram-negative bacteria, pathogenesis depend on structures assembled in the bacterial outer membrane. We have been working to understand the mechanism by which beta-barrel and other integral membrane proteins are assembled into bacterial outer membranes.The BAM complex is the core machinery for the assembly of membrane proteins, and inhibition of BAM complex activity is lethal to bacteria. In Escherichia coli, the BAM coplex is formed from four lipoproteins (BamB, BamC, BamD and BamE) attached to the integral membrane protein BamA to modulate its function, with the BamCDE module spanning the outer membrane. An additional, enigmatic module of the beta-barrel membrane protein assembly is the translocation and assembly module (the TAM). Composed of an outer membrane protein (TamA) and an integral inner membrane protein (TamB), the TAM is not essential for cell viability under laboratory conditions. However, the tamA and tamB genes are often hits in genetic screens of virulence, and have been well-conserved through evolution suggesting selection for an important function. Recent work demonstrating the reconstitution of active forms of the BAM complex and the TAM, and new assays for the assembly of a diverse array of outer membrane protein substrates will be discussed. Comparative genome analysis will be presented that demonstrates evolutionary tinkering in the interplay between the various modules (BamAB, BamCDE and TamAB) of the beta-barrel membrane protein assembly machinery. Finally, new data from neutron imaging and super-resolution microscopy will be presented which details the molecular organization of assembly precincts in the outer membrane: specialized clusters of BAM complexes that suggest current models for the mechanism of beta-barrel assembly need revision.

Bacterial Porin Disrupts Mitochondrial Membrane Potential and Sensitizes Host Cells to Apoptosis

The bacterial PorB porin, an ATP-binding β-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (ΔΨm). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of β-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of ΔΨm. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce ΔΨm loss and apoptosis, demonstrating that dissipation of ΔΨm is a requirement for cell death caused by neisserial infection.

Mitochondrial Inheritance Is Required for MEN-Regulated Cytokinesis in Budding Yeast

Mitochondrial inheritance, the transfer of mitochondria from mother to daughter cell during cell division, is essential for daughter cell viability. The mitochore, a mitochondrial protein complex containing Mdm10p, Mdm12p, and Mmm1p, is required for mitochondrial motility leading to inheritance in budding yeast. We observe a defect in cytokinesis in mitochore mutants and another mutant (mmr1Delta gem1Delta) with impaired mitochondrial inheritance. This defect is not observed in yeast that have no mitochondrial DNA or defects in mitochondrial protein import or assembly of beta-barrel proteins in the mitochondrial outer membrane. Deletion of MDM10 inhibits contractile-ring closure, but does not inhibit contractile-ring assembly, localization of a chromosomal passenger protein to the spindle during early anaphase, spindle alignment, nucleolar segregation, or nuclear migration during anaphase. Release of the mitotic exit network (MEN) component, Cdc14p, from the nucleolus during anaphase is delayed in mdm10Delta cells. Finally, hyperactivation of the MEN by deletion of BUB2 restores defects in cytokinesis in mdm10Delta and mmr1Delta gem1Delta cells and reduces the fidelity of mitochondrial segregation between mother and daughter cells in wild-type and mdm10Delta cells. Our studies identify a novel MEN-linked regulatory system that inhibits cytokinesis in response to defects in mitochondrial inheritance in budding yeast.

Probing the Barrier Function of the Outer Membrane with Chemical Conditionality

A key function of biological membranes is to exclude toxic small molecules while allowing influx of nutrients. Cells achieve this by controlling the composition of different types of proteins and lipids within the membrane by a process called membrane biogenesis. We have recently proposed a strategy to identify genes involved in membrane biogenesis in Gram-negative bacteria such as Escherichia coli by selecting for suppressors of mutations that render the outer membrane (OM) leaky. We predicted that different small molecules could select different suppressors because mutations that answer a specific selection will correct the membrane permeability defect to different degrees depending on the structure of the small molecule. We have tested this hypothesis by selecting for resistance to bile acids in an imp4213 strain, which contains a compromised OM owing to a defect in lipopolysaccharide biogenesis. We report here that a suppressor mutation in yaeT , which specifies an essential protein involved in the assembly of beta-barrel proteins in the OM, confers resistance to a specific subset of bile acids in the imp4213 strain. YaeT is conserved from bacteria to man because it is involved in OM biogenesis in mitochondria and chloroplasts. These results demonstrate that structurally different toxic small molecules select different, and highly specific, genetic solutions for correcting membrane-permeability defects. The remarkable chemical specificity of the imp4213 suppressors provides insights into the molecular nature of the OM permeability barrier.