Zn 2+ plays essential roles in biology, and cells have adopted exquisite mechanisms for regulating steady-state Zn 2+ levels. Although much is known about total Zn 2+ in cells, very little is known about its subcellular distribution. Yet defining the location of Zn 2+ and how it changes with signaling events is essential for elucidating how cells regulate this essential ion. Here we create fluorescent sensors genetically targeted to the endoplasmic reticulum (ER) and Golgi to monitor steady-state Zn 2+ levels as well as flux of Zn 2+ into and out of these organelles. These studies reveal that ER and Golgi contain a concentration of free Zn 2+ that is 100 times lower than the cytosol. Both organelles take up Zn 2+ when cytosolic levels are elevated, suggesting that the ER and Golgi can sequester elevated cytosolic Zn 2+ and thus have the potential to play a role in influencing Zn 2+ toxicity. ER Zn 2+ homeostasis is perturbed by small molecule antagonists of Ca 2+ homeostasis and ER Zn 2+ is released upon elevation of cytosolic Ca 2+ pointing to potential exchange of these two ions across the ER. This study provides direct evidence that Ca 2+ signaling can influence Zn 2+ homeostasis and vice versa, that Zn 2+ dynamics may modulate Ca 2+ signaling.

Folding and insertion of integral β-barrel proteins in the outer membrane is an essential process for Gram-negative bacteria that requires the β-barrel Assembly Machinery (BAM). Efficient OMP folding and insertion appears to require a consensus C-terminal signal in OMPs characterized by terminal F or W residues. The BAM complex is embedded in the outer membrane and, in E. coli, consists of the β-barrel BamA and four lipoproteins BamBCDE. BamA and BamD are broadly distributed across all species of Gram-negative bacteria whereas the other components are present in only a subset of species. BamA and BamD are also essential for viability suggesting that these two proteins constitute the functional core of the bacterial BAM complex. Here we present the crystal structure of BamD from the thermophilic bacteria Rhodothermus marinus refined to 2.15 Å resolution. The protein contains 5 tetratricopeptide repeats (TPRs) organized into two offset tandems, each capped by a terminal helix. The N-terminal domain contains three TPRs and displays remarkable structural similarity with proteins that recognize targeting signals in extended conformations. The C-terminal domain harbors the remaining 2 TPRs and previously described mutations that impair binding to other BAM components map to this domain. Therefore, the structure suggests a model where the C-terminal domain provides a scaffold for interaction with BAM components, while the N-terminal domain participates in interaction with the substrates, either recognizing the C-terminal consensus sequence or binding unfolded OMP intermediates.

Background:The ␤-barrel assembly machine (BAM) is essential for outer membrane protein (OMP) biogenesis and Gram-negative bacterial survival. Results: BamB binds polypeptide translocation-associated domain 3 (POTRA3) of BamA. Conclusion: BamB, binding at the hinge in the periplasmic domain of BamA, is poised to modulate BAM conformational changes. Significance: The BAM complex architecture illuminates the mechanism of OMP assembly.

The assembly of β-barrel Outer Membrane Proteins (OMPs) in the outer membrane is essential for Gram-negative bacteria. The process requires the β-Barrel Assembly Machine (BAM), a multiprotein complex that, in E. coli, is composed of the OMP BamA and four lipoproteins BamB-E. Whereas BamA and BamD are essential, deletion of BamB, C or E produce membrane permeability defects. Here we present the high-resolution structure of BamB from Pseudomonas aeruginosa. This protein can complement the deletion of bamB in E. coli indicating that they are functionally equivalent. Conserved structural features include an eight-bladed β-propeller fold stabilized by tryptophan docking motifs with a central pore about 8 Å in diameter at the narrowest point. This pore distinguishes BamB from related β-propellers, such as quinoprotein dehydrogenases. However, a double mutation designed to block this pore was fully functional indicating that the opening is not essential. Two loops protruding from the bottom of the propeller are conserved and mediate binding to BamA. Conversely, an additional loop only present in E. coli BamB is not required for function. A cluster of highly conserved residues in a groove between blades 6 and 7 is crucial for proper BamB folding or biogenesis. It has been proposed that BamB may bind nascent OMPs by β-augmentation to its propeller outer strands, or recognize the aromatic residue signature at the C-terminus of OMPs. However, Isothermal Titration Calorimetry experiments and structural analysis do not support these proposals. The structural and mutagenesis analysis suggests that the main function of BamB is to bind and modulate BamA, rather than directly interact with nascent OMPs.

Colicins are Escherichia coli–specific bacteriocins that translocate across the outer bacterial membrane by a poorly understood mechanism. Group A colicins typically parasitize the proton-motive force–linked Tol system in the inner membrane via porins after first binding an outer membrane protein receptor. Recent studies have suggested that the pore-forming group A colicin N (ColN) instead uses lipopolysaccharide as a receptor. Contrary to this prevailing view, using diffusion-precipitation assays, native state MS, isothermal titration calorimetry, single-channel conductance measurements in planar lipid bilayers, and in vivo fluorescence imaging, we demonstrate here that ColN uses OmpF both as its receptor and translocator. This dual function is achieved by ColN having multiple distinct OmpF-binding sites, one located within its central globular domain and another within its disordered N terminus. We observed that the ColN globular domain associates with the extracellular surface of OmpF and that lipopolysaccharide (LPS) enhances this binding. Approximately 90 amino acids of ColN then translocate through the porin, enabling the ColN N terminus to localize within the lumen of an OmpF subunit from the periplasmic side of the membrane, a binding mode reminiscent of that observed for the nuclease colicin E9. We conclude that bifurcated engagement of porins is intrinsic to the import mechanism of group A colicins.