Outer membrane protein biogenesis in Gram-negative bacteria

Gram-negative bacteria contain a double membrane which serves for both protection and for providing nutrients for viability. The outermost of these membranes is called the outer membrane (OM), and it contains a host of fully integrated membrane proteins which serve essential functions for the cell, including nutrient uptake, cell adhesion, cell signalling and waste export. For pathogenic strains, many of these outer membrane proteins (OMPs) also serve as virulence factors for nutrient scavenging and evasion of host defence mechanisms. OMPs are unique membrane proteins in that they have a β-barrel fold and can range in size from 8 to 26 strands, yet can still serve many different functions for the cell. Despite their essential roles in cell survival and virulence, the exact mechanism for the biogenesis of these OMPs into the OM has remained largely unknown. However, the past decade has witnessed significant progress towards unravelling the pathways and mechanisms necessary for moulding a nascent polypeptide into a functional OMP within the OM. Here, we will review some of these recent discoveries that have advanced our understanding of the biogenesis of OMPs in Gram-negative bacteria, starting with synthesis in the cytoplasm to folding and insertion into the OM.     

The Escherichia coli outer membrane protein OmpA acquires secondary structure prior to its integration into the membrane

Almost all proteins that reside in the outer membrane (OM) of Gram-negative bacteria contain a membrane-spanning segment that folds into a unique β barrel structure and inserts into the membrane by an unknown mechanism. To obtain further insight into outer membrane protein (OMP) biogenesis, we revisited the surprising observation reported over 20 years ago that the Escherichia coli OmpA β barrel can be assembled into a native structure in vivo when it is expressed as two noncovalently linked fragments. Here, we show that disulfide bonds between β strand 4 in the N-terminal fragment and β strand 5 in the C-terminal fragment can form in the periplasmic space and greatly increase the efficiency of assembly of “split” OmpA, but only if the cysteine residues are engineered in perfect register (i.e., they are aligned in the fully folded β barrel). In contrast, we observed only weak disulfide bonding between β strand 1 in the N-terminal fragment and β strand 8 in the C-terminal fragment that would form a closed or circularly permutated β barrel. Our results not only demonstrate that β barrels begin to fold into a β-sheet-like structure before they are integrated into the OM but also help to discriminate among the different models of OMP biogenesis that have been proposed.     

Co-translational assembly and localized translation of nucleoporins in nuclear pore complex biogenesis

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GAIP participates in budding of membrane carriers at the trans-Golgi network

Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans -Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans -Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans -Golgi network.     

Outer membrane lipoprotein Lpp is Gram-negative bacterial cell surface receptor for cationic antimicrobial peptides

Antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms. Here we demonstrate that the outer membrane lipoprotein, Lpp, of Enterobacteriaceae interacts with and promotes susceptibility to the bactericidal activities of AMPs. The oligomeric Lpp was specifically recognized by several cationic α-helical AMPs, including SMAP-29, CAP-18, and LL-37; AMP-mediated bactericidal activities were blocked by anti-Lpp antibody blocking. Blebbing of the outer membrane and increase in membrane permeability occurred in association with the coordinate internalization of Lpp and AMP. Interestingly, the specific binding of AMP to Lpp was resistant to divalent cations and salts, which were able to inhibit the bactericidal activities of some AMPs. Furthermore, using His-tagged Lpp as a ligand, we retrieved several characterized AMPs, including SMAP-29 and hRNase 7, from a peptide library containing crude mammalian cell lysates. Overall, this study explores a new mechanism and target of antimicrobial activity and provides a novel method for screening of antimicrobials for use against drug-resistant bacteria.     

Palmitoylation is required for membrane association of activated but not inactive invertebrate visual Gqalpha

The invertebrate visual G protein, iGqα plays a central role in invertebrate phototransduction by relaying signals from rhodopsin to phospholipase C leading to membrane depolarization. Previous studies have shown reversible association of iGqα with rhabdomeric membranes regulated by light. To address the mechanism of membrane association we cloned iGqα from a Loligo pealei photoreceptor cDNA library and expressed it in HEK293T cells. Mutations were introduced to eliminate putative sites for palmitoylation at cysteines in positions 3 and 4. Membrane and soluble fractions were prepared from cells where iGqα was either activated or maintained in the GDP-bound form, followed by identification of iGqα through immunoblot analysis. The wild-type iGqα was entirely membrane-bound and shown to be post-translationally modified by palmitoylation. The mutant iGqα (C3,4A) was not palmitoylated yet it was found to be membrane-associated in the inactive state, however, approximately half of the protein became soluble when activated. These results suggest that palmitoylation is not required for membrane association of iGqα in the inactive state but is important in maintaining the stable membrane association of activated iGqα–GTP. The mechanism by which iGqα moves away from the membrane into the cytosol in response to prolonged light-stimulation in the native squid eye appears, therefore, to involve both activation and depalmitoylation processes.     

Non-invasive topology analysis of membrane proteins in the secretory pathway

We present a novel method to experimentally visualize in vivo the topology of transmembrane proteins residing in the endoplasmic reticulum (ER) membrane or passing through the secretory pathway on their way to their final destination. This approach, so-called redox-based topology analysis (ReTA), is based on fusion of transmembrane proteins with redox-sensitive GFP (roGFP) and ratiometric imaging. The ratio images provide direct information on the orientation of roGFP relative to the membrane as the roGFP fluorescence alters with changes in the glutathione redox potential across the ER membrane. As proof of concept, we produced binary read-outs using oxidized roGFP inside the ER lumen and reduced roGFP on the cytosolic side of the membrane for both N- and C-terminal fusions of single and multi-spanning membrane proteins. Further, successive deletion of hydrophobic domains from the C-terminus of the K/HDEL receptor ERD2 resulted in alternating localization of roGFP and a topology model for At ERD2 with six transmembrane domains.     

Covalent linkage of lipoprotein to peptidoglycan is not essential for outer membrane stability inProteus mirabilis

Incubation of hexadecameric glyceraldehyde 3-phosphate dehydrogenase of Scenedesmus obliquus with dithiothreitol caused a transient increase in NADPH-dependent activity. When ATP was also included in the incubation, the induced NADPH-dependent activity was stabilised. The rate of induction of NADPH-dependent activity increased hyperbolically with respect to ATP concentration. The effect of binding of ATP to the enzyme was two-fold; not only did it stimulate the dithiothreitol-promoted formation of an enzyme species with high NADPH-dependent activity it also prevented the inactivation of this species. The high concentration of ATP required for the stimulation of the NADPH-dependent activity compared with those for NADPH suggest that activation by ATP is unlikely to be of physiological significance.     

Galactosyltransferase activity is not localized to the brush border membrane of human small intestine

A recent report [Rothet al. (1985)J. Cell Biol. 100: 118–125], using immunocytochemical techniques, calimed that human duodenal galactosyltransferase is located predominantly on the external aspect of enterocyte brush border membranes. Analytical subcellular fractionation by sucrose density gradient centrifugation of human jejunum biopsy homogenates demonstrated that galactosyltransferase activity is localized to the Golgi fraction (equilibrium density of 1.14 g cm^–3) and is not found in significant amounts in the brush border membrane (equilibrium density of 1.22 g cm^–3).     

The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria

Integral proteins in the outer membrane of mitochondria control all aspects of organelle biogenesis, being required for protein import, mitochondrial fission, and, in metazoans, mitochondrial aspects of programmed cell death. How these integral proteins are assembled in the outer membrane had been unclear. In bacteria, Omp85 is an essential component of the protein insertion machinery, and we show that members of the Omp85 protein family are also found in eukaryotes ranging from plants to humans. In eukaryotes, Omp85 is present in the mitochondrial outer membrane. The gene encoding Omp85 is essential for cell viability in yeast, and conditional omp85 mutants have defects that arise from compromised insertion of integral proteins like voltage-dependent anion channel (VDAC) and components of the translocase in the outer membrane of mitochondria (TOM) complex into the mitochondrial outer membrane.     

Two palmitoyl acyltransferases involved sequentially in the biogenesis of the inner membrane complex of Toxoplasma gondii

The phylum Apicomplexa includes a number of significant human pathogens like Toxoplasma gondii and Plasmodium species. These obligate intracellular parasites possess a membranous structure, the inner membrane complex (IMC), composed of flattened vesicles apposed to the plasma membrane. Numerous proteins associated with the IMC are anchored via a lipid post‐translational modification termed palmitoylation. This acylation is catalysed by multi‐membrane spanning protein S‐acyl‐transferases (PATs) containing a catalytic Asp‐His‐His‐Cys (DHHC) motif, commonly referred to as DHHCs. Contrasting the redundancy observed in other organisms, several PATs are essential for T. gondii tachyzoite survival; 2 of them, TgDHHC2 and TgDHHC14 being IMC‐resident. Disruption of either of these TgDHHCs results in a rapid collapse of the IMC in the developing daughter cells leading to dramatic morphological defects of the parasites while the impact on the other organelles is limited to their localisation but not to their biogenesis. The acyl‐transferase activity of TgDHHC2 and TgDHHC14 is involved sequentially in the formation of the sub‐compartments of the IMC. Investigation of proteins known to be palmitoylated and localised to these sub‐compartments identified TgISP1/3 as well as TgIAP1/2 to lose their membrane association revealing them as likely substrates of TgDHHC2, while these proteins are not impacted by TgDHHC14 depletion.     

The end of the beginning: structural studies of ribosomal proteins

Work on the structural biology of ribosomes has progressed rapidly over the past few years. It has come to a stage at which the structures of the individual components are no longer of interest, except for those that still present ambiguous information about their structure because of conformational dynamics, as well as for those that show very little homology with their counterparts from other species or other kingdoms. The recently solved structure of protein L7/L12 and its proposed modes of dimerization have helped to understand the structural flexibility of this protein, which occurs as two dimers in the ribosome. The structure provides a missing link for many previous biochemical and functional studies.     

WNK1 is an assembly factor for the human ER membrane protein complex

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A lipoprotein signal peptide plus a cysteine residue at the amino-terminal end of the periplasmic protein β-lactamase is sufficient for its lipid modification, processing and membrane localization in Escherichia coli

Abstract By genetic exchange and in vitro mutagenesis a hybrid β-lactamase was constructed that contained the pCloDF13-encoded bacteriocin release protein signal peptide plus a cysteine residue coupled to the mature portion of β-lactamase. Immunoblotting, labelling with [³H]palmitate in the presence and absence of globomycin, and pulse-chase experiments revealed that this hybrid construct is modified with lipid and processed into a lipid-modified β-lactamase. Subcellular localization studies revealed that this hybrid is localized both in the cytoplasmic and outer membranes of Escherichia coli cells. A mutant derivative with an incomplete lipobox (LVG instead of LVAC⁺¹) was not processed and was found in the cytoplasmic membranes     

Assessment of the integral membrane protein topology in living cells

The bimolecular fluorescence complementation (BiFC) phenomenon has been successfully applied for in vivo protein-protein interaction studies and protein tagging analysis. Here we report a novel BiFC-based technique for investigation of integral membrane protein topology in living plant cells. This technique relies on the formation of a fluorescent complex between a non-fluorescent fragment of the yellow fluorescent protein (YFP) targeted into a specific cellular compartment and a counterpart fragment attached to the integral membrane protein N- or C-terminus or inserted into the internal loop(s). We employed this technique for topological studies of beet yellows virus-encoded p6 membrane-embedded movement protein, a protein with known topology, and the potato mop-top virus-encoded integral membrane TGBp2 protein with predicted topology. The results confirm that p6 is a type III integral transmembrane protein. Using a novel method, the central hydrophilic region of TGBp2 was localized into the ER lumen, whereas the N- and C-termini localized to the cytosol. We conclude that the BiFC-based reporter system for membrane protein topology analysis is a relatively fast and efficient method that can be used for high-throughput analysis of proteins integrated into the endoplasmic reticulum in living plant cells.     

Outer membrane protein A of E. coli folds into detergent micelles, but not in the presence of monomeric detergent.

Outer membrane protein A (OmpA) of Escherichia coli is a beta-barrel membrane protein that unfolds in 8 M urea to a random coil. OmpA refolds upon urea dilution in the presence of certain detergents or lipids. To examine the minimal requirements for secondary and tertiary structure formation in beta-barrel membrane proteins, folding of OmpA was studied as a function of the hydrophobic chain length, the chemical structure of the polar headgroup, and the concentration of a large array of amphiphiles. OmpA folded in the presence of detergents only above a critical minimal chain length of the apolar chain as determined by circular dichroism spectroscopy and a SDS-PAGE assay that measures tertiary structure formation. Details of the chemical structure of the polar headgroup were unimportant for folding. The minimal chain length required for folding correlated with the critical micelle concentration in each detergent series. Therefore, OmpA requires preformed detergent micelles for folding and does not adsorb monomeric detergent to its perimeter after folding. Formation of secondary and tertiary structure is thermodynamically coupled and strictly dependent on the interaction with aggregated amphiphiles.