Gram-negative bacteria have an outer membrane (OM) that functions as a barrier to protect the cell from toxic compounds such as antibiotics and detergents. The OM is a highly asymmetric bilayer composed of phospholipids, glycolipids, and proteins. Assembly of this essential organelle occurs outside the cytoplasm in an environment that lacks obvious energy sources such as ATP, and the mechanisms involved are poorly understood. We describe the identification of a multiprotein complex required for the assembly of proteins in the OM of Escherichia coli. We also demonstrate genetic interactions between genes encoding components of this protein assembly complex and imp, which encodes a protein involved in the assembly of lipopolysaccharides (LPS) in the OM. These genetic interactions suggest a role for YfgL, one of the lipoprotein components of the protein assembly complex, in a homeostatic control mechanism that coordinates the overall OM assembly process.

Integral ␤-barrel proteins (OMPs) are a major class of outer membrane proteins in Gram-negative bacteria. In Escherichia coli, these proteins are synthesized in the cytoplasm, translocated across the inner membrane via the Sec machinery, and assembled in the outer membrane through an unknown mechanism that requires the outer membrane YaeT complex and the periplasmic chaperones SurA, DegP, and Skp. Here, we have established the relationship between these three chaperones providing insight into the mechanism of OMP biogenesis using depletion analysis. Depletion of SurA alone results in a marked decrease in outer membrane density, while the loss of DegP and Skp has no effect on outer membrane composition. Furthermore, we demonstrate that SurA and YaeT interact directly in vivo. Based on these results, we suggest that SurA is the primary chaperone responsible for the periplasmic transit of the bulk mass of OMPs to the YaeT complex. The role of Skp and DegP is amplified in the absence of SurA. Evidence presented suggests that DegP/Skp function to rescue OMPs that fall off the SurA pathway. The seemingly redundant periplasmic chaperones do function in parallel, but the relative importance of the primary function of each pathway depends on whether or not cells are under stress.

A major role of the outer membrane (OM) of Gram-negative bacteria is to provide a protective permeability barrier for the cell, and proper maintenance of the OM is required for cellular viability. OM biogenesis requires the coordinated assembly of constituent lipids and proteins via dedicated OM assembly machineries. We have previously shown that, in Escherichia coli, the multicomponent YaeT complex is responsible for the assembly of OM ␤-barrel proteins (OMPs). This complex contains the OMP YaeT and three OM lipoproteins. Here, we report another component of the YaeT complex, the OM lipoprotein small protein A (SmpA). Strains carrying loss-of-function mutations in smpA are viable but exhibit defects in OMP assembly. Biochemical experiments show that SmpA is involved in maintaining complex stability. Taken together, these experiments establish an important role for SmpA in both the structure and function of the YaeT complex.

BACKGROUND Increased secretion of growth hormone leads to gigantism in children and acromegaly in adults; the genetic causes of gigantism and acromegaly are poorly understood. METHODS We performed clinical and genetic studies of samples obtained from 43 patients with gigantism and then sequenced an implicated gene in samples from 248 patients with acromegaly. RESULTS We observed microduplication on chromosome Xq26.3 in samples from 13 patients with gigantism; of these samples, 4 were obtained from members of two unrelated kindreds, and 9 were from patients with sporadic cases. All the patients had disease onset during early childhood. Of the patients with gigantism who did not carry an Xq26.3 microduplication, none presented before the age of 5 years. Genomic characterization of the Xq26.3 region suggests that the microduplications are generated during chromosome replication and that they contain four protein-coding genes. Only one of these genes, GPR101, which encodes a G-protein–coupled receptor, was overexpressed in patients’ pituitary lesions. We identified a recurrent GPR101 mutation (p.E308D) in 11 of 248 patients with acromegaly, with the mutation found mostly in tumors. When the mutation was transfected into rat GH3 cells, it led to increased release of growth hormone and proliferation of growth hormone–producing cells. CONCLUSIONS We describe a pediatric disorder (which we have termed X-linked acrogigantism [X-LAG]) that is caused by an Xq26.3 genomic duplication and is characterized by early-onset gigantism resulting from an excess of growth hormone. Duplication of GPR101 probably causes X-LAG. We also found a recurrent mutation in GPR101 in some adults with acromegaly. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others.)

The outer membrane of most Gram-negative bacteria is made up of LPS, and in nearly all bacteria that contain LPS it is essential for the life of the organism. The lipid portion of this molecule, lipid A, also known as endotoxin, is a potent activator of the innate immune response. More than 50 genes are required to synthesize LPS and assemble it at the cell surface. Enormous progress has been made in elucidating the structure and biosynthesis of LPS, but until recently the cellular components required for its transport from its site of synthesis in the inner membrane to its final cellular location at the cell surface remained elusive. Here we describe the identification of a protein complex that functions to assemble LPS at the surface of the cell. This complex contains two proteins: Imp, already identified as an essential outer-membrane protein implicated in LPS assembly; and another protein, RlpB, heretofore identified only as a rare lipoprotein. We show that RlpB is also essential for cell viability and that the Imp͞RlpB complex is responsible for LPS reaching the outer surface of the outer membrane.essential lipoprotein ͉ Gram-negative bacteria ͉ outer-membrane biogenesis T he cell envelope of Gram-negative bacteria such as Escherichia coli is composed of the inner (cytoplasmic) membrane (IM), the outer membrane (OM), and the periplasmic space in between, where the bacterial peptidoglycan (cell wall) is located. The OM is an asymmetric lipid bilayer with phospholipids forming the inner leaf let and LPS forming the outer leaf let (1). ␤-Barrel OM proteins (OMPs) are inserted into the OM whereas lipoproteins are anchored to the inner leaf let of the OM through posttranslationally attached lipid moieties. The OM serves as a permeability barrier that protects the cells against toxic compounds such as antibiotics and detergents in their environment (2).The components of the OM (proteins and lipids) are synthesized inside the cell or at the inner leaflet of the IM. They need to be transported to and assembled at the OM in the correct orientation to maintain this barrier function during cell growth and division. Proteins involved in transporting these components across the IM have been identified and characterized (3-5). Much less is known, however, about how the OM components are transported across the aqueous periplasmic space and inserted into the OM. The recently identified Lol system targets lipoproteins to the OM through a periplasmic carrier protein, LolA, and an OM receptor, LolB (6, 7). Periplasmic factors involved in OMP folding have also been identified (8, 9). However, the mechanism(s) of how these factors facilitate transport of the OMPs across the periplasmic space is not known. Recently, a multiprotein complex involved in OMP assembly was identified by using a combination of genetic and biochemical approaches (10-12). This complex contains an OMP, YaeT, and three previously uncharacterized lipoproteins, YfgL, YfiO, and NlpB.An essential gene imp was recently shown to be involved in OM biogenesis. In ...

To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.

Estrogens have been shown to have positive and negative effects on anxiety and depressive-like behaviors, perhaps explained by the existence of two distinct estrogen receptor (ER) systems, ERalpha and ERbeta. The ERbeta agonist, diarylpropionitrile (DPN) has been shown to have anxiolytic properties in rats. DPN exists as a racemic mixture of two enantiomers, R-DPN and S-DPN. In this study, we compared R-DPN and S-DPN for their in vitro binding affinity, ability to activate transcription in vitro at an estrogen response element, and in vivo endocrine and behavioral responses. In vitro binding studies using recombinant rat ERbeta revealed that S-DPN has a severalfold greater relative binding affinity for ERbeta than does R-DPN. Furthermore, cotransfection of N-38 immortalized hypothalamic cells with an estrogen response element-luc reporter and ERbeta revealed that S-DPN is a potent activator of transcription in vitro, whereas R-DPN is not. Subsequently, we examined anxiety-like behaviors using the open-field test and elevated plus maze or depressive-like behaviors, using the forced swim test. Ovariectomized young adult female Sprague Dawley rats treated with racemic DPN, S-DPN, and the ERbeta agonist, WAY-200070, showed significantly decreased anxiety-like behaviors in both the open-field and elevated plus maze and significantly less depressive-like behaviors in the forced swim test compared with vehicle-, R-DPN-, or propylpyrazoletriol (ERalpha agonist)-treated animals. In concordance with the relative binding affinity and transcriptional potency, these results demonstrate that the S-enantiomer is the biologically active form of DPN. These studies also indicate that estrogen's positive effects on mood, including its anxiolytic and antidepressive actions, are due to its actions at ERbeta.

The assembly of β-barrel proteins into membranes is mediated by an evolutionarily conserved machine. This process is poorly understood because no stable partially folded barrel substrates have been characterized. Here, we slowed the folding of the Escherichia coli β-barrel protein, LptD, with its lipoprotein plug, LptE. We identified a late-stage intermediate in which LptD is folded around LptE, and both components interact with the two essential β-barrel assembly machine (Bam) components, BamA and BamD. We propose a model in which BamA and BamD act in concert to catalyze folding, with the final step in the process involving closure of the ends of the barrel with release from the Bam components. Because BamD and LptE are both soluble proteins, the simplest model consistent with these findings is that barrel folding by the Bam complex begins in the periplasm at the membrane interface.outer membrane | Bam complex | β-barrel | protein folding T he assembly of β-barrel membrane proteins into the outer membrane (OM) of Gram-negative bacteria, mitochondria, and chloroplasts is facilitated by conserved cellular machinery (1-4). The β-barrel assembly machine (Bam) folds and inserts integral membrane proteins into the OM of Gram-negative organisms (5). Bam is a five-protein complex consisting of the essential protein BamA, a β-barrel itself, and four lipoproteins, BamB, -C, -D, and -E, of which only BamD is essential (4-8). The Bam complex recognizes a large number of different substrates, but how each component catalyzes the folding and insertion of such structurally diverse substrates is unclear.How β-barrels are assembled into membranes is not obvious. Where and how folding occurs is unclear because intermediates could contain both exposed polar amides and hydrophobic residues until the barrel has completed its fully hydrophobic exterior. By contrast, α-helical membrane proteins have internally satisfied hydrogen bonds, making stepwise assembly from stable secondary structural elements possible. Although Bam has been shown to accelerate membrane β-barrel assembly (9-11), the transient nature of folding intermediates has made accumulating such discrete species for characterization difficult (12-15). If structurally defined folding intermediates were to exist long enough for characterization, they could reveal crucial aspects of the folding process.Here, we studied the assembly of an essential, slow-folding β-barrel, LptD. LptD is one of two components of the OM translocon that transports lipopolysaccharide to the cell surface (16-18). The other component, LptE, is a lipoprotein that forms a plug inside the LptD barrel (19)(20)(21)(22). LptD also contains two disulfide bonds (23), and its assembly involves the formation of consecutive disulfide bonds that after barrel folding rearrange to form nonconsecutive disulfide bonds (24). The assembly of LptD is orders-ofmagnitude slower (∼20 min versus seconds) than that of other barrel substrates (24-26). Because of the slow rate of folding and our ability to use oxidation sta...