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At least 109 records · Page 6Linked to original sources
PubMed2006

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.

Bacterial Outer Membrane Proteins↗
PubMed1996

Membrane fusion in organelle biogenesis.

Intracellular membrane fusion is a fascinating reaction that is crucial for cellular function. Several components of the membrane fusion machinery have been identified, although a precise understanding of the fusion mechanism is lacking. More recent studies are revealing novel proteins that regulate membrane fusion during organelle assembly.

Adenosine Triphosphatases↗
PubMed1998

Sec-dependent membrane protein biogenesis: SecYEG, preprotein hydrophobicity and translocation kinetics control the stop-transfer function.

Preprotein translocase catalyzes membrane protein integration as well as complete translocation. Membrane proteins must interrupt their translocation and be laterally released from the translocase into the lipid bilayer. We have analyzed the translocation arrest and lateral release activities of Escherichia coli preprotein translocase with an in vitro reaction and the preprotein proOmpA carrying a synthetic stop-transfer sequence. Membrane protein integration is catalytic, occurs with kinetics similar to those of proOmpA itself and only requires the functions of SecYEG and SecA. Though a strongly hydrophobic segment will direct the protein to leave the translocase and enter the lipid bilayer, a protein with a segment of intermediate hydrophobicity partitions equally between the translocated and membrane-integrated states. Analysis of the effects of PMF, varied ATP concentrations or synthetic translocation arrest show that the stop-translocation efficiency of a mildly hydrophobic segment depends on the translocation kinetics. In contrast, the lateral partitioning from translocase to lipids depends solely on temperature and does not require SecA ATP hydrolysis or SecA membrane cycling. Thus translocation arrest is controlled by the SecYEG translocase activity while lateral release and membrane integration are directed by the hydrophobicity of the segment itself. Our results suggest that a greater hydrophobicity is required for efficient translocation arrest than for lateral release into the membrane.

Adenosine Triphosphatases↗
PubMed2006

Homeostatic restitution of cell membranes. Nuclear membrane lipid biogenesis and transport of protein from cytosol to intranuclear spaces.

Our studies on homeostatic restitution of cellular and subcellular membranes showed that vesicular intracellular transport is engaged in systematic and coordinated replacement of lipids and proteins in the membranes of the secretory, non-dividing epithelial cells (Slomiany et al., J. Physiol. Pharmacol. 2004; 55: 837-860). In this report, we present evidence on the homeostatic restitution of lipids in the biomembranes that constitute nuclear envelopes. We investigated nuclear membranes lipid synthesis by employing purified intact nuclei (IN), the outer nuclear membrane (ONM), the inner nuclear membrane (INM) and the cell cytosol (CC). In contrast to Endoplasmic Reticulum (ER) which in the presence of CC generates new biomembrane that forms ER vesicles transporting ER products to Golgi, the IN, ONM and INM are not producing transport vesicles. Instead, the newly synthesized lipids remain in the nuclear membranes. The membranes (INM, ONM) of IN incubated with CC become enriched with newly synthesized phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylinositol phosphates (PIPs) and phosphatidic acid (PA). The incubation of separated ONM and INM with CC also enriched the membranes with IN specific lipids identified above. Moreover, the incubation of IN or its membranes with CC afforded retention of numerous CC proteins on the nuclear membrane. Here, we concentrated on 30kDa CC protein that displayed affinity to nuclear membrane PIP2. The 30kDa CC protein bound to PIP2 of IN, INM, and ONM. With IN, initially the PIP2-30kDa CC protein complex was detected on ONM, after 30-120 min of incubation, was found on INM and in nuclear contents. At the same time when the 30 kDa protein was released from INM and found in nuclear contents, the PIP2 of INM and ONM became undetectable, while the lipid extract from the membrane displaced from IN contained labeled PI only. Since ONM is an uninterrupted continuum of ER and INM, we speculate that the synthesis of the lipids in the ER, in the region adjacent to nucleus, is defining nuclear outer and inner biomembrane composition, is responsible for transport of the cytosolic protein into the nucleus and, replenishment of ER membrane used for vesicular transport.

Animals↗
PubMed2006

Localization of the phosphoethanolamine methyltransferase of the human malaria parasite Plasmodium falciparum to the Golgi apparatus.

Phosphatidylcholine is the most abundant phospholipid in the membranes of Plasmodium falciparum, the agent of severe human malaria. The synthesis of this phospholipid occurs via two routes, the CDP-choline pathway, which uses host choline as a precursor, and the plant-like serine decarboxylase-phosphoethanolamine methyltransferase (SDPM) pathway, which uses host serine as a precursor. Although various components of these pathways have been identified, their cellular locations remain unknown. We have previously reported the identification and characterization of the phosphoethanolamine methyltransferase, Pfpmt, of P. falciparum and shown that it plays a critical role in the synthesis of phosphatidylcholine via the SDPM pathway. Here we provide the first evidence that the transmethylation step of the SDPM pathway occurs in the parasite Golgi apparatus. We show that the level of Pfpmt protein in the infected erythrocyte is regulated in a stage-specific fashion, with high levels detected during the trophozoite stage at the peak of parasite membrane biogenesis. Confocal microscopy revealed that Pfpmt is not cytoplasmic. Immunoelectron microscopy revealed that Pfpmt localizes to membrane structures that extend from the nuclear membrane but that it only partially co-localizes with the endoplasmic reticulum marker BiP. Using transgenic parasites expressing green fluorescent protein targeted to different cellular compartments, a complete co-localization was detected with Rab6, a marker of the Golgi apparatus. Together these studies provide the first evidence that the transmethylation step of the SDPM pathway of P. falciparum occurs in the Golgi apparatus and indicate an important role for this organelle in parasite membrane biogenesis.

Animals↗
PubMed2006

Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival.

Many pathogenic organisms produce pore-forming toxins as virulence factors. Target cells however mount a response to such membrane damage. Here we show that toxin-induced membrane permeabilization leads to a decrease in cytoplasmic potassium, which promotes the formation of a multiprotein oligomeric innate immune complex, called the inflammasome, and the activation of caspase-1. Further, we find that when rendered proteolytic in this context caspase-1 induces the activation of the central regulators of membrane biogenesis, the Sterol Regulatory Element Binding Proteins (SREBPs), which in turn promote cell survival upon toxin challenge possibly by facilitating membrane repair. This study highlights that, in addition to its well-established role in triggering inflammation via the processing of the precursor forms of interleukins, caspase-1 has a broader role, in particular linking the intracellular ion composition to lipid metabolic pathways, membrane biogenesis, and survival.

Aeromonas↗
PubMed2004

In the Archaea Haloferax volcanii, membrane protein biogenesis and protein synthesis rates are affected by decreased ribosomal binding to the translocon.

In the haloarchaea Haloferax volcanii, ribosomes are found in the cytoplasm and membrane-bound at similar levels. Transformation of H. volcanii to express chimeras of the translocon components SecY and SecE fused to a cellulose-binding domain substantially decreased ribosomal membrane binding, relative to non-transformed cells, likely due to steric hindrance by the cellulose-binding domain. Treatment of cells with the polypeptide synthesis terminator puromycin, with or without low salt washes previously shown to prevent in vitro ribosomal membrane binding in halophilic archaea, did not lead to release of translocon-bound ribosomes, indicating that ribosome release is not directly related to the translation status of a given ribosome. Release was, however, achieved during cell starvation or stationary growth, pointing at a regulated manner of ribosomal release in H. volcanii. Decreased ribosomal binding selectively affected membrane protein levels, suggesting that membrane insertion occurs co-translationally in Archaea. In the presence of chimera-incorporating sterically hindered translocons, the reduced ability of ribosomes to bind in the transformed cells modulated protein synthesis rates over time, suggesting that these cells manage to compensate for the reduction in ribosome binding. Possible strategies for this compensation, such as a shift to a post-translational mode of membrane protein insertion or maintained ribosomal membrane-binding, are discussed.

Archaea↗
PubMed2001

Role of sphingolipids in the biogenesis of membrane domains.

In recent years, a huge interest in sphingolipid- and cholesterol-enriched membrane domains has risen, after their involvement in fundamental membrane-associated events such as signal transmission, cell adhesion and lipid/protein sorting was postulated. Theoretical considerations and several experimental data suggest that sphingolipids play an important role in the biogenesis and function of domains. In fact, their physicochemical features, different from those of other membrane lipids, allow their interaction either with other sphingolipids or with other membrane components and external ligands. Owing to these features, sphingolipids may undergo segregation and represent a nucleation point for co-clustering with other lipids and proteins in a complex, functional domain. Moreover, sphingolipids confer dynamic properties on domains, a fundamental feature for the modulation of their postulated functions.

Animals↗
PubMed1999

Affinity of the periplasmic chaperone Skp of Escherichia coli for phospholipids, lipopolysaccharides and non-native outer membrane proteins. Role of Skp in the biogenesis of outer membrane protein.

The Skp protein of Escherichia coli has been proposed to be a periplasmic molecular chaperone involved in the biogenesis of outer membrane proteins. In this study, evidence is obtained that Skp exists in two different states characterized by their different sensitivity to proteases. The conversion between these states can be modulated in vitro by phospholipids, lipopolysaccharides and bivalent cations. Skp is able to associate with and insert into phospholipid membranes in vitro, indicating that it may associate with phospholipids in the inner and/or outer membrane in vivo. In addition, it interacts specifically with outer membrane proteins that are in their non-native state. We propose that Skp is required in vivo for the efficient targeting of unfolded outer membrane proteins to the membrane.

Bacterial Outer Membrane Proteins↗
PubMedDate not supplied

Polytopic membrane protein folding and assembly in vitro and in vivo.

The insertion and folding of proteins in biological membranes during protein synthesis in vivo is fundamental to membrane biogenesis. At present, however, certain molecular aspects of this process can only be understood by complementary studies in vitro. We bring together in vitro and in vivo results, highlighting how the studies inform each other and increase our knowledge of the folding and assembly of polytopic membrane proteins. A notable recent advance is the high-resolution crystal structure of the protein machinery responsible for membrane protein insertion into the endoplasmic reticulum. This provides an opportunity to combine in vitro and in vivo studies at a more sophisticated level and address mechanistic aspects of polytopic protein insertion and folding. Quality control is another important aspect of membrane biogenesis, and we give an overview of the current understanding of this process, focusing on cystic fibrosis as a well-studied paradigm. Mutations in the associated membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), can cause the quality control mechanisms to prevent the mutant protein reaching its normal site of action, the cell surface. In vitro studies of CFTR shed light on the possible origins of other clinically relevant folding mutants and highlight the potential synergy between in vitro and in vivo approaches.

Cell Membrane↗
PubMed1999

Regulation of protein biogenesis at the endoplasmic reticulum membrane.

The biogenesis of most secretory and membrane proteins involves targeting the nascent protein to the endoplasmic reticulum (ER), translocation across or integration into the ER membrane and maturation into a functional product. The essential machinery that directs these events for model secretory and membrane proteins has been identified, shifting the focus of studies towards the molecular mechanisms by which these core components function. By contrast, regulatory mechanisms that allow certain proteins to serve multiple functions within a cell remain entirely unexplored. This article examines each stage of protein biogenesis as a potential site of regulation that could be exploited by the cell to effectively increase the diversity of functional gene expression.

Animals↗
PubMed1976

Biogenesis of mitochondrial membranes in Neurospora crassa during cellular differentiation: ultrastructural changes accompanying differentiation.

The ultrastructural characteristics of Neurospora cells during dedifferentiation and redifferentiation of conidiospores into vegetative cells have been determined. This germination process occurs between 2 and 5 h after inoculation; by 3-5 h, approximately 50% of the cells have germinated. The cells enter the exponential phase of dry-weight gain between 4 and 5 h after inoculation. Several unusual structures are observed in Neurospora cells during germination. Whorled structures are frequently seen in the cytoplasm during germination, and occasionally at other times. They appear to be derived from the cytoplasmic membrane. Whorled structures of different appearance were observed in the mitochondria between 2 and 4 h after inoculation. Their number was related to the level of metabolizable carbohydrate, and was higher in 15% glucose-than in 2% sucrose-supplemented medium, and very low in medium containing 15% mannitol, or 2% sucrose+13% 2-deoxyglucose, or no added carbohydrate. The mitochondrial inclusions were osmiophilic and could be removed by treatment with 90% aqueous acetone in the cold, indicating that they were composed at least in part of lipid. The strong dependence of the number of mitochondrial inclusions on time and on carbohydrate supplementation, suggests that there is a physiological basis for these structures and that they reflect changes occurring in the mitochondria at times significant to cellular differentiation.

Carbohydrate Metabolism↗
PubMed2003

Genome expression analysis in yeast reveals novel transcriptional regulation by inositol and choline and new regulatory functions for Opi1p, Ino2p, and Ino4p.

In Saccharomyces cerevisiae, genes encoding phospholipid-synthesizing enzymes are regulated by inositol and choline (IC). The current model suggests that when these precursors become limiting, the transcriptional complex Ino2p-Ino4p activates the expression of these genes, whereas repression requires Opi1p and occurs when IC are available. In this study, microarray-based expression analysis was performed to assess the global transcriptional response to IC in a wild-type strain and in the opi1delta, ino2delta, and ino4delta null mutant strains. Fifty genes were either activated or repressed by IC in the wild-type strain, including three already known IC-repressed genes. We demonstrated that the IC response was not limited to genes involved in membrane biogenesis, but encompassed various metabolic pathways such as biotin synthesis, one-carbon compound metabolism, nitrogen-containing compound transport and degradation, cell wall organization and biogenesis, and acetyl-CoA metabolism. The expression of a large number of IC-regulated genes did not change in the opi1delta, ino2delta, and ino4delta strains, thus implicating new regulatory elements in the IC response. Our studies revealed that Opi1p, Ino2p, and Ino4p have dual regulatory activities, acting in both positive and negative transcriptional regulation of a large number of genes, most of which are not regulated by IC and only a subset of which is involved in membrane biogenesis. These data provide the first global response profile of yeast to IC and reveal novel regulatory mechanisms by these precursors.

Basic Helix-Loop-Helix Proteins↗