Lipid-protein interactions drive membrane protein topogenesis in accordance with the positive inside rule.

Transmembrane domain orientation within some membrane proteins is dependent on membrane lipid composition. Initial orientation occurs within the translocon, but final orientation is determined after membrane insertion by interactions within the protein and between lipid headgroups and protein extramembrane domains. Positively and negatively charged amino acids in extramembrane domains represent cytoplasmic retention and membrane translocation forces, respectively, which are determinants of protein orientation. Lipids with no net charge dampen the translocation potential of negative residues working in opposition to cytoplasmic retention of positive residues, thus allowing the functional presence of negative residues in cytoplasmic domains without affecting protein topology.

Study of polytopic membrane protein topological organization as a function of membrane lipid composition.

A protocol is described using lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by the substituted-cysteine accessibility method as applied to transmembrane domains (SCAM). SCAM is adapted to follow changes in membrane protein topology as a function of changes in membrane lipid composition. The strategy described can be adapted to any membrane system.

Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli.

Phosphatidylcholine (PC) has been widely used in place of naturally occurring phosphatidylethanolamine (PE) in reconstitution of bacterial membrane proteins. However, PC does not support native structure or function for several reconstituted transport proteins. Lactose permease (LacY) of Escherichia coli, when reconstituted in E. coli phospholipids, exhibits energy-dependent uphill and energy-independent downhill transport function and proper conformation of periplasmic domain P7, which is tightly linked to uphill transport function. LacY expressed in cells lacking PE and containing only anionic phospholipids exhibits only downhill transport and lacks native P7 conformation. Reconstitution of LacY in the presence of E. coli-derived PE, but not dioleoyl-PC, results in uphill transport. We now show that LacY exhibits uphill transport and native conformation of P7 when expressed in a mutant of E. coli in which PC completely replaces PE even though the structure is not completely native. E. coli-derived PC and synthetic PC species containing at least one saturated fatty acid also support the native conformation of P7 dependent on the presence of anionic phospholipids. Our results demonstrate that the different effects of PE and PC species on LacY structure and function cannot be explained by differences in the direct interaction of the lipid head groups with specific amino acid residues alone but are due to more complex effects of the physical and chemical properties of the lipid environment on protein structure. This conclusion is supported by the effect of different lipids on the proper folding of domain P7, which indirectly influences uphill transport function.

Mitochondrial outer membrane proteome of Trypanosoma brucei reveals macromolecular transport machineries and novel factors required to maintain mitochondrial morphology

The mitochondrial outer membrane (MOM) separates the mitochondria from the cytoplasm, serving both as a barrier and as a gateway. Protein complexes — believed to be universally conserved in all eukaryotes — reside in the MOM to orchestrate and control metabolite exchange, lipid metabolism and uptake of biopolymers such as protein and RNA. African trypanosomes are the causative agent of the sleeping sickness in humans. The parasites are among the earliest diverging eukaryotes that have bona fide mitochondria capable of oxidative phosphorylation. Trypanosomes have unique mitochondrial biology that concerns their mitochondrial metabolism and their unusual mitochondrial morphology that differs to great extent between life stages. Another striking feature is the organization of the mitochondrial genome that does not encode any tRNA genes, thus all tRNAs needed for mitochondrial translation have to be imported. However, the MOM of T. brucei is essentially unchartered territory. It lacks a canonical protein import machinery and facilitation of tRNA translocation remains completely elusive. Using biochemical fractionation and label-free quantitative mass spectrometry for correlated protein abundance-profiling we were able to identify a cluster of 82 candidate proteins that can be localized to the trypanosomal MOM with high confidence. This enabled us to identify a highly unusual, potentially archaic protein import machinery that might also transport tRNAs. Moreover, two-thirds of the identified polypeptides present on the MOM have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the MOM of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of insect-stage parasites and therefore directly or indirectly are involved in the regulation of mitochondrial morphology.

Cholesterol metabolism, transport, and hepatic regulation in dairy cows during transition and early lactation

The transition from the nonlactating to the lactating state represents a critical period for dairy cow lipid metabolism because body reserves have to be mobilized to meet the increasing energy requirements for the initiation of milk production. The purpose of this study was to provide a comprehensive overview on cholesterol homeostasis in transition dairy cows by assessing in parallel plasma, milk, and hepatic tissue for key factors of cholesterol metabolism, transport, and regulation. Blood samples and liver biopsies were taken from 50 multiparous Holstein dairy cows in wk 3 antepartum (a.p.), wk 1 postpartum (p.p.), wk 4 p.p., and wk 14 p.p. Milk sampling was performed in wk 1, 4, and 14 p.p. Blood and milk lipid concentrations [triglycerides (TG), cholesterol, and lipoproteins], enzyme activities (phospholipid transfer protein and lecithin:cholesterol acyltransferase) were analyzed using enzymatic assays. Hepatic gene expression patterns of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGC) synthase 1 (HMGCS1) and HMGC reductase (HMGCR), sterol regulatory element-binding factor (SREBF)-1 and -2, microsomal triglyceride transfer protein (MTTP), ATP-binding cassette transporter (ABC) A1 and ABCG1, liver X receptor (LXR) α and peroxisome proliferator activated receptor (PPAR) α and γ were measured using quantitative RT-PCR. Plasma TG, cholesterol, and lipoprotein concentrations decreased from wk 3 a.p. to a minimum in wk 1 p.p., and then gradually increased until wk 14 p.p. Compared with wk 4 p.p., phospholipid transfer protein activity was increased in wk 1 p.p., whereas lecithin:cholesterol acyltransferase activity was lowest at this period. Total cholesterol concentration and mass, and cholesterol concentration in the milk fat fraction decreased from wk 1 p.p. to wk 4 p.p. Both total and milk fat cholesterol concentration were decreased in wk 4 p.p. compared with wk 1 and 14 p.p. The mRNA abundance of genes involved in cholesterol synthesis (SREBF-2, HMGCS1, and HMGCR) markedly increased from wk 3 a.p. to wk 1 p.p., whereas SREBF-1 was downregulated. The expression of ABCA1 increased from wk 3 a.p. to wk 1 p.p., whereas ABCG1 was increased in wk 14 p.p. compared with other time points. In conclusion, hepatic expression of genes involved in the biosynthesis of cholesterol as well as the ABCA1 transporter were upregulated at the onset of lactation, whereas plasma concentrations of total cholesterol, phospholipids, lipoprotein-cholesterol, and TG were at a minimum. Thus, at the gene expression level, the liver seems to react to the increased demand for cholesterol after parturition. Whether the low plasma cholesterol and TG levels are due to impaired hepatic export mechanisms or reflect an enhanced transfer of these compounds into the milk to provide essential nutrients for the newborn remains to be elucidated.

Lipid-dependent topogenesis and function of membrane proteins

Membranes are essential for the integrity and function of the cell. The collective property of the lipid bilayer is critical in providing an optimal functioning environment for membrane proteins. The simple yet well-characterized bacterium Escherichia coli serves an ideal model system to study the function of specific lipids since its lipid content can be easily manipulated. The most abundant lipid in E. coli membrane is phosphatidylethanolamine (PE, 70-80%). A PE-lacking E. coli mutant displays a complex mixture of deficient phenotypes, suggesting a profound role for PE in different aspects of cell function. A novel role of PE as a topological and functional determinant for membrane proteins has been established using lactose permease (LacY) as a model protein. PE is found to be required for energy-dependent uphill transport process of LacY. In PE-lacking membranes, LacY undergoes a dramatic conformational change, and the first half of the protein adopts an inverted topology with respect to the bilayer plane. ^ The work reported here was initiated to understand the molecular properties of lipids that enable their function as topological and functional determinants for membrane proteins. A glycolipid, monoglucosyldiacylglycerol (MGlcDAG) which shares physicochemical similarities with PE, was introduced to PE-lacking E. coli membranes. The introduction of MGlcDAG suppresses many of the PE-deficient phenotypes, and in particular supports the function and native topology of LacY. ^ The lipid-sensitive topogenic signals encoded in the amino acid sequence of LacY were also identified. Native LacY adopts an inverted topology when synthesized without PE, but mutation of specific acidic residues in the cytoplasmic extra-membrane domains can prevent this inversion and supports a native topological organization of LacY in PE-lacking membranes. These results suggest that it is the interplay between the collective charge properties of the lipid bilayer and extra-membrane loops of protein that determines the final orientation of transmembrane domains. By comparing the similarities as well as differences between these two lipids, we established how specific physical and chemical properties of lipids influence various cell functions and elucidated the molecular basis for the novel role of lipids in determining membrane protein topology. ^

A relationship between aerosol transport and compound-specific d13C land plant biomarker and pollen records

We examined near-surface, late Holocene deep-sea sediments at nine sites on a north-south transect from the Congo Fan (4°S) to the Cape Basin (30°S) along the Southwest African continental margin. Contents, distribution patterns and molecular stable carbon isotope signatures of long-chain n-alkanes (C27-C33) and n-alkanols (C22-C32) are indicators of land plant vegetation of different biosynthetic types, which can be correlated with concentrations and distributions of pollen taxa in the same sediments. Calculated clusters of wind trajectories and satellite Aerosol Index imagery afford information on the source areas for the lipids and pollen on land and their transport pathways to the ocean sites. This multidisciplinary approach on an almost continental scale provides clear evidence of latitudinal differences in lipid and pollen composition paralleling the major phytogeographic zonations on the adjacent continent. Dust and smoke aerosols are mainly derived from the western and central South African hinterland dominated by deserts, semi-deserts and savannah regions rich in C4 and CAM plants. The northern sites (Congo Fan area and northern Angola Basin), which get most of their terrestrial material from the Congo Basin and the Angolan highlands, may also receive some material from the Chad region. Very little aerosol from the African continent is transported to the most southerly sites in the Cape Basin. As can be expected from the present position of the phytogeographic zones, the carbon isotopic signatures of the n-alkanes and n-alkanols both become isotopically more enriched in 13C from north to south. The results of the study suggest that this combination of pollen data and compound-specific isotope geochemical proxies can be effectively applied in the reconstruction of past continental phytogeographic developments.

Lipid biomarkers in urface sediments of the Southeast Atlantic

Surface sediments from the eastern South Atlantic were investigated for their lipid biomarker contents and bulk organic geochemical characteristics to identify sources, transport pathways and preservation processes of organic components. The sediments cover a wide range of depositional settings with large differences in mass accumulation rates. The highest marine organic carbon (OC) contributions are detected along the coast, especially underlying the Benguela upwelling system. Terrigenous OC contributions are highest in the Congo deep-sea fan. Lipid biomarker fluxes are significantly correlated to the extent of oxygen exposure in the sediment. Normalization to total organic carbon (TOC) contents enabled the characterization of regional lipid biomarker production and transport mechanisms. Principal component analyses revealed five distinct groups of characteristic molecular and bulk organic geochemical parameters. Combined with information on lipid sources, the main controlling mechanisms of the spatial lipid distributions in the surface sediments are defined, indicating marine productivity related to river-induced mixing and oceanic upwelling, wind-driven deep upwelling, river-supply of terrigenous organic material, shallow coastal upwelling and eolian supply of plant-waxes.

Lipid composition of particulate matter measured on water bottle samples during POLARSTERN cruise ARK-XI/1

The lipid composition of particulate matter in oceanic environments can provide informations on the nature and origin of the organic matter as well as on their transformation processes. Molecular characteristics for lipids in the Arctic environment have been used as indicators of the sources and transformation of organic particulate matter (Smith et al., 1997; Fahl and Stein, 1997, 1999). However, the features of the lipid composition of particulate matter in the Arctic with its high seasonality of ice Cover and primary productivity has been studied insufficiently. Lipids are one of the most important compounds of organic matter. On the one hand, the composition of lipids is a result of the variability of biological sources (phyto- and zooplankton, higher plants, bacteria etc.). On the other hand, the lipid composition of particulate matter is undergone significant alteration during vertical transport. The organic matter balance in the Arctic marginal seas, such as the Kara and Laptev seas, is characterized by the significant supply of dissolved and particulate material by the major Eurasian rivers - Ob, Yenisei and Lena (Cauwet and Sidorov, 1996; Gordeev et al., 1996, Martin et al., 1993). In relation to the world's ocean the primary productivity values are lower in the Arctic seas due to the ice-cover. However local increased values of primary productivity can be connected with the melting processes inducing increased phytoplankton growth near ice-edge (Nelson et al., 1989; Fahl and Stein, 1997) and enhanced river supply of nutrients, These features can influence the proportion of allochtonous and autochtonous components of the organic matter in the Arctic marginal seas (Fahl and Stein, 1997; Stein and Fahl, 1999). Furthermore, increased lipid contents in aquatic environments were found near density discontinuities (Parish et al., 1988). Although being less informative than lipid studies on the molecular level the character of lipid composition analysis on the group could also be used for studying of particulate organic matter and its transformation in sedimentation processes in the Arctic. In this paper the investigation of the characteristics of lipid composition performed by Alexandrova and Shevchenko (1997) in Arctic seas was continued.

Transport of Pru p 3 across gastrointestinal epithelium - an essential step towards the induction of food allergy?

Background Since intestinal absorption of food protein can trigger an allergic reaction, the effect of plant food allergen on intestinal epithelial cell permeability and its ability to cross the epithelial monolayer was evaluated. Objective To study the interaction of Pru p 3 with intestinal epithelium, its natural entrance, analyzing transport kinetics and cellular responses that trigger. Methods This was achieved using Pru p 3, the peach LTP, as a model. Enterocytic monolayers were established by culturing Caco 2 cells, as a model of enterocytes, on permeable supports that separate the apical and basal compartments. Pru p 3 was added to the apical compartment, the transepithelial resistance (TEER) was measured, and the transport was quantified. Results The peach allergen that crossed the cell monolayer was detected in the cell fraction and in the basal medium by immunodetection with specific antibodies and the quantity was measured by ELISA assay. Pru p 3 was able to cross the monolayer without disturbing the integrity of the tight junctions. This transport was significantly higher than that of a non-allergenic peach LTP, LTP1, and occurred via lipid raft pathway. The incubation of Caco 2 cells with Pru p 3 and LTP1 produced the expression of epithelial-specific cytokines TSLP, IL33 and IL25. Conclusion These results suggest that Pru p 3 was able to cross the cell monolayer by the transcellular route and then induce the production of Th2 cytokines. The results of the present study represent a step towards clarifying the importance of Pru p 3 as a sensitizer. Clinical relevance The capacity of food allergens to cross the intestinal monolayer could explain their high allergenic capacity and its fast diffusion through the body associating to severe symptoms.

Two Endoplasmic Reticulum (ER) Membrane Proteins That Facilitate ER-to-Golgi Transport of Glycosylphosphatidylinositol-anchored Proteins

Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes in Saccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for “delayed GPI-anchored protein transport”), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Δ dgt1Δ cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting that LAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non–GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Δ dgt1Δ cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Δ dgt1Δ cells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.