Lipids and topological rules governing membrane protein assembly

Membrane protein folding and topogenesis are tuned to a given lipid profile since lipids and proteins have co-evolved to follow a set of interdependent rules governing final protein topological organization. Transmembrane domain (TMD) topology is determined via a dynamic process in which topogenic signals in the nascent protein are recognized and interpreted initially by the translocon followed by a given lipid profile in accordance with the Positive Inside Rule. The net zero charged phospholipid phosphatidylethanolamine and other neutral lipids dampen the translocation potential of negatively charged residues in favor of the cytoplasmic retention potential of positively charged residues (Charge Balance Rule). This explains why positively charged residues are more potent topological signals than negatively charged residues. Dynamic changes in orientation of TMDs during or after membrane insertion are attributed to non-sequential cooperative and collective lipid–protein charge interactions as well as long-term interactions within a protein. The proportion of dual topological conformers of a membrane protein varies in a dose responsive manner with changes in the membrane lipid composition not only in vivo but also in vitro and therefore is determined by the membrane lipid composition. Switching between two opposite TMD topologies can occur in either direction in vivo and also in liposomes (designated as fliposomes) independent of any other cellular factors. Such lipid-dependent post-insertional reversibility of TMD orientation indicates a thermodynamically driven process that can occur at any time and in any cell membrane driven by changes in the lipid composition. This dynamic view of protein topological organization influenced by the lipid environment reveals previously unrecognized possibilities for cellular regulation and understanding of disease states resulting from mis-folded proteins. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Functional and topological analysis of phosphatidylcholine synthase from Sinorhizobium meliloti

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of eubacteria. It can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation pathway or the phosphatidylcholine synthase (Pcs) pathway. Pcs belongs to the CDP-alcohol phosphotransferase superfamily and synthesizes PC and CMP in one step from CDP-diacylglycerol and choline. In this study, we aligned sequences of characterized Pcs enzymes to identify conserved amino acid residues. Alanine scanning mutagenesis was performed on 55 of these conserved residues. The mutation of nine residues caused a drastic to complete loss (< 20% of wild type activity) of Pcs activity. Six of these essential residues were subjected to further mutagenesis studies replacing them by amino acids with similar properties or size. A topological analysis of sinorhizobial Pcs showed the presence of eight transmembrane helices, with the C- and N-terminus located in the cytoplasm. The majority of the conserved residues is predicted to be either located within the cytoplasmic loops or on the cytoplasmic side of the membrane which can be expected for an enzyme using one membrane-associated and one soluble substrate.     

A retrospective: Use of Escherichia coli as a vehicle to study phospholipid synthesis and function

Although the study of individual phospholipids and their synthesis began in the 1920s first in plants and then mammals, it was not until the early 1960s that Eugene Kennedy using Escherichia coli initiated studies of bacterial phospholipid metabolism. With the base of information already available from studies of mammalian tissue, the basic blueprint of phospholipid biosynthesis in E. coli was worked out by the late 1960s. In 1970s and 1980s most of the enzymes responsible for phospholipid biosynthesis were purified and many of the genes encoding these enzymes were identified. By the late 1990s conditional and null mutants were available along with clones of the genes for every step of phospholipid biosynthesis. Most of these genes had been sequenced before the complete E. coli genome sequence was available. Strains of E. coli were developed in which phospholipid composition could be changed in a systematic manner while maintaining cell viability. Null mutants, strains in which phospholipid metabolism was artificially regulated, and strains synthesizing foreign lipids not found in E. coli have been used to this day to define specific roles for individual phospholipid. This review will trace the findings that have led to the development of E. coli as an excellent model system to study mechanisms underlying the synthesis and function of phospholipids that are widely applicable to other prokaryotic and eukaryotic systems. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Stabilization of the Tertiary Structure of the Cholera Toxin A1 Subunit Inhibits Toxin Dislocation and Cellular Intoxication

Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. The catalytic subunit of CT (CTA1) then crosses the ER membrane and enters the cytosol in a process that involves the quality control mechanism of ER-associated degradation. The molecular details of this dislocation event have not been fully characterized. Here, we report that thermal instability in the CTA1 subunit—specifically, the loss of CTA1 tertiary structure at 37 °C—triggers toxin dislocation. Biophysical studies found that glycerol preferentially stabilized the tertiary structure of CTA1 without having any noticeable effect on the thermal stability of its secondary structure. The thermal disordering of CTA1 tertiary structure normally preceded the perturbation of its secondary structure, but in the presence of 10% glycerol the temperature-induced loss of CTA1 tertiary structure occurred at higher temperatures in tandem with the loss of CTA1 secondary structure. The glycerol-induced stabilization of CTA1 tertiary structure blocked CTA1 dislocation from the ER and instead promoted CTA1 secretion into the extracellular medium. This, in turn, inhibited CT intoxication. Glycerol treatment also inhibited the in vitro degradation of CTA1 by the core 20S proteasome. Collectively, these findings indicate that toxin thermal instability plays a key role in the intoxication process. They also suggest the stabilization of CTA1 tertiary structure is a potential goal for novel antitoxin therapeutic agents.     

Phosphatidylcholine biosynthesis and function in bacteria

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of the domain Bacteria. Usually, PC can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation (Pmt) pathway or the phosphatidylcholine synthase (Pcs) pathway. The three subsequent enzymatic methylations of phosphatidylethanolamine are performed by a single phospholipid N-methyltransferase in some bacteria whereas other bacteria possess multiple phospholipid N-methyltransferases each one performing one or several distinct methylation steps. Phosphatidylcholine synthase condenses choline directly with CDP-diacylglycerol to form CMP and PC. Like in eukaryotes, bacterial PC also functions as a biosynthetic intermediate during the formation of other biomolecules such as choline, diacylglycerol, or diacylglycerol-based phosphorus-free membrane lipids. Bacterial PC may serve as a specific recognition molecule but it affects the physicochemical properties of bacterial membranes as well. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Nuclear localization of profilin III-ArpM1 complex in mouse spermiogenesis

Actin-related proteins (Arps) have been reported to be localized in the cell nucleus, and implicated in the regulation of chromatin and nuclear structure, as well as being involved in cytoplasmic functions. We demonstrate here that mouse ArpM1, which closely resembles the conventional actin, is expressed exclusively in the testis, particularly in haploid germ cells. ArpM1 protein first appears in the round spermatid and changes its localization dynamically in the nucleus during spermiogenesis. By co-immunoprecipitation analysis, profilin III was identified as ArpM1-interacting protein. Our findings suggest that the testis-specific profilin III-ArpM1 complex may be involved in conformational changes in the organization of the sperm-specific nucleus. STRUCTURED SUMMARY:     

Transmembrane Segments Form Tertiary Hairpins in the Folding Vestibule of the Ribosome

Folding of membrane proteins begins in the ribosome as the peptide is elongated. During this process, the nascent peptide navigates along 100 Å of tunnel from the peptidyltransferase center to the exit port. Proximal to the exit port is a “folding vestibule” that permits the nascent peptide to compact and explore conformational space for potential tertiary folding partners. The latter occurs for cytosolic subdomains but has not yet been shown for transmembrane segments. We now demonstrate, using an accessibility assay and an improved intramolecular crosslinking assay, that the helical transmembrane S3b–S4 hairpin (“paddle”) of a voltage-gated potassium (Kv) channel, a critical region of the Kv voltage sensor, forms in the vestibule. S3–S4 hairpin interactions are detected at an early stage of Kv biogenesis. Moreover, this vestibule hairpin is consistent with a closed-state conformation of the Kv channel in the plasma membrane.     

N-glycosylation is required for efficient secretion of a novel human secreted glycoprotein, hPAP21

The present study reported the isolation and characterization of a novel human secreted protein, named as hPAP21 (human protease-associated domain-containing protein, 21 kDa), encoded by the hypothetical gene chromosome 2 open reading frame 7 (C2orf7) that contains signal peptide in its N-terminus, without transmembrane domain, except for PA domain in its middle. Western blotting assay indicated that the c-Myc tagged hPAP21 could be secreted into culture medium in the transfected Chinese hamster ovary cells. However, the molecular weights, whatever intracellular (28 kDa) or extracellular (30 kDa) forms, are larger than that of the prediction. To define whether the glycosylation was important process for its secretion, endoglycosidase H (Endo H) and PNGase F (PNG F) were employed to evaluate the effect of glycosylation types on secretion of hPAP21. Interestingly, the extracellular forms were primarily sensitive to PNG F, not Endo H, implying that complex N-glycosylation could be required for the secretion of hPAP21. Furthermore, N-glycosylation of Asn171 was confirmed as potential crucial process for the secretory protein via site-directed mutagenesis assay. All data will be contributed to the understanding of molecular functions of hPAP21.     

Role of the carboxyl terminal stop transfer sequence of UGT1A6 membrane protein in ER targeting and translocation of upstream lumenal domain

We investigated the role of the stop transfer sequence of human UGT1A6 in ER assembly and enzyme activity. We found that this sequence was able to address and translocate the upstream lumenal domain into microsomal membranes in vitro co- and posttranslationally. The signal activity of this sequence was further demonstrated in HeLa cells by its ability to target and maintain the CD4 protein deleted from both the N-terminal signal peptide and C-terminal transmembrane domain into the ER. We showed that total or partial deletion of the stop transfer sequence of UGT1A6 severely impaired enzyme activity highlighting its importance in both membrane assembly and function.     

Modulation of the in vitro activity of lysosomal phospholipase A1 by membrane lipids

Lysosomal phospholipases play a critical role for degradation of cellular membranes after their lysosomal segregation. We investigated the regulation of lysosomal phospholipase A1 by cholesterol, phosphatidylethanolamine, and negatively-charged lipids in correlation with changes of biophysical properties of the membranes induced by these lipids. Lysosomal phospholipase A1 activity was determined towards phosphatidylcholine included in liposomes of variable composition using a whole-soluble lysosomal fraction of rat liver as enzymatic source. Phospholipase A1 activity was then related to membrane fluidity, lipid phase organization and membrane potential as determined by fluorescence depolarization of DPH, 31P NMR and capillary electrophoresis. Phospholipase A1 activity was markedly enhanced when the amount of negatively-charged lipids included in the vesicles was increased from 10 to around 30% of total phospholipids and the intensity of this effect depended on the nature of the acidic lipids used (ganglioside GM1相似文献   

The synthesis of a DOPA analogue, 1-amino-2-(3, 4-dihydroxyphenyl)-ethylphosphonic acid and distribution studies in mice bearing the Harding-Passey melanoma

The aminophosphonic acid analogue of DOPA, DL-1-amino-2-(3,4-dihydroxy-phenyl) ethylphosphonic acid (ADEP) has been synthesised. The compound was of low toxicity; a single dose of 2 g/kg given to mice subcutaneously was not lethal.[³H]ADEP was injected subcutaneously into mice carrying the established Harding-Passey melanoma, and the distribution of the tritium determined. The highest initial concentration of radioactivity was in the kidneys, adrenal glands and eyes. Isotope content fell to low values in all tissues within 8 days or less but the tumour retained radioactivity for a longer period than did the other tissues examined.ADEP served as a substrate for mushroom tyrosinase.     

Metabolism, function and mass spectrometric analysis of bis(monoacylglycero)phosphate and cardiolipin

Polyglycerophospholipids (PGPLs) such as bismonoacylglycerophosphate (BMP) and cardiolipin are important membrane phospholipid species for the maintenance of membrane integrity. While BMP serves as membrane curvature regulator in multivesicular bodies for efficient lysosomal enzyme function, cardiolipin stabilizes the electron transfer complex in the inner mitochondrial membrane, which is crucial for physiological ATP production. Beside their membrane modulatory functions PGPLs play an important role in various signaling events. Although a number of disease associations were found for PGPL species, detailed information about their molecular role still remains unknown. This article reviews the known biological functions of PGPLs and the existing mass spectrometric methods. We discuss the different analytical strategies and how ESI–MS/MS can expand our understanding of PGPL homeostasis.     

Immunocytochemical analysis for intracellular dynamics of C1GalT associated with molecular chaperone, Cosmc

The core 1 structure Galβ1-3GalNAcα1-Ser/Thr (T antigen), the major constituent of O-glycan core structure, is synthesized by cooperation of core 1 synthase (C1GalT) and its specific molecular chaperone, Cosmc. The chaperone function of Cosmc has been well investigated biochemically. In this study, we established monoclonal antibodies specifically recognizing either C1GalT or Cosmc, respectively, and investigated the sub-cellular localization of each protein to elucidate how they cooperate to synthesize the core 1 structure.A sequential immunocytochemical analysis of the human colon cancer cell line, LSB, demonstrated different localization of two proteins. C1GalT was localized in Golgi apparatus, while Cosmc was localized in endoplasmic reticulum. In contrast, the LSC cells, which do not have core 1 synthase activity due to a missense mutation in the Cosmc gene, did not express the C1GalT protein. Although the treatment with a proteasome inhibitor, lactacystin, of LSC cells resulted in the increased expression of C1GalT protein, the distribution of C1GalT was not in Golgi apparatus as seen in LSB cells. On the contrary, overexpression of Cosmc but not C1GalT lead to precise localization of C1GalT protein, which distributed in Golgi apparatus and recovered the core 1 synthase activity in LSC cells. These results suggest that the intracellular dynamics of C1GalT is controlled by its specific molecular chaperon, Cosmc, in association with core 1 synthase activity.     

The nucleotide exchange factor activity of Grp170 may explain the non-lethal phenotype of loss of Sil1 function in man and mouse

Recent genetic work characterized homozygous mutations in the SIL1 gene as cause for the neurodegeneration that is associated with Marinesco-Sj?gren syndrome in man and the woozy mouse mutant. All reported mutations were expected to result in loss of Sil1 function. Sil1 has previously been shown to act as nucleotide exchange factor for the molecular chaperone immunoglobulin heavy chain binding protein (BiP) in the lumen of the endoplasmic reticulum (ER). In the yeast ER Lhs1p was shown to be able to substitute for Sil1p and to represent an alternative nucleotide exchange activity. Therefore, by analogy the mammalian ortholog of Lhs1p, Grp170, was suggested to be able to compensate for the loss of Sil1 function in many mammalian organs. Here we characterize mammalian Grp170 as alternative nucleotide exchange factor for BiP, thus providing a likely explanation for the non-lethal phenotype of the homozygous human and murine SIL1 mutations.     

Disrupted plasma membrane localization and loss of function reveal regions of human equilibrative nucleoside transporter 1 involved in structural integrity and activity

Human Equilibrative Nucleoside Transporter 1 (hENT1) is an integral membrane protein that transports nucleosides and analog drugs across cellular membranes. Very little is known about intracellular processing and localization of hENT1. Here we show that disruption of a highly conserved triplet (PWN) near the N-terminus, or the last eight C-terminal residues (two hydrophobic triplets separated by a positive arginine) result in loss of plasma membrane localization and/or transport function. To understand the role of specific residues within these regions, we studied the localization patterns of N- or C-terminal deletion and/or substitution mutants of GFP-hENT1 using confocal microscopy. Quantification of GFP-hENT1 (mutant and wildtype) protein at the plasma membrane was conducted using nitrobenzylthioinosine (NBTI) binding. Functionality of the GFP-hENT1 mutants was determined by heterologous expression in Xenopus laevis oocytes followed by measurement of uridine uptake. Mutation of the proline within the PWN motif disrupts plasma membrane localization. C-terminal mutations (primarily within the hydrophobic triplets) lead to hENT1 retention within the cell (e.g. in the ER). Some mutants still localize to the plasma membrane but show reduced transport activity. These data suggest that these two regions contribute to the structural integrity and thus correct processing and function of hENT1.     

Lipid domains in bacterial membranes and the action of antimicrobial agents

There has been increasing interest in recent years in describing the lateral organization of membranes and the formation of membrane domains. Much of the focus in this area has been on the formation of cholesterol-rich domains in mammalian membranes. However, it is likely that there are domains in all biological membranes. One of the challenges has been to define the chemical composition, lifetime and size of these domains. There is evidence that bacteria have domains that are enriched in cardiolipin. In addition, the formation of lipid domains can be induced in bacteria by clustering negatively charged lipids with polycationic substances. Many antimicrobial compounds have multiple positive charges. Such polycationic compounds can sequester anionic lipids to induce lipid phase separation. The molecular interactions among lipids and their lateral packing density will be different in a domain from its environment. This will lead to phase boundary defects that will lower the permeability barrier between the cell and its surroundings. The formation of these clusters of anionic lipids may also alter the stability or composition of existing membrane domains that may affect bacterial function. Interestingly many antimicrobial agents are polycationic and therefore likely have some effect in promoting lipid phase segregation between anionic and zwitterionic lipids. However, this mechanism is expected to be most important for substances with sequential positive charges contained within a flexible molecule that can adapt to the arrangement of charged groups on the surface of the bacterial cell. When this mechanism is dominant it can allow the prediction of the bacterial species that will be most affected by the agent as a consequence of the nature of the lipid composition of the bacterial membrane.     

Inactivation of microsomal triglyceride transfer protein impairs the normal redistribution but not the turnover of newly synthesized glycerolipid in the cytosol,endoplasmic reticulum and Golgi of primary rat hepatocytes

The requirements for microsomal triglyceride transfer protein (MTP) during the turnover and transfer of glycerolipids from intracellular compartments into secretory very low-density lipoprotein (VLDL) were studied by pre-labelling lipids with [³H]glycerol and [¹⁴C]oleate in primary cultures of rat hepatocytes. The intracellular redistribution of pre-labelled glycerolipids was then compared at the end of subsequent chase periods during which the MTP inhibitor BMS-200150 was either present or absent in the medium. Inhibition of MTP resulted in a decreased output of VLDL triacylglycerol (TAG) and a delayed removal of labelled TAG from the cytosol and from the membranes of the smooth endoplasmic reticulum (SER), the cis- and the trans-Golgi. Inactivation of MTP did not decrease the bulk lipolytic turnover of cellular TAG as reflected by changes in its [³H]glycerol:[¹⁴C]oleate ratios. However, a larger proportion of the resultant TAG fatty acids was re-esterified and remained with the membranes of the various subcellular fractions rather than emerging as VLDL. The effects of BMS-200150 on the pattern of phospholipid (PL) mechanism and redistribution suggested that inhibition of MTP prevented the normal lipolytic transfer of PL-derived fatty acids out of the SER, cis- and trans-Golgi membrane pools. Finally, changes in the ¹⁴C specific radioactivities of the cytosolic and membrane pools of TAG suggested that inhibition of MTP prevented a normal influx of relatively unlabelled fatty acids into these pools during the chase period.     

Mechanism of dimerization of the human melanocortin 1 receptor

The melanocortin 1 receptor (MC1R) is a dimeric G protein-coupled receptor expressed in melanocytes, where it regulates the amount and type of melanins produced and determines the tanning response to ultraviolet radiation. We have studied the mechanisms of MC1R dimerization. Normal dimerization of a deleted mutant lacking the seventh transmembrane fragment and the C-terminal cytosolic extension excluded coiled-coil interactions as the basis of dimerization. Conversely, the electrophoretic pattern of wild type receptor and several Cys → Ala mutants showed that four disulfide bonds are established between the monomers. Disruption of any of these bonds abolished MC1R function, but only the one involving Cys35 was essential for traffic to the plasma membrane. A quadruple Cys35-267-273-275Ala mutant migrating as a monomer in SDS-PAGE in the absence of reducing agents was able to dimerize with WT, suggesting that in addition to disulfide bond formation, dimerization involves non-covalent interactions, likely of domain swap type.     

Altered lipid A structures and polymyxin hypersensitivity of Rhizobium etli mutants lacking the LpxE and LpxF phosphatases

The lipid A of Rhizobium etli, a nitrogen-fixing plant endosymbiont, displays significant structural differences when compared to that of Escherichia coli. An especially striking feature of R. etli lipid A is that it lacks both the 1- and 4′-phosphate groups. The 4′-phosphate moiety of the distal glucosamine unit is replaced with a galacturonic acid residue. The dephosphorylated proximal unit is present as a mixture of the glucosamine hemiacetal and an oxidized 2-aminogluconate derivative. Distinct lipid A phosphatases directed to the 1 or the 4′-positions have been identified previously in extracts of R. etli and Rhizobium leguminosarum. The corresponding structural genes, lpxE and lpxF, respectively, have also been identified. Here, we describe the isolation and characterization of R. etli deletion mutants in each of these phosphatase genes and the construction of a double phosphatase mutant. Mass spectrometry confirmed that the mutant strains completely lacked the wild-type lipid A species and accumulated the expected phosphate-containing derivatives. Moreover, radiochemical analysis revealed that phosphatase activity was absent in membranes prepared from the mutants. Our results indicate that LpxE and LpxF are solely responsible for selectively dephosphorylating the lipid A molecules of R. etli. All the mutant strains showed an increased sensitivity to polymyxin relative to the wild-type. However, despite the presence of altered lipid A species containing one or both phosphate groups, all the phosphatase mutants formed nitrogen-fixing nodules on Phaseolus vulgaris. Therefore, the dephosphorylation of lipid A molecules in R. etli is not required for nodulation but may instead play a role in protecting the bacteria from cationic antimicrobial peptides or other immune responses of plants.