Membrane Topology and Insertion of Membrane Proteins: Search for Topogenic Signals

Integral membrane proteins are found in all cellular membranes and carry out many of the functions that are essential to life. The membrane-embedded domains of integral membrane proteins are structurally quite simple, allowing the use of various prediction methods and biochemical methods to obtain structural information about membrane proteins. A critical step in the biosynthetic pathway leading to the folded protein in the membrane is its insertion into the lipid bilayer. Understanding of the fundamentals of the insertion and folding processes will significantly improve the methods used to predict the three-dimensional membrane protein structure from the amino acid sequence. In the first part of this review, biochemical approaches to elucidate membrane protein topology are reviewed and evaluated, and in the second part, the use of similar techniques to study membrane protein insertion is discussed. The latter studies search for signals in the polypeptide chain that direct the insertion process. Knowledge of the topogenic signals in the nascent chain of a membrane protein is essential for the evaluation of membrane topology studies.     

Dynamics of Axonal Microtubules Regulate the Topology of New Membrane Insertion into the Growing Neurites

Nerve growth depends on the delivery of cell body–synthesized material to the growing neuronal processes. The cellular mechanisms that determine the topology of new membrane addition to the axon are not known. Here we describe a technique to visualize the transport and sites of exocytosis of cell body– derived vesicles in growing axons. We found that in Xenopus embryo neurons in culture, cell body–derived vesicles were rapidly transported all the way down to the growth cone region, where they fused with the plasma membrane. Suppression of microtubule (MT) dynamic instability did not interfere with the delivery of new membrane material to the growth cone region; however, the insertion of vesicles into the plasma membrane was dramatically inhibited. Local disassembly of MTs by focal application of nocodazole to the middle axonal segment resulted in the addition of new membrane at the site of drug application. Our results suggest that the local destabilization of axonal MTs is necessary and sufficient for the delivery of membrane material to specific neuronal sites.     

Light-Harvesting Chlorophyll a/b Protein : Membrane Insertion, Proteolytic Processing, Assembly into LHC II, and Localization to Appressed Membranes Occurs in Chloroplast Lysates

The apoprotein of the light-harvesting chlorophyll a/b protein (LHCP) is a major integral thylakoid membrane protein that is normally complexed with chlorophyll and xanthophylls and serves as the antenna complex of photosystem II. LHCP is encoded in the nucleus and synthesized in the cytosol as a higher molecular weight precursor that is subsequently imported into chloroplasts and assembled into thylakoids. In a previous study it was established that the LHCP precursor can integrate into isolated thylakoid membranes. The present study demonstrates that under conditions designed to preserve thylakoid structure, the inserted LHCP precursor is processed to mature size, assembled into the LHC II chlorophyll-protein complex, and localized to the appressed thylakoid membranes. Under these conditions, light can partially replace exogenous ATP in the membrane integration process.     

Membrane proteins of Rhodopseudomonas spheroides III. Isolation,purification, and characterization of cell envelope proteins

Cell envelopes (cell wall and cell membrane) from aerobically grown cells of Rhodopseudomonas spheroides were isolated and purified by a combination of differential centrifugation and centrifugation through 40% sucrose. Cell envelope protein from aerobically grown cells was resolved by dodecyl sulphate-polyacrylamide gel electrophoresis. Biochemical characterization of selected envelope membrane proteins demonstrated heterogeneity between different protein species. Amino acid analyses of individual proteins revealed between 50–60 mole% nonpolar residues.Envelope membranes derived from anaerobically grown cells were also isolated and purified by a combination of differential centrifugation, column chromatography on Sepharose 2B, and centrifugation in 40% sucrose. The dodecyl sulphate-polyacrylamide gel patterns of anaerobic and aerobic envelope membrane proteins were very similar and the results suggest a common protein structure.     

Mutant Strains of Rhodopseudomonas spheroides Lacking δ-Aminolevulinate Synthase: Growth, Heme, and Bacteriochlorophyll Synthesis

Two mutant strains of Rhodopseudomonas spheroides were described which lacked delta-aminolevulinate synthase activity. They required delta-aminolevulinate for growth; they did not respond to protoporphyrin or magnesium photoporphyrin, and only poorly to hemin. Synthesis of cytochromes and heme by mutant H-4 was dependent upon delta-aminolevulinate; this strain did not form bacteriochlorophyll either with or without delta-aminolevulinate and, consequently, grew only under aerobic conditions. Mutant H-5 formed bacteriochlorophyll in response to delta-aminolevulinate and grew both anaerobically in the light and aerobically in the dark; the amount of delta-aminolevulinate needed for optimal anaerobic growth was higher than that required aerobically. Synthesis of bacteriochlorophyll and heme by suspensions of mutant H-5 incubated anaerobically in the light was dependent upon delta-aminolevulinate; bacteriochlorophyll production was completely inhibited by high aeration and by puromycin. The mutants differed in their ability to take up radioactive delta-aminolevulinate from the external environment; mutant H-5 was less active than mutant H-4 or the wild type. It was suggested that R. spheroides made only one form of delta-aminolevulinate synthase, which provided delta-aminolevulinate for bacteriochlorophyll and heme synthesis.     

Epitope Mapping of Antibodies Suggests the Novel Membrane Topology of B-Cell Receptor Associated Protein 31 on the Cell Surface of Embryonic Stem Cells: The Novel Membrane Topology of BAP31

When located in the endoplasmic reticulum (ER) membrane, B-cell receptor associated protein 31 (BAP31) is involved in the export of secreted proteins from the ER to the plasma membrane. In a previous study, we generated two monoclonal antibodies (mAbs), 297-D4 and 144-A8, that bound to surface molecules on human embryonic stem cells (hESCs), but not to surface molecules on mouse embryonic stem cells (mESCs). Subsequent studies revealed that the mAbs recognized BAP31 on the surface of hESCs. To investigate the membrane topology of BAP31 on the cell surface, we first examined the epitope specificity of 297-D4 and 144-A8, as well as a polyclonal anti-BAP31 antibody (α-BAP31). We generated a series of GST-fused BAP31 mutant proteins in which BAP31 was serially deleted at the C- terminus. GST-fused BAP31 mutant proteins were then screened to identify the epitopes targeted by the antibodies. Both 297-D4 and 144-A8 recognized C-terminal residues 208–217, while α-BAP31 recognized C-terminal residues 165–246, of BAP31 on hESCs, suggesting that the C-terminal domain of BAP31 is exposed on the cell surface. The polyclonal antibody α-BAP31 bound to mESCs, which confirmed that the C-terminal domain of BAP31 is also exposed on the surface of these cells. Our results show for the first time the novel membrane topology of cell surface-expressed BAP31 as the extracellular exposure of the BAP31 C-terminal domain was not predicted from previous studies.     

A Soluble Protein Factor is Required in Vitro for Membrane Insertion of the Thylakoid Precursor Protein, pLHCP

The precursor to the light-harvesting chlorophyll a/b protein of photosystem II can insert into isolated thylakoid membranes if reaction mixtures also contain ATP and a soluble extract of chloroplasts. Optimization of this insertion process and the initial characterization of the soluble chloroplastic component are presented. With a fixed amount of precursor, maximum integration rates occurred during the first 30 minutes at pH 8.0 and 30°C when the soluble chloroplast extract was increased eight-fold over the stoichiometric amount. Under these conditions, insertion was routinely about 60% of that which occurred during import into intact chloroplasts. Integration also increased virtually linearly with increasing amounts of precursor. However, assays revealed that at least 40% of the in vitro-synthesized pLHCP was pelletable and inactive. The soluble chloroplastic component exhibited characteristics expected of a protein. It was inactivated by heat, protease, and N-ethylmaleimide, but was insensitive to ribonuclease. The soluble component migrated on a Sephacryl S-200 gel filtration column as a single peak with an M_r of approximately 65,000. The proteinaceous nature of this factor suggests a similarity to soluble factors required for protein transport/integration in other membrane systems.     

Membrane Insertion and Biogenesis of the Turnip Crinkle Virus p9 Movement Protein

Plant viral infection and spread depends on the successful introduction of a virus into a cell of a compatible host, followed by replication and cell-to-cell transport. The movement proteins (MPs) p8 and p9 of Turnip crinkle virus are required for cell-to-cell movement of the virus. We have examined the membrane association of p9 and found that it is an integral membrane protein with a defined topology in the endoplasmic reticulum (ER) membrane. Furthermore, we have used a site-specific photo-cross-linking strategy to study the membrane integration of the protein at the initial stages of its biosynthetic process. This process is cotranslational and proceeds through the signal recognition particle and the translocon complex.Cell-to-cell transport of plant virus requires the virally encoded movement proteins (MPs). These proteins specialize in the translocation of the viral genome or, in some cases, the virions from the replication/encapsidation site to adjacent cells. This process takes place through the plasmodesmata (PD), the small pores formed by prolongations of the endoplasmic reticulum (ER) membranes trapped within the center of the plasma membrane-lined cytoplasmic cylinder that connect plant cells. MPs belong to different protein families with unique functional and structural characteristics. The most studied MP is p30 from the Tobacco mosaic virus, a 30-kDa RNA-binding protein (4) with two putative transmembrane (TM) segments (2) that has so far been considered an integral membrane protein (13, 42). At an early stage of infection, p30 associates with the ER network (18, 59). Given that the ER is continuous through PD, it was suggested that the movement complex transports cell to cell via the PD. On the other hand, passage through the connecting structure largely remains a mystery, although it seems reasonable that the process again occurs in close juxtaposition to the ER-derived membrane (desmotubule) that runs through the PD (12, 35). Many other plant viruses have a cell-to-cell transport system based not on one but on two (double-gene block [DGB]) or even three (triple-gene block [TGB]) MPs. In some of these cases it has been shown that at least one MP is closely associated with the ER membrane (28, 34, 41, 50, 55). Thus, it has been assumed that other MPs associate similarly with membranes.The targeting and insertion of an integral membrane protein can occur either posttranslationally, in which the protein is completely synthesized on cytosolic ribosomes before being inserted, or cotranslationally, in which protein synthesis and integration into the ER membrane are coupled. In the latter case, the targeting of the ribosome-mRNA-nascent chain complex to the membrane depends on the signal recognition particle (SRP) and its interaction with the membrane-bound SRP receptor (11), which is located in close proximity to the translocon. The translocon, a multiprotein complex composed of the Sec61α, -β, and -γ subunits (16) and the translocating chain-associated membrane protein (TRAM) (15) in eukaryotic cells, facilitates the translocation of soluble proteins into the ER lumen and the insertion of integral membrane proteins into the lipid bilayer (24).Plant virus infection depends on the proper targeting and association or insertion of the movement proteins with or into the ER membrane. In this report, we investigate the insertion into, topology of, and targeting to the membrane of the p9 MP from Turnip crinkle virus (TCV). This is a positive-sense single-stranded RNA virus that belongs to the Carmovirus genus and thus to the DGB. Its 4-kb genome encodes five open reading frames (ORFs) (3, 17). Translation of the first two yields p28 and p88, both implicated in viral RNA synthesis. In the central region, two overlapping ORFs encode the small proteins p8 and p9, which have been shown to be involved in cell-to-cell movement (6, 17, 31). The RNA-binding protein p8 (17, 58) overlaps the distal 3′ region of the replicase p88. The 3′ region of the genome encodes the viral coat protein p38, and its 5′ end overlaps p9 (3).A strong interaction with the membrane is expected for p9 due to the close similarities in the genomic arrangement of TCV (57) with other carmoviruses, like Carnation mottle virus (CarMV) and Melon necrotic spot virus (MNSV). Both CarMV and MNSV have two small MPs, one an RNA-binding protein (39, 53, 54) and the other a cotranslationally inserted integral membrane protein (34, 47, 55). In this study, we present evidence of the integration of TCV p9 into ER-derived microsomal membranes. Using an in vitro translation system based on a model integral membrane protein, we have been able to identify two membrane-spanning domains. Additionally, the membrane topology of the p9 MP was analyzed in vitro and found to have an N terminus (N-t)/C terminus (C-t) luminal orientation. Finally, using a site-directed photo-cross-linking approach, we demonstrated that the mechanism of p9 insertion into the ER membrane involves SRP and the translocon.     

Bacteriochlorophyll and Heme Synthesis in Rhodopseudomonas spheroides: Possible Role of Heme in Regulation of the Branched Biosynthetic Pathway

Synthesis of heme, measured by incorporation of iron-59, and of bacteriochlorophyll was studied with wild-type and mutant strains of Rhodopseudomonas spheroides. The wild type formed heme from glycine and succinate at one-fortieth the rate of bacteriochlorophyll under anaerobic-light conditions. Added delta-aminolevulinate stimulated heme synthesis 10-fold without increasing bacteriochlorophyll production. Heme synthesis from glycine and succinate was increased when the magnesium branch of the biosynthetic path was curtailed by mutation or by p-fluorophenylalanine or 8-azaguanine. Synthesis of bacteriochlorophyll by the wild type from glycine and succinate stopped immediately after addition of puromycin, but heme production continued for a period. Porphyrins and other precursors did not appear upon addition of puromycin alone, but simultaneous addition of o-phenanthroline resulted in the accumulation of coproporphyrin. Production of this porphyrin by a mutant strain with impaired ability to form heme was unaffected by puromycin. Heme synthesis from glycine and succinate or from delta-aminolevulinate was decreased by limitation of methionine; it is suggested that coproporphyrin accumulation from glycine and succinate under conditions of methionine deficiency results from relief of feedback inhibition of delta-aminolevulinate synthase by heme. The development of delta-aminolevulinate synthase activity in response to low aeration is prevented by addition of delta-aminolevulinate. This repressive action of the latter is abolished when its conversion to heme is impeded by mutation or by methionine deficiency. It is suggested that heme, the quantitatively minor end product of the branched biosynthetic pathway, may regulate the flow of common intermediates when utilization of protoporphyrin by the magnesium branch is diminished. This regulation may be exerted by feedback inhibition of delta-aminolevulinate synthase and also by repression of enzyme formation.     

发菜类囊体膜色素蛋白复合物分离及其谱性质的研究

Nostoc flagelliforme Born. et Flah is highly adapted to drought stress, cold and light stresses, and suitable for growing in the unfavorable areas. This paper presents the results of the analysis of the membrane (mainly thylakoid membrane) lipids from N. flagelliforme in order to investigate the relationship between membrane lipid composition and stress resistance to this cyanobacteria. The membrane lipids are composed of monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), sulfoquinovosyl diacylglycerol (SQDG) and phosphatidylglycerol (PG). The major fatty acids in these lipids are palmitic (16∶0), palmitoleic (16∶1), stearic (18∶0), oleic (18∶1), linoleic (18∶2) and linolenic (18∶3) acids. In N. flagelliforme, polyunsaturated fatty acids account for 73% of the total fatty acids, much higher than that of the other cyanobacteria reported so far. Among which 16∶1 and 18∶3 are as high as 28.9% and 34.3% respectively. The high resistance of N. flagelliforme to abnormal conditions may be associated with the extent of unsaturation of fatty acids. In addition, the wild N. flagelliforme treated with water for 30 min and cultured for 24 h and the lipid and fatty acid composition were found to be not affected by water-absorption.     

发菜类囊体膜色素蛋白复合物分离及其光谱性质的研究

采用改进的Ａｌｌｅｎ’ｓ的绿胶系统,首次对陆生蓝藻发菜（Ｎｏｓｔｏｃ ｆｌａｇｅｌｌｉｆｏｒｍｅ Ｂｏｒｎ．ｅｔ Ｆｌａｈ．）类囊体膜色素蛋白复合物进行了分离,共分离出了１１条绿色的色素蛋白复合物条带。两条浅黄色的条带。其中７条绿色条带属于ＰＳⅠ组分,４条绿色条带属于ＰＳⅡ组分,１条浅黄色条带经光谱分析初步认定为类胡萝卜素蛋白复合物,而另一条浅黄色条带为游离色素。     

Insertion of a MalE β-Galactosidase Fusion Protein into the Envelope of Escherichia coli Disrupts Biogenesis of Outer Membrane Proteins and Processing of Inner Membrane Proteins

The synthesis of a membrane-bound MalE β-galactosidase hybrid protein, when induced by growth of Escherichia coli on maltose, leads to inhibition of cell division and eventually a reduced rate of mass increase. In addition, the relative rate of synthesis of outer membrane proteins, but not that of inner membrane proteins, was reduced by about 50%. Kinetic experiments demonstrated that this reduction coincided with the period of maximum synthesis of the hybrid protein (and another maltose-inducible protein, LamB). The accumulation of this abnormal protein in the envelope therefore appeared specifically to inhibit the synthesis, the assembly of outer membrane proteins, or both, indicating that the hybrid protein blocks some export site or causes the sequestration of some limiting factor(s) involved in the export process. Since the MalE protein is normally located in the periplasm, the results also suggest that the synthesis of periplasmic and outer membrane proteins may involve some steps in common. The reduced rate of synthesis of outer membrane proteins was also accompanied by the accumulation in the envelope of at least one outer membrane protein and at least two inner membrane proteins as higher-molecular-weight forms, indicating that processing (removal of the N-terminal signal sequence) was also disrupted by the presence of the hybrid protein. These results may indicate that the assembly of these membrane proteins is blocked at a relatively late step rather than at the level of primary recognition of some site by the signal sequence. In addition, the results suggest that some step common to the biogenesis of quite different kinds of envelope protein is blocked by the presence of the hybrid protein.