Specific Removal of Proteins from the Envelope of Escherichia coli by Protease Treatments

When the envelope fraction of Escherichia coli was treated by trypsin, about 40% of total envelope proteins were removed from the fraction without changing its phospholipid content. Analysis of envelope proteins by acrylamide gel electrophoresis in 0.5% sodium dodecyl sulfate revealed that trypsin treatment was very specific; one of the major proteins (molecular weight, 38,000) and all proteins of molecular weight greater than 70,000 were completely removed by the treatment. On the other hand, three other major proteins were found to be resistant to the treatment, including protein Y, which was previously shown to be related to deoxyribonucleic acid replication. The trypsin treatment of the envelope fractions composed of a five electron-dense layered structure formed vesicles with a triple-layered membrane (two electron-dense layers). Pronase treatment of the envelope fraction removed about 60% of the envelope proteins without changing its phospholipid content. A major protein of molecular weight of 58,000 was found to be the only protein resistant to the Pronase treatment. Application of these treatments is useful for purification and structural studies of envelope proteins.     

Increased degradation rates of protein synthesized in hepatoma cells in the presence of amino acid analogues.

1. Reuber H35 hepatoma cells incorporate the arginine analogue canavanine into cell protein when arginine is omitted from the incubation medium. 2. By labelling arginine-containing proteins with (14-C)leucine and then canavanine-containing proteins with (3-H)leucine in the same cells, it is possible to measure the degradation of both types of protein during a subsequent 'chase' period. With this technique it has been shown that canavanine-containing proteins are degraded at a rate severalfold greater than normal proteins. Comparable results were found when 6-fluorotryptophan was used as an analogue to tryptophan. 3. Control experiments in which the labelling order was reversed or where the animo acid and its analogue were incubated in separate cell cultures support the conclusion that abberrant proteins are rapidly degraded in vivo.     

Proteins Released from Cell Envelopes of Pseudomonas aeruginosa on Exposure to Ethylenediaminetetraacetate: Comparison with Dimethylformamide-Extractable Proteins

Isolated cell envelopes of Pseudomonas aeruginosa were treated with N,N'-dimethylformamide (DMF) or with ethylenediaminetetraacetate (EDTA). DMF solubilized 73% of the dry weight of the cell envelope, 76% of the protein, 78% of the carbohydrate, and 76% of the phosphorus. Electron microscopy showed that DMF caused extensive alterations in the appearance of the cell envelope with blebs and bleblike vesicles predominating. After incubation with EDTA, the cell envelopes appeared to have lost material, but still retained the cell-like morphology. Analysis of DMF-solubilized proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed 16 protein bands. There were three major proteins that predominated, however, with molecular masses of 43,000 (protein A), 16,500 (protein B), and 72,000 daltons (protein C). Evidence is presented that protein A and protein B are glycoproteins. Gel electrophoresis of EDTA-solubilized material revealed that a number of proteins were released from the cell envelope. However, electrophoresis of an isolated protein-lipopolysaccharide complex released by EDTA showed that protein A and protein B were the major protein components of this complex. These data suggest that protein A and protein B are components of the outer cell wall membrane of P. aeruginosa. There is suggestive evidence that these proteins may play a role in maintaining the structural integrity of the cell envelope. Whether these proteins also have enzymatic activity could not be discerned from the present study, although it is possible that they may be associated with the terminal stages of lipopolysaccharide synthesis.     

Large-Molecular-Weight Precursors of Sindbis Virus Proteins

Infection of chicken embryo fibroblasts with a temperature-sensitive mutant of Sindbis virus at the nonpermissive temperature leads to the accumulation of a large-molecular-weight protein. We have shown that this protein contains (14)C-arginine tryptic peptides present in the three virion proteins. We have also found that a slightly smaller protein which is detected in Sindbis-infected BHK cells contains the (14)C-arginine tryptic peptides of the two envelope proteins but not those of the capsid protein. Pulse-chase experiments indicate that the Sindbis virus protein in BHK cells is cleaved to the envelope proteins.     

Incorporation of canavanine into rat pars intermedia proteins inhibits the maturation of pro-opiomelanocortin, the common precursor to adrenocorticotropin and beta-lipotropin

Pro-opiomelanocortin, the common glycoprotein precursor to adrenocorticotropin and beta-lipotropin, is the most abundant protein synthesized in rat neurointermediate lobes. Dissected rat neurointermediate lobes were incubated in the presence of canavanine, an analog of arginine, to determine (a) whether canavanine could be incorporated into pro-opiomelanocortin molecules and (b) if incorporation occurs, whether there is any effect on the processing mechanism of the prohormone. Preincubation of rat neurointermediate lobes for 16 h in the presence of 10 mM canavanine results in the production of pro-opiomelanocortin molecules in which most, if not all, the arginine residues have been replaced by canavanine. Identification of canavanine-containing pro-opiomelanocortin forms was done by two-dimensional electrophoresis, tryptic and chymotryptic peptide mapping, as well as by analysis, on polyacrylamide gels in the presence of sodium dodecyl sulfate, of the fragments resulting from a partial digestion with chymotrypsin. During pulse-chase experiments, canavanine-containing pro-opiomelanocortin molecules were found to be processed at a much slower rate than the normal precursor forms: after a 2-h chase, conversion of approximately 25% of the analog-containing prophormone was observed compared to 83% of the nonanalog-containing precursors. Moreover, the small proportion of canavanine-containing precursor molecules which had undergone cleavage during the chase yielded atypical large molecular weight peptides. These results indicate that canavanine incorporation into neurointermediate lobe proteins considerably slows down the conversion of pro-opiomelanocortin into its different end products.     

Formation of Sindbis Virus Proteins: Identification of a Precursor for One of the Envelope Proteins

Exposure of Sindbis virus-infected chicken embryo cells to a short pulse of radioactive amino acids revealed the formation of primarily three proteins: the nucleocapsid (C) of the virus, one of the viral envelope proteins (E1), and a glycoprotein that did not appear in the virion. This third protein (PE2) has now been identified as a precursor of the other viral envelope protein (E2) on the basis of two observations: (i) the simultaneous disappearance of radioactive PE2 and appearance of labeled E2 in pulse-chase experiments, and (ii) the identity of (14)C-arginine tryptic peptides in fingerprints of the two proteins. The nucleocapsid was the most heavily labeled protein in the cell and appeared in the virus during the short pulse. The two (14)C-labeled envelope proteins, although having different kinetics of labeling in the cell, appeared simultaneously in the virus only after the chase. Addition of pactamycin, a drug inhibiting initiation of protein synthesis, preferentially inhibited the formation of capsid protein Assuming that Sindbis virus proteins are formed initially as a single polypeptide, our studies locate the nucleocapsid at the amino-terminal end of the polypeptide chain.     

Analysis of Pseudomonas aeruginosa Cell Envelope Proteome by Capture of Surface-Exposed Proteins on Activated Magnetic Nanoparticles

We report on specific magneto-capturing followed by Multidimensional Protein Identification Technology (MudPIT) for the analysis of surface-exposed proteins of intact cells of the bacterial opportunistic pathogen Pseudomonas aeruginosa. The magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction allowed the MudPIT identification of the captured and neighboring proteins. Remarkably, we identified 63 proteins captured directly by nanoparticles and 67 proteins embedded in the cell envelope fragments. For a high number of proteins, our analysis strongly indicates either surface exposure or localization in an envelope district. The localization of most identified proteins was only predicted or totally unknown. This novel approach greatly improves the sensitivity and specificity of the previous methods, such as surface shaving with proteases that was also tested on P. aeruginosa. The magneto-capture procedure is simple, safe, and rapid, and appears to be well-suited for envelope studies in highly pathogenic bacteria.     

Tegument Proteins of Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a common, medically relevant human herpesvirus. The tegument layer of herpesvirus virions lies between the genome-containing capsids and the viral envelope. Proteins within the tegument layer of herpesviruses are released into the cell upon entry when the viral envelope fuses with the cell membrane. These proteins are fully formed and active and control viral entry, gene expression, and immune evasion. Most tegument proteins accumulate to high levels during later stages of infection, when they direct the assembly and egress of progeny virions. Thus, viral tegument proteins play critical roles at the very earliest and very last steps of the HCMV lytic replication cycle. This review summarizes HCMV tegument composition and structure as well as the known and speculated functions of viral tegument proteins. Important directions for future investigation and the challenges that lie ahead are identified and discussed.     

被膜蛋白糖基化在HIV感染中的作用

在HIV感染过程中,病毒被膜蛋白糖基化起着重要作用。它使病毒粒子具有高度糖基化的表面,帮助HIV逃避人体免疫细胞识别和攻击。在病毒入侵时,被膜糖蛋白与宿主细胞表面的受体结合,并进行一系列构象变化,使病毒粒子顺利地与宿主细胞膜融合。介绍近年来对被膜蛋白糖基化过程与HIV成熟、感染和逃避免疫应答等方面分子水平作用机理的深入了解,这些作用机理将会有助于艾滋病疫苗的研制和以“糖链为靶”药物的开发。     

Enzymatic Radioiodination of the Envelope Proteins of Avian Myeloblastosis Virus

Purified avian myeloblastosis virus was iodinated by the lactoperoxidase method. Disruption of the labeled virions and chromatography of the viral proteins showed that radioiodine was associated only with the glycoproteins of the viral envelope. Reaction of the radiolabeled virus with antiviral antibody followed by mild detergent treatment and subsequent recovery of immune complexes showed this technique to be useful for the isolation of viral envelope antigens.     

Distinct Pathways Mediate the Sorting of Tail-Anchored Proteins to the Plastid Outer Envelope

Background

Tail-anchored (TA) proteins are a distinct class of membrane proteins that are sorted post-translationally to various organelles and function in a number of important cellular processes, including redox reactions, vesicular trafficking and protein translocation. While the molecular targeting signals and pathways responsible for sorting TA proteins to their correct intracellular destinations in yeasts and mammals have begun to be characterized, relatively little is known about TA protein biogenesis in plant cells, especially for those sorted to the plastid outer envelope.

Methodology/Principal Findings

Here we investigated the biogenesis of three plastid TA proteins, including the 33-kDa and 34-kDa GTPases of the translocon at the outer envelope of chloroplasts (Toc33 and Toc34) and a novel 9-kDa protein of unknown function that we define here as an outer envelope TA protein (OEP9). Using a combination of in vivo and in vitro assays we show that OEP9 utilizes a different sorting pathway than that used by Toc33 and Toc34. For instance, while all three TA proteins interact with the cytosolic OEP chaperone/receptor, AKR2A, the plastid targeting information within OEP9 is distinct from that within Toc33 and Toc34. Toc33 and Toc34 also appear to differ from OEP9 in that their insertion is dependent on themselves and the unique lipid composition of the plastid outer envelope. By contrast, the insertion of OEP9 into the plastid outer envelope occurs in a proteinaceous-dependent, but Toc33/34-independent manner and membrane lipids appear to serve primarily to facilitate normal thermodynamic integration of this TA protein.

Conclusions/Significance

Collectively, the results provide evidence in support of at least two sorting pathways for plastid TA outer envelope proteins and shed light on not only the complex diversity of pathways involved in the targeting and insertion of proteins into plastids, but also the molecular mechanisms that underlie the delivery of TA proteins to their proper intracellular locations in general.     

Low Density Membranes Are Associated with RNA-binding Proteins and Thylakoids in the Chloroplast of Chlamydomonas reinhardtii

Chloroplast subfractions were tested with a UV cross-linking assay for proteins that bind to the 5′ untranslated region of the chloroplast psbC mRNA of the green alga Chlamydomonas reinhardtii. These analyses revealed that RNA-binding proteins of 30–32, 46, 47, 60, and 80 kD are associated with chloroplast membranes. The buoyant density and the acyl lipid composition of these membranes are compatible with their origin being the inner chloroplast envelope membrane. However, unlike previously characterized inner envelope membranes, these membranes are associated with thylakoids. One of the membrane-associated RNA-binding proteins appears to be RB47, which has been reported to be a specific activator of psbA mRNA translation. These results suggest that translation of chloroplast mRNAs encoding thylakoid proteins occurs at either a subfraction of the chloroplast inner envelope membrane or a previously uncharacterized intra-chloroplast compartment, which is physically associated with thylakoids.     

Kinetics of Incorporation of Structural Proteins into Sindbis Virions

The morphogenesis of Sindbis virus was studied by determining the kinetics with which newly synthesized nucleocapsid and envelope proteins appeared in virions released into the extracellular medium. Assembly of the nucleocapsid was more rapid than modification of the cellular membrane by the addition of the viral envelope protein. However, both viral structural proteins were efficiently incorporated into virions; a 0.5-hr pulse-labeling period resulted in the release of maximally labeled virus during the next hour. When protein synthesis was inhibited, release of virus soon declined even though large amounts of both viral structural proteins were present within the cell and ribonucleic acid replication was unaffected.     

Immunocytochemical Identification of Spore Proteins in Two Microsporidia, with Emphasis on Extrusion Apparatus

Microsporidia can form small spores with a unique invasive apparatus featuring a long polar tube whose extrusion allows entry of infectious sporoplasm into a host cell. The reactivity of mouse polyclonal antibodies raised against sporal proteins from two microsporidian species belonging to different genera ( Glugea atherinae and Encephalitozoon cuniculi ) was studied by western blotting and indirect immunofluorescence. Whole protein antisera provided a few cross-reactions relatable to some proteins of the spore envelope or polar tube. Ultrastructural immunocytochemistry with murine antibodies against protein bands separated by sodium dodecylsulphate polyacrylamide gel electrophoresis allowed the assignment of several proteins to the polar tube (34, 75 and 170 kDa in Glugea , 35, 55 and 150 kDa in Encephalitozoon ). Antigenic similarities were detected for the Glugea 34 kDa and Encephalitozoon 35 kDa polar tube proteins. Species-specific proteins were shown to be located in either the lamellar polaroplast of Glugea or the spore envelope of Encephalitozoon.     

Avian Tumor Virus Proteins and RNA in Uninfected Chicken Embryo Cells

The content of proteins P19 and P15 (mol wt 19,000 and 15,000, respectively) of avian leukovirus in various types of uninfected chicken embryos has been determined by radioimmunoassay. All chicken embryos examined, including embryos which have thus far been classified as group specific (gs) antigen negative by complement fixation tests, contained these viral proteins as well as P27 as previously reported. The embryos known as “gs antigen-positive” type contained about five times as much of these viral proteins as did the “gs antigen-negative” type. The ratio of the three viral proteins was similar for all types of embryos, suggesting that the genes for these proteins are coordinately controlled. In contrast to the relatively high levels of viral internal proteins in gs antigen-negative cells, the amounts of virus-specific RNA detectable by molecular hybridization were extremely low. The levels of helper activity, which presumably reflect the level of viral envelope glycoprotein, were also generally low or undetectable in these cells. Thus, the expression of the gene for envelope glycoprotein does not appear to be controlled coordinately with the genes for viral internal proteins.     

Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins.     

昆虫病毒的增强蛋白

在某些昆虫病毒的包涵体中相继发现了增强蛋白．这类蛋白可以增强核型多角体病毒对昆虫幼虫及体外培养的昆虫细胞的感染力,缩短杀虫时间,提高杀虫效率;还可以增强其他生物杀虫剂（如：Bt）的毒力．关于增强蛋白的作用机制有两种观点：降解围食膜以利于病毒粒子侵染细胞或直接作用于细胞质膜促进病毒粒子的套膜与之融合．该蛋白是由病毒编码的,现已克隆出第一个增强蛋白基因,该基因含有一个2703bP的开放阅读框,其核苷酸序列分析业已完成．增强蛋白的发现有望对病毒杀虫剂的推广产生积极影响．     

Integral Membrane Proteins of the Nuclear Envelope Are Dispersed throughout the Endoplasmic Reticulum during Mitosis

We have analyzed the fate of several integral membrane proteins of the nuclear envelope during mitosis in cultured mammalian cells to determine whether nuclear membrane proteins are present in a vesicle population distinct from bulk ER membranes after mitotic nuclear envelope disassembly or are dispersed throughout the ER. Using immunofluorescence staining and confocal microscopy, we compared the localization of two inner nuclear membrane proteins (laminaassociated polypeptides 1 and 2 [LAP1 and LAP2]) and a nuclear pore membrane protein (gp210) to the distribution of bulk ER membranes, which was determined with lipid dyes (DiOC₆ and R6) and polyclonal antibodies. We found that at the resolution of this technique, the three nuclear envelope markers become completely dispersed throughout ER membranes during mitosis. In agreement with these results, we detected LAP1 in most membranes containing ER markers by immunogold electron microscopy of metaphase cells. Together, these findings indicate that nuclear membranes lose their identity as a subcompartment of the ER during mitosis. We found that nuclear lamins begin to reassemble around chromosomes at the end of mitosis at the same time as LAP1 and LAP2 and propose that reassembly of the nuclear envelope at the end of mitosis involves sorting of integral membrane proteins to chromosome surfaces by binding interactions with lamins and chromatin.     

Restricted Mobility of Cell Surface Proteins in the Polar Regions of Escherichia coli

The polar regions of the Escherichia coli murein sacculus are metabolically inert and stable in time. Because the sacculus and the outer membrane are tightly associated, we investigated whether polar inert murein could restrict the mobility of other cell envelope elements. Cells were covalently labeled with a fluorescent reagent, chased in dye-free medium, and observed by microscopy. Fluorescent material was more efficiently retained at the cell poles than at any other location. The boundary between high and low fluorescence intensity areas was rather sharp. Labeled material consisted mostly of cell envelope proteins, among them the free and murein-bound forms of Braun's lipoprotein. Our results indicate that the mobility of at least some cell envelope proteins is restrained at regions in correspondence with underlying areas of inert murein.