G proteins Go green: a plant G protein signaling FAQ sheet

Plants, like animals, use signal transduction pathways based on heterotrimeric guanine nucleotide-binding proteins (G proteins) to regulate many aspects of development and cell signaling. Some components of G protein signaling are highly conserved between plants and animals and some are not. This Viewpoint compares key aspects of G protein signal transduction in plants and animals and describes the current knowledge of this system in plants, the questions that still await exploration, and the value of research on plant G proteins to scientists who do not study plants. Pathways in Science's Signal Transduction Knowledge Environment Connections Maps database provide details about the emerging roles of G proteins in several cellular processes of plants.     

Partial reversion in yeast: genetic evidence for a new type of bifunctional protein

Two kinds of phenotypic expression in purine biosynthesis result from recessive mutation to adel2 in baker's yeast. The mutants are adenine-specific, blocked in the conversion of inosine 5'-phosphate to adenylosuccinic acid; their response to inhibition of pathway activity by adenine is considerably reduced. Allelic partial reversions can restore prototrophy without correcting the regulatory defect imparted by the primary mutation. The separation of the two properties of the locus by allelic mutation supports the hypothesis that the locus specifies a protein of two independent functions, enzymatic and regulatory.     

Pheromone signaling mechanisms in yeast: a prototypical sex machine

The actions of many extracellular stimuli are elicited by complexes of cell surface receptors, heterotrimeric guanine nucleotide-binding proteins (G proteins), and mitogen-activated protein (MAP) kinase complexes. Analysis of haploid yeast cells and their response to peptide mating pheromones has produced important advances in our understanding of G protein and MAP kinase signaling mechanisms. Many of the components, their interrelationships, and their regulators were first identified in yeast. Current analysis of the pheromone response pathway (see the Connections Maps at Science's Signal Transduction Knowledge Environment) will benefit from new and powerful genomic, proteomic, and computational approaches that will likely reveal additional general principles that are applicable to more complex organisms.     

G protein signaling from activated rat frizzled-1 to the beta-catenin-Lef-Tcf pathway

The frizzled receptors, which mediate development and display seven hydrophobic, membrane-spanning segments, are cell membrane-localized. We constructed a chimeric receptor with the ligand-binding and transmembrane segments from the beta2-adrenergic receptor (beta2AR) and the cytoplasmic domains from rat Frizzled-1 (Rfz1). Stimulation of mouse F9 clones expressing the chimera (beta2AR-Rfz1) with the beta-adrenergic agonist isoproterenol stimulated stabilization of beta-catenin, activation of a beta-catenin-sensitive promoter, and formation of primitive endoderm. The response was blocked by inactivation of pertussis toxin-sensitive, heterotrimeric guanine nucleotide-binding proteins (G proteins) and by depletion of Galphaq and Galphao. Thus, G proteins are elements of Wnt/Frizzled-1 signaling to the beta-catenin-lymphoid-enhancer factor (LEF)-T cell factor (Tcf) pathway.     

Erythrocyte G protein-coupled receptor signaling in malarial infection

Erythrocytic mechanisms involved in malarial infection are poorly understood. We have found that signaling via the erythrocyte beta2-adrenergic receptor and heterotrimeric guanine nucleotide-binding protein (Galphas) regulated the entry of the human malaria parasite Plasmodium falciparum. Agonists that stimulate cyclic adenosine 3',5'-monophosphate production led to an increase in malarial infection that could be blocked by specific receptor antagonists. Moreover, peptides designed to inhibit Galphas protein function reduced parasitemia in P. falciparum cultures in vitro, and beta-antagonists reduced parasitemia of P. berghei infections in an in vivo mouse model. Thus, signaling via the erythrocyte beta2-adrenergic receptor and Galphas may regulate malarial infection across parasite species.     

酵母菌发酵半干罗非鱼挥发性成分分析

采用顶空-固相微萃取(HS-SPME)技术结合气相色谱-质谱(GC-MS)技术对未发酵半干罗非鱼和经酵母菌发酵半干罗非鱼中的挥发性物质进行研究,优化了固相微萃取的条件.结果显示:样品在50℃条件下.采用聚二甲基硅氧烷/二乙烯基苯(PDMS/DVB)涂层的萃取头萃取50 min,可得到较好谱图.通过气相色谱-质谱分析,在未发酵罗非鱼与酵母菌发酵罗非鱼中分别检测出74,59种挥发性物质.酵母发酵半干罗非鱼与未发酵半干罗非鱼的挥发性物质有显著差异.酵母发酵半干罗非鱼中苯乙醉相对含量明显高于未发酵半干罗非鱼.     

Structure-based assembly of protein complexes in yeast

Images of entire cells are preceding atomic structures of the separate molecular machines that they contain. The resulting gap in knowledge can be partly bridged by protein-protein interactions, bioinformatics, and electron microscopy. Here we use interactions of known three-dimensional structure to model a large set of yeast complexes, which we also screen by electron microscopy. For 54 of 102 complexes, we obtain at least partial models of interacting subunits. For 29, including the exosome, the chaperonin containing TCP-1, a 3'-messenger RNA degradation complex, and RNA polymerase II, the process suggests atomic details not easily seen by homology, involving the combination of two or more known structures. We also consider interactions between complexes (cross-talk) and use these to construct a structure-based network of molecular machines in the cell.     

Variations on conserved signaling pathways in biocontrol and development:G protein and MAPK genes of Trichoderma. atroviride and T. virens

Filamentous fungi employ conserved eukaryotic signaling pathway to detect and respond to environmental signals, including the presence of the host. Genetic experiment in which a particular signaling protein is lost, or its activity enhanced, have defined some of the function of heterotrimeric G proteins and MAP kinases in development and virulence. A hallmark of these studies is that orthologs in different species may have different functions. Antagonistic fungal-fungal interactions form …     

Pot1, the putative telomere end-binding protein in fission yeast and humans

Telomere proteins from ciliated protozoa bind to the single-stranded G-rich DNA extensions at the ends of macronuclear chromosomes. We have now identified homologous proteins in fission yeast and in humans. These Pot1 (protection of telomeres) proteins each bind the G-rich strand of their own telomeric repeat sequence, consistent with a direct role in protecting chromosome ends. Deletion of the fission yeast pot1+ gene has an immediate effect on chromosome stability, causing rapid loss of telomeric DNA and chromosome circularization. It now appears that the protein that caps the ends of chromosomes is widely dispersed throughout the eukaryotic kingdom.     

Experimental models of primitive cellular compartments: encapsulation, growth, and division

The clay montmorillonite is known to catalyze the polymerization of RNA from activated ribonucleotides. Here we report that montmorillonite accelerates the spontaneous conversion of fatty acid micelles into vesicles. Clay particles often become encapsulated in these vesicles, thus providing a pathway for the prebiotic encapsulation of catalytically active surfaces within membrane vesicles. In addition, RNA adsorbed to clay can be encapsulated within vesicles. Once formed, such vesicles can grow by incorporating fatty acid supplied as micelles and can divide without dilution of their contents by extrusion through small pores. These processes mediate vesicle replication through cycles of growth and division. The formation, growth, and division of the earliest cells may have occurred in response to similar interactions with mineral particles and inputs of material and energy.     

Intervacuole exchange in the yeast zygote: a new pathway in organelle communication

A new pathway of vesicle traffic between organelles has been identified. The vacuoles (lysosomes) of Saccharomyces cerevisiae zygotes rapidly exchange their contents at a specific point in the cell cycle. With the use of fluorescence microscopy, "tracks" were observed that connect the original parental vacuoles to the newly forming bud vacuoles. These observations suggest that vacuole-derived vesicles rapidly move along the tracks in both directions, equilibrating vacuole contents. This rapid vesicle movement may be responsible for vacuole formation in newly developing cells.     

糜子籽粒贮藏蛋白的组分分析

【目的】研究糜子籽粒贮藏蛋白组分,为糜子种质资源研究和品种改良提供理论依据。【方法】分步提取13个糜子品种籽粒的清蛋白、球蛋白、醇溶蛋白和谷蛋白,利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)技术,对各蛋白组分的亚基组成进行分析。【结果】参试样品籽粒清蛋白平均含量为13.67 mg/g,球蛋白为8.12 mg/g,谷蛋白为9.73 mg/g,醇溶蛋白为2.70 mg/g;在4种组分中,品种之间醇溶蛋白含量差异最大。SDS-PAGE图谱显示,染色后清蛋白、球蛋白、谷蛋白和剩余蛋白条带清晰可见;在14.4~94.0 ku,清蛋白有10~12亚基,谷蛋白有10或11亚基,球蛋白有9~11亚基,剩余蛋白含量高,成分比较丰富。【结论】利用SDS-PAGE方法可以得到糜子籽粒清蛋白、球蛋白和谷蛋白的清晰条带,而醇溶蛋白基本没有条带;相比而言,球蛋白的条带清晰且多态性高。     

酵母菌种的筛选及其对反刍动物的影响

随着酵母类饲料添加剂在反刍动物中应用的日益增加,酵母菌对瘤胃作用的生物学机理及酵母菌与瘤胃内微生物的相互作用逐渐成为研究热点。文章针对目前国内外新型酵母菌种的筛选工作,在影响新型酵母菌筛选的因素,如何利用分子生物学技术选择新型酵母菌种、以及新型酵母对反刍动物的作用机理等方面进行了分析,为进一步研究提供理论依据。     

An in vivo map of the yeast protein interactome

Protein interactions regulate the systems-level behavior of cells; thus, deciphering the structure and dynamics of protein interaction networks in their cellular context is a central goal in biology. We have performed a genome-wide in vivo screen for protein-protein interactions in Saccharomyces cerevisiae by means of a protein-fragment complementation assay (PCA). We identified 2770 interactions among 1124 endogenously expressed proteins. Comparison with previous studies confirmed known interactions, but most were not known, revealing a previously unexplored subspace of the yeast protein interactome. The PCA detected structural and topological relationships between proteins, providing an 8-nanometer-resolution map of dynamically interacting complexes in vivo and extended networks that provide insights into fundamental cellular processes, including cell polarization and autophagy, pathways that are evolutionarily conserved and central to both development and human health.     

Two-state allosteric behavior in a single-domain signaling protein

Protein actions are usually discussed in terms of static structures, but function requires motion. We find a strong correlation between phosphorylation-driven activation of the signaling protein NtrC and microsecond time-scale backbone dynamics. Using nuclear magnetic resonance relaxation, we characterized the motions of NtrC in three functional states: unphosphorylated (inactive), phosphorylated (active), and a partially active mutant. These dynamics are indicative of exchange between inactive and active conformations. Both states are populated in unphosphorylated NtrC, and phosphorylation shifts the equilibrium toward the active species. These results support a dynamic population shift between two preexisting conformations as the underlying mechanism of activation.     

HRR25, a putative protein kinase from budding yeast: association with repair of damaged DNA

In simple eukaryotes, protein kinases regulate mitotic and meiotic cell cycles, the response to polypeptide pheromones, and the initiation of nuclear DNA synthesis. The protein HRR25 from the budding yeast Saccharomyces cerevisiae was defined by the mutation hrr25-1. This mutation resulted in sensitivity to continuous expression of the HO double-strand endonuclease, to methyl methanesulfonate, and to x-irradiation. Homozygotes of hrr25-1 were unable to sporulate and disruption and deletion of HRR25 interfered with mitotic and meiotic cell division. Sequence analysis revealed two distinctive regions in the protein. The NH2-terminus of HRR25 contains the hallmark features of protein kinases, whereas the COOH-terminus is rich in proline and glutamine. Mutations in HRR25 at conserved residues found in all protein kinases inactivated the gene, and these mutants exhibited the hrr25 null phenotypes. Taken together, the hrr25 mutant phenotypes and the features of the gene product indicate that HRR25 is a distinctive member of the protein kinase superfamily.     

Selective phagocytosis: a new concept in protein catabolism

The clearance of different metabolic products derived from two plasma proteins, prothrombin and fibrinogen, was studied with the aid of the isolated, perfused rat liver. Active thrombin and fibrin were rapidly cleared by the Kupffer cells. Inactive thrombin and a partially degraded fibrin molecule were also cleared but at much slower rates. This difference in clearance rates suggests the presence of a high degree of selectivity in the clearance of altered plasma proteins.     

Two G protein oncogenes in human endocrine tumors

Somatic mutations in a subset of growth hormone (GH)-secreting pituitary tumors convert the gene for the alpha polypeptide chain (alpha s) of Gs into a putative oncogene, termed gsp. These mutations, which activate alpha s by inhibiting its guanosine triphosphatase (GTPase) activity, are found in codons for either of two amino acids, each of which is completely conserved in all known G protein alpha chains. The likelihood that similar mutations would activate other G proteins prompted a survey of human tumors for mutations that replace either of these two amino acids in other G protein alpha chain genes. The first gene so far tested, which encodes the alpha chain of Gi2, showed mutations that replaced arginine-179 with either cysteine or histidine in 3 of 11 tumors of the adrenal cortex and 3 of 10 endocrine tumors of the ovary. The mutant alpha i2 gene is a putative oncogene, referred to as gip2. In addition, gsp mutations were found in 18 of 42 GH-secreting pituitary tumors and in an autonomously functioning thyroid adenoma. These findings suggest that human tumors may harbor oncogenic mutations in various G protein alpha chain genes.     

Disruption of signaling by Yersinia effector YopJ, a ubiquitin-like protein protease

Homologs of the Yersinia virulence effector YopJ are found in both plant and animal bacterial pathogens, as well as plant symbionts. These YopJ family members were shown to act as cysteine proteases. The catalytic triad of the protease was required for inhibition of the mitogen-activated protein kinase (MAPK) and nuclear factor kappaB (NF-kappaB) signaling in animal cells and for induction of localized cell death in plants. The substrates for YopJ were shown to be highly conserved ubiquitin-like molecules, which are covalently added to numerous regulatory proteins. YopJ family members exert their pathogenic effect on cells by disrupting this posttranslational modification.