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Phosphorylation-dependent interactions between enzymes of plant metabolism and 14-3-3 proteins

PLANT JOURNAL 1999年第1期18卷 1-12页

作者： Moorhead, G Douglas, P Cotelle, V Harthill, J Morrice, N Meek, S Deiting, U Stitt, M Scarabel, M Aitken, A MacKintosh, C Univ Dundee Dept Biochem MRC Prot Phosphorylat Unit Dundee DD1 5EH Scotland Heidelberg Univ Inst Bot D-69120 Heidelberg Germany Univ Edinburgh Dept Biochem Edinburgh EH8 9XD Midlothian Scotland

Far-Western overlays of soluble extracts of cauliflower revealed many proteins that bound to digoxygenin (DIG)labelled 14-3-3 proteins. Binding to DIG-14-3-3s was prevented by prior dephosphorylation of the extract proteins or by competition with 14-3-3-binding phosphopeptides, indicating that the 14-3-3 proteins bind to phosphorylated sites. The proteins that bound to the DIG-14-3-3s were also immunoprecipitated from extracts with anti-14-3-3 antibodies, demonstrating that they were bound to endogenous plant 14-3-3 proteins. 14-3-3-binding proteins were purified from cauliflower extracts, in sufficient quantity for amino acid sequence analysis, by affinity chromatography on immobilised 14-3-3 proteins and specific elution with a 14-3-3-binding phosphopeptide. Purified 14-3-3-binding proteins included sucrose-phosphate synthase, trehalose-6-phosphate synthase, glutamine synthetases, a protein (LIM17) that has been implicated in early floral development, an approximately 20 kDa protein whose mRNA is induced by NaCl, and a calcium-dependent protein kinase that was capable of phosphorylating and rendering nitrate reductase (NR) sensitive to inhibition by 14-3-3 proteins. In contrast to the phosphorylated NR-14-3-3 complex which is activated by dissociation with 14-3-3-binding phosphopeptides, the total sugar-phosphate synthase activity in plant extracts was inhibited by up to 40% by a 14-3-3-binding phosphopeptide and the phosphopeptide-inhibited activity was reactivated by adding excess 14-3-3 proteins. Thus, 14-3-3 proteins are implicated in regulating several aspects of primary N and C metabolism. The procedures described here will be valuable for determining how the phosphorylation and 14-3-3-binding status of defined target proteins change in response to extracellular stimuli.

关键词： 14-3-3蛋白质类氨基酸序列芸苔属/酶学芸苔属/代谢碳水化合物代谢载体蛋白质类/分离和提纯载体蛋白质类/代谢色谱法亲和分子序列数据磷蛋白类/分离和提纯磷蛋白类/代谢磷酰化植物蛋白质类/化学植物蛋白质类/分离和提纯植物蛋白质类/代谢沉淀素试验蛋白质类/化学蛋白质类/分离和提纯蛋白质类/代谢酪氨酸单氧化酶

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Non-bilayer lipids stimulate the activity of the reconstituted bacterial protein translocase

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第4期275卷 2472-2478页

作者： van der Does, C Swaving, J van Klompenburg, W Driessen, AJM Univ Groningen Dept Microbiol NL-9751 NN Haren Netherlands Univ Groningen Biomol Sci & Biotechnol Inst NL-9751 NN Haren Netherlands

To determine the phospholipid requirement of the preprotein translocase in vitro, the Escherichia coli SecYEG complex was purified in a delipidated form using the detergent dodecyl maltoside, SecYEG was reconstituted into liposomes composed of defined synthetic phospholipids, and proteoliposomes were analyzed for their preprotein translocation and SecA translocation ATPase activity. The activity strictly required the presence of anionic phospholipids, whereas the non-bilayer lipid phosphatidylethanolamine was found stimulatory. The latter effect could also be induced by dioleoylglycerol, a lipid that adopts a non-bilayer conformation, Phosphatidylethanolamine derivatives that prefer the bilayer state were unable to stimulate translocation, In the absence of SecG, activity was reduced, but the phospholipid requirement was unaltered. Remarkably, nonbilayer lipids were found essential for the activity of the Bacillus subtilis SecYEG complex. Optimal activity required a mixture of anionic and non-bilayer Lipids at concentrations that correspond to concentrations found in the natural membrane.

关键词：腺苷三磷酸酶类/分离和提纯腺苷三磷酸酶类/代谢细菌蛋白质类载体蛋白质类/分离和提纯载体蛋白质类/代谢酶激活大肠杆菌/酶学大肠杆菌蛋白质类膜转运蛋白质类磷脂类/代谢

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Compartmentalization of the cell cortex by septins is required for maintenance of cell polarity in yeast

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MOLECULAR CELL 2000年第5期5卷 841-851页

作者： Barral, Y Mermall, V Mooseker, MS Snyder, M Yale Univ Dept Mol Cellular & Dev Biol New Haven CT 06520 USA Yale Univ Sch Med Dept Cell Biol New Haven CT 06520 USA Yale Univ Sch Med Dept Pathol New Haven CT 06520 USA

Formation and maintenance of specialized plasma membrane domains are crucial for many biological processes, such as cell polarization and signaling. During isotropic bud growth, the yeast cell periphery is divided into two domains: the bud surface, an active site of exocytosis and growth, and the relatively quiescent surface of the mother cell. We found that cells lacking septins at the bud neck failed to maintain the exocytosis and morphogenesis factors Spa2, Sec3, Sec5, and Myo2 in the bud during isotropic growth. Furthermore, we found that septins were required for proper regulation of actin patch stability;septin-defective cells permitted to enter isotropic growth lost actin and growth polarity. We propose that septins maintain cell polarity by specifying a boundary between cortical domains.

关键词：肌动蛋白类/分离和提纯载体蛋白质类/分离和提纯细胞区室化细胞周期蛋白质类细胞分裂/生理学细胞膜/超微结构细胞极性细胞质/超微结构细胞支架蛋白质类胞吐作用真菌蛋白质类/分离和提纯形态发生肌球蛋白重链肌球蛋白Ⅱ型肌球蛋白Ⅴ型蛋白酪氨酸激酶类/代谢酿酒酵母菌/超微结构酿酒酵母蛋白质类非洲粟酒人体酵母蛋白质类

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Isolation and characterization of peroxisome proliferator-activated receptor (PPAR) interacting protein (PRIP) as a coactivator for PPAR

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第18期275卷 13510-13516页

作者： Zhu, YJ Kan, LX Qi, C Kanwar, YS Yeldandi, AV Rao, MS Reddy, JK Northwestern Univ Sch Med Dept Pathol Chicago IL 60611 USA

We previously isolated and identified steroid receptor coactivator-1 (SRC-1) and peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP/PPARBP) as coactivators for PPAR, using the ligand-binding domain of PPAR gamma as bait in a yeast two-hybrid screening. As part of our continuing effort to identify cofactors that influence the transcriptional activity of PPARs, we now report the isolation of a novel coactivator from mouse, designated PRIP (peroxisome proliferator-activated receptor interacting protein), a nuclear protein with 2068 amino acids and encoded by 13 exons. Northern analysis showed that PRIP mRNA is ubiquitously expressed in many tissues of adult mice. PRIP contains two LXXLL signature motifs. The amino-terminal LXYLL motif (amino acid position 892 to 896) of PRIP was found to be necessary for nuclear receptor interaction, but the second LXYLL motif (amino acid position 1496 to 1500) appeared unable to bind PPAR gamma. Deletion of the last 12 amino acids from the carboxyl terminus of PPAR gamma resulted in the abolition of the interaction between PRIP and PPAR gamma, PRIP also binds to PPAR alpha;RAR alpha;RXR alpha, ER, and TR beta 1, and this binding is increased in the presence of specific ligands. PRIP acts as a strong coactivator for PPAR gamma in the yeast and also potentiates the transcriptional activities of PPAR gamma and RXR alpha in mammalian cells. A truncated form of PRIP (amino acids 786-1132) acts as a dominant-negative repressor, suggesting that PRIP is a genuine coactivator.

关键词：氨基酸序列载体蛋白质类/遗传学载体蛋白质类/分离和提纯载体蛋白质类/代谢克隆分子中介复合体亚基1 分子序列数据突变核蛋白质类/遗传学核蛋白质类/分离和提纯核蛋白质类/代谢受体胞质和核/代谢转录因子/代谢动物小鼠

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The human WASP-interacting protein, WIP, activates the cell polarity pathway in yeast

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第24期274卷 17103-17108页

作者： Vaduva, G Martinez-Quiles, N Anton, IM Martin, NC Geha, RS Hopper, AK Ramesh, N Penn State Univ Coll Med Dept Biochem & Mol Biol Hershey PA 17033 USA Harvard Univ Childrens Hosp Sch Med Dept PediatDiv Immunol Boston MA 02115 USA Univ Louisville Sch Med Dept Biochem Louisville KY 40292 USA

WIP, the Wiskott-Aldrich syndrome protein-interacting protein, is a human protein involved in actin polymerization and redistribution in lymphoid cells. The mechanism by which WIP reorganizes actin cytoskeleton is unknown. WIP is similar to yeast verprolin, an actin- and myosin-interacting protein required for polarized morphogenesis. To determine whether WIP and verprolin are functional homologues, we analyzed the function of WIP in yeast. WIP suppresses the growth defects of VRP1 missense and null mutations as well as the defects in cytoskeletal organization and endocytosis observed in vrp1-1 cells. The ability of WIP to replace verprolin is dependent on its WH2 actin binding domain and a putative profilin binding domain. Immunofluorescence localization of WIP in yeast cells reveals a pattern consistent with its function at the cortical sites of growth. Thus, like verprolin, WIP functions in yeast to link the polarity development pathway and the actin cytoskeleton to generate cytoskeletal asymmetry. A role for WIP in cell polarity provides a framework for unifying, under a common paradigm, distinct molecular defects associated with immunodeficiencies like Wiskott-Aldrich syndrome.

关键词：氨基酸序列结合部位载体蛋白质类/遗传学载体蛋白质类/分离和提纯细胞区室化细胞极性/遗传学收缩蛋白质类细胞支架蛋白质类细胞骨架/生理学胞吞作用/生理学进化分子真菌蛋白质类/遗传学遗传互补测验细胞内信号肽和蛋白质类微丝蛋白质类/遗传学微丝蛋白质类/代谢分子序列数据突变误义肌动蛋白抑制蛋白质类蛋白质结合酿酒酵母蛋白质类序列同源性氨基酸抑制遗传 Wiskott-Aldrich综合征酵母菌人类

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Isolation of a 40-kDa Huntingtin-associated protein

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JOURNAL OF BIOLOGICAL CHEMISTRY 2001年第5期276卷 3188-3194页

作者： Peters, MF Ross, CA Johns Hopkins Univ Sch Med Dept Psychiat Mol Neurobiol LabDiv Neurobiol Baltimore MD 21205 USA Johns Hopkins Univ Sch Med Dept Neurosci Baltimore MD 21205 USA Johns Hopkins Univ Sch Med Program Cellular Mol Med Baltimore MD 21205 USA

Huntington's disease is caused by an expanded CAG trinucleotide repeat coding for a polyglutamine stretch within the huntingtin protein. Currently, the function of normal huntingtin and the mechanism by which expanded huntingtin causes selective neurotoxicity remain unknown. Clues may come from the identification of huntingtin-associated proteins (HAPs). Here, we show that huntingtin copurifies with a single novel 40-kDa protein termed HAP40. HAP40 is encoded by the open reading frame factor VIII-associated gene A (F8A) located within intron 22 of the factor VIII gene. In transfected cell extracts, HAP40 coimmunoprecipitates with full-length huntingtin but not with an N-terminal huntingtin fragment. Recombinant HAP40 is cytoplasmic in the presence of huntingtin but is actively targeted to the nucleus in the absence of huntingtin. These data indicate that HAP40 is likely to contribute to the function of normal huntingtin and is a candidate for involvement in the aberrant nuclear localization of mutant huntingtin found in degenerating neurons in Huntington's disease.

关键词：氨基酸序列载体蛋白质类/遗传学载体蛋白质类/免疫学载体蛋白质类/分离和提纯疾病模型动物杭廷顿病/代谢分子序列数据分子量神经组织蛋白质类/遗传学神经组织蛋白质类/免疫学神经组织蛋白质类/分离和提纯核蛋白质类/遗传学核蛋白质类/免疫学核蛋白质类/分离和提纯动物人类小鼠大鼠

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A SPECIFIC STAIN FOR THE DETECTION OF NONHEME IRON PROTEINS IN POLYACRYLAMIDE GELS

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ANALYTICAL BIOCHEMISTRY 1992年第2期207卷 317-320页

作者： LEONG, LM TAN, BH HO, KK NATL UNIV SINGAPORE FAC SCIDEPT BOTSINGAPORE 0511SINGAPORE

Nonheme iron proteins can be visualized as blue bands in native polyacrylamide gels using a staining method that is both simple and rapid. The reaction of potassium ferricyanide with protein-bound iron atoms to form royal blue complexes occurs almost instantaneously and is sensitive enough to detect 1 microgram of analytical-grade ferritin and 2 micrograms of purified ferredoxin from cyanobacteria. No special treatment of reagents or apparatus was necessary. On comparison, this stain was found to be more specific than the Ferene S stain, not detecting bovine serum albumin even when present as a hundredfold excess over ferritin. The method was found to be effective for isoelectric focusing gels as well.

关键词：血蛋白质类/分析血蛋白质类/分离和提纯载体蛋白质类/分析载体蛋白质类/分离和提纯生色化合物电泳聚丙烯酰氨凝胶/方法铁氧化还原蛋白类/分析铁氧化还原蛋白类/分离和提纯铁氰化物铁蛋白质类/分析铁蛋白质类/分离和提纯胎血/化学马铁/分析铁结合蛋白质类等电聚焦/方法微量化学/方法肌红蛋白/分析肌红蛋白/分离和提纯品红类染料血清白蛋白牛/分析血清白蛋白牛/分离和提纯染色与标记转铁蛋白/分析转铁蛋白/分离和提纯转铁结合蛋白质类三嗪类动物牛人类

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MULTIPLE FOLATE TRANSPORT-SYSTEMS IN L1210 CELLS

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ADVANCES IN ENZYME REGULATION 1992年第C期32卷 3-+页

作者： FAN, J VITOLS, KS HUENNEKENS, FM DepartmentofMolecularandExperimentalMedicine ScrippsResearchInstituteLaJollaCA92037.

Biotin derivatives of methotrexate (biotin-SS-MTX) and folate (biotin-SS-folate), in which the functional components are joined by a dissociable disulfide-containing spacer, have been synthesized, purified by DEAE-Trisacryl chromatography, and characterized by HPLC, elemental analysis and mass spectrometry. These compounds provide a convenient means for the single-step purification of the folate transporters from L1210 cells. Parental L1210 murine leukemia cells, which contain only the microM transporter (the reduced folate/MTX transport protein) were treated with the N-hydroxysulfosuccinimide ester of biotin-SS-MTX, and a detergent extract of the plasma membranes was exposed to streptavidin-agarose beads to adsorb the labeled protein. Dithiothreitol cleavage of the disulfide linkage released the transporter, which migrated as a well-defined component (43 kDa) on SDS-PAGE gels; no other proteins were present. An L1210 subline (JF), obtained by adapting cells to grow on nanomolar concentrations of folate, contains both the microM transporter and the nM transporter (high-affinity folate binding protein). When these cells were treated with the N-hydroxysulfosuccimide ester of biotin-SS-folate and processed as described above, analysis on SDS-PAGE gels revealed the presence of two proteins, the microM transporter (43 kDa) and the nM transporter (39 kDa). Both transporters were characterized with respect to amino acid content; blocked N-termini precluded Edman sequencing. Treatment of the nM transporter with peptide:N-glycosidase F produced a smaller component (32 kDa); the microM transporter, conversely, was unchanged by this procedure. When the microM transporter in parental L1210 cells was labeled with fluorescein-MTX and then treated with phosphoinositol-specific phospholipase C (PI-PLC), no change in fluorescence was detected. Alternatively, when the nM transporter in the JF subline was labeled with fluorescein-folate and then treated with PI-PLC, complete loss of f

关键词：生物素/类似物和衍生物生物素/化学合成生物素/代谢载体蛋白质类/化学载体蛋白质类/分离和提纯载体蛋白质类/代谢色谱法亲和/方法叶酸/类似物和衍生物叶酸/化学合成叶酸/代谢白血病L1210/代谢甲氨蝶呤/类似物和衍生物甲氨蝶呤/化学合成甲氨蝶呤/代谢受体细胞表面/分离和提纯受体细胞表面/代谢肿瘤细胞培养的动物小鼠

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Distinct membrane binding properties of N- and C-terminal domains of Escherichia coli SecA ATPase

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第32期275卷 25000-25007页

作者： Dapic, V Oliver, D Wesleyan Univ Dept Mol Biol & Biochem Middletown CT 06459 USA

SecA is a motor protein that drives protein translocation at the Escherichia coli translocon. SecA membrane binding has been shown to occur with high affinity at SecYE and low affinity at anionic phospholipids. To dissect SecA-membrane interaction with reference to SecA structure, the membrane binding properties of N- and C-terminal SecA domains, denoted SecA-N664 and SecA-619C, respectively, were characterized. Remarkably, only SecA-N664 bound to the membrane with high affinity, whereas SecA-619C bound with low affinity in a nonsaturable manner through partitioning with phospholipids. Moreover, SecA-N664 and SecA-619C associated with each other to reconstitute wild type binding affinity. Corroborative results were also obtained from membrane binding competition and subcellular fractionation studies along with binding studies to membranes prepared from strains overproducing SecYE protein. Together, these findings indicate that the specific interaction of SecA with SecYE occurs through its N-terminal domain and that the C-terminal domain, although important in SecA membrane cycling at a later stage of translocation, appears to initially assist SecA membrane binding by interaction with phospholipids. These results provide the first evidence for distinct membrane binding characteristics of the two SecA primary domains and their importance for optimal binding activity, and they are significant for understanding SecA dynamics at the translocon.

关键词：腺苷三磷酸酶类/化学腺苷三磷酸酶类/分离和提纯腺苷三磷酸酶类/代谢氨基酸取代细菌蛋白质类/化学细菌蛋白质类/代谢结合部位载体蛋白质类/化学载体蛋白质类/分离和提纯载体蛋白质类/代谢细胞膜/酶学大肠杆菌/酶学大肠杆菌/遗传学大肠杆菌蛋白质类基因细菌动力学膜转运蛋白质类诱变定点磷脂类/药理学重组蛋白质类/化学重组蛋白质类/分离和提纯重组蛋白质类/代谢

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A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation

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MOLECULAR CELL 2000年第2期5卷 299-309页

作者： McKie, AT Marciani, P Rolfs, A Brennan, K Wehr, K Barrow, D Miret, S Bomford, A Peters, TJ Farzaneh, F Hediger, MA Hentze, MW Simpson, RJ Univ London Kings Coll Guys Kings & St Thomas Sch Med Dept Mol Med London SE5 England Univ London Kings Coll Guys Kings & St Thomas Sch Med Dept Clin Biochem London SE5 England Univ London Kings Coll Guys Kings & St Thomas Sch Med Inst Liver Studies London SE5 England European Mol Biol Lab Gene Express Programme D-69117 Heidelberg Germany Brigham & Womens Hosp Membrane Biol Program Boston MA 02115 USA Brigham & Womens Hosp Div Renal Boston MA 02115 USA Harvard Univ Sch Med Inst Med Boston MA 02115 USA Univ Barcelona Fac Biol Dept Fisiol E-08028 Barcelona Spain

Iron absorption by the duodenal mucosa is initiated by uptake of ferrous Fe(ll) iron across the brush border membrane and culminates in transfer of the metal across the basolateral membrane to the portal vein circulation by an unknown mechanism. We describe here the isolation and characterization of a novel cDNA (Ireg1) encoding a duodenal protein that is localized to the basolateral membrane of polarized epithelial cells. Ireg1 mRNA and protein expression are increased under conditions of increased iron absorption, and the 5' UTR of the Ireg1 mRNA contains a functional iron-responsive element (IRE). IREG1 stimulates iron efflux following expression in Xenopus oocytes. We conclude that IREG1 represents the long-sought duodenal iron export protein and is upregulated in the iron overload disease, hereditary hemochromatosis.

关键词：氨基酸序列碱基序列生物转运载体蛋白质类/遗传学载体蛋白质类/分离和提纯载体蛋白质类/代谢阳离子转运蛋白质类细胞区室化细胞极性克隆分子十二指肠/代谢正铁化合物/代谢基因表达调控肠吸收/生理学肠黏膜/代谢铁/代谢膜蛋白质类/遗传学膜蛋白质类/分离和提纯膜蛋白质类/代谢模型生物学分子序列数据门静脉/代谢 RNA 信使/遗传学反应元件序列同源性氨基酸序列同源性核酸转铁蛋白/缺乏动物小鼠

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