Nuclear translocation of the calcium-binding protein ALG-2 induced by the RNA-binding protein RBM22

By yeast two-hybrid screening using the calcium-binding protein ALG-2 as bait a new target of ALG-2 was identified, the RNA-binding protein RBM22. In order to confirm these interactions in vivo we prepared fluorescent constructs by using the monomeric red fluorescent protein to label ALG-2 and the enhanced green fluorescent protein to label RBM22. Confocal microscopy of NIH 3T3 cells transfected with either ALG-2 or RBM22 expression constructs encoding fluorescent fusion proteins alone revealed that the majority of ALG-2 was localized in the cytoplasm whereas RBM22 was located in the nucleus. When cells were co-transfected with expression vectors encoding both fusion proteins ALG-2 was found in the nucleus indicating that RBM22 which can shuttle between the cytoplasm and the nucleus may play a role in nuclear translocation of ALG-2. Using zebrafish as a model mRNA homologues of ALG-2 and RBM22 were microinjected into the blastodisc-yolk margin of zebrafish embryos at the 1-cell stage followed by monitoring the fusion proteins during development of the zebrafish. Hereby, we observed that ALG-2 alone evenly distributed within the cell, whereas in the presence of RBM22 the two proteins co-localized within the nucleus. More than 95% of the two proteins co-localized within the same area in the nucleus suggesting a functional interaction between the Ca(2+)-signaling protein ALG-2 and the RNA-binding protein RBM22.     

Splicing factor hSlu7 contains a unique functional domain required to retain the protein within the nucleus

Precursor-mRNA splicing removes the introns and ligates the exons to form a mature mRNA. This process is carried out in a spliceosomal complex containing >150 proteins and five small nuclear ribonucleoproteins. Splicing protein hSlu7 is required for correct selection of the 3' splice site. Here, we identify by bioinformatics and mutational analyses three functional domains of the hSlu7 protein that have distinct roles in its subcellular localization: a nuclear localization signal, a zinc-knuckle motif, and a lysine-rich region. The zinc-knuckle motif is embedded within the nuclear localization signal in a unique functional structure that is not required for hSlu7's entrance into the nucleus but rather to maintain hSlu7 inside it, preventing its shuttle back to the cytoplasm via the chromosomal region maintenance 1 pathway. Thus, the zinc-knuckle motif of hSlu7 determines the cellular localization of the protein through a nucleocytoplasmic-sensitive shuttling balance. Altogether, this indicates that zinc-dependent nucleocytoplasmic shuttling might be the possible molecular basis by which hSlu7 protein levels are regulated within the nucleus.     

Peflin and ALG-2, members of the penta-EF-hand protein family, form a heterodimer that dissociates in a Ca2+-dependent manner

Peflin, a newly identified 30-kDa Ca(2+)-binding protein, belongs to the penta-EF-hand (PEF) protein family, which includes the calpain small subunit, sorcin, grancalcin, and ALG-2 (apoptosis-linked gene 2). We prepared a monoclonal antibody against human peflin. The antibody immunoprecipitated a 22-kDa protein as well as the 30-kDa protein from the lysate of Jurkat cells. Western blotting of the immunoprecipitates revealed that the 22-kDa protein corresponds to ALG-2. This was confirmed by Western blotting of the immunoprecipitates of epitope-tagged peflin or ALG-2 whose cDNA expression constructs were transfected to human embryonic kidney (HEK) 293 cells. Gel filtration of the cytosolic fraction of Jurkat cells revealed co-elution of peflin and ALG-2 in fractions eluting earlier than recombinant ALG-2, further supporting the notion of heterodimerization of the two PEF proteins. Surprisingly, peflin dissociated from ALG-2 in the presence of Ca(2+). Peflin and ALG-2 co-localized in the cytoplasm, but ALG-2 was also detected in the nuclei as revealed by immunofluorescent staining and subcellular fractionation. Peflin was recovered in the cytosolic fraction in the absence of Ca(2+) but in the membrane/cytoskeletal fraction in the presence of Ca(2+). These results suggest that peflin has features common to those of other PEF proteins (dimerization and translocation to membranes) and may modulate the function of ALG-2 in Ca(2+) signaling.     

The C protein of wild-type measles virus has the ability to shuttle between the nucleus and the cytoplasm

Measles virus (MV) C protein is a small and basic non-structural protein, but its function is not well understood. We have found that a FLAG-tagged wild-type MV C protein expressed from cDNA was accumulated exclusively in the nucleus. To analyze the amino acid sequence important for the nuclear localization of C protein, a plasmid expressing C protein fused to the enhanced green fluorescent protein (EGFP) was generated. Mutation analysis revealed that (41)PPARKRRQ(48), belonging to the classical nuclear localization signal was important for nuclear localization. Analysis of the amino acid sequence of C protein revealed that it has a nuclear export signal (NES)-like sequence, (76)LEKAMTTLKL(85). Addition of the putative NES to the EGFP resulted in the translocation of EGFP to the cytoplasm. The Rev(1.4)-EGFP nuclear export assay showed that this putative NES has a CRM1-dependent NES activity. C-EGFP accumulated in HeLa nuclei could be translocated to NIH3T3 nuclei in heterokaryon assays. In MV-infected cells, C-EGFP was accumulated in the nuclei in early phase but in the cytoplasm in late phase. These results indicate that the putative NES is functional and that C protein has the ability to shuttle between the nucleus and the cytoplasm.     

Energy requirements for biosynthesis of DNA in Escherichia coli. Role of membrane-bound energy-transducing ATPase (coupling factor).

A mutant of Escherichia coli missing energy-transducing ATPase and known to be defective in a variety of membrane functions from earlier studies (Yamamoto, T. H., Mével-Ninio, M. and Valentine, R. C. (1973) Biochim. Biophys. Acta 314, 267-275; Thipayathasana, P. and Valentine, R. C. (1974) Biochim. Biophys. Acta 347, 464-468; Mével-Ninio, M. and Yamamoto, T. (1974) Biochim. Biophys. Acta 357, 63-66) has been found to be blocked for anaerobic DNA synthesis. The rate of anaerobic DNA synthesis in the mutant, measured as radioactive adenine incorporation into the alkali-resistant fraction of whole cells, is about 1/6 the rate of DNA synthesis in the wild type culture under similar conditions. Addition of NO-3- or O-2 restores DNA biosynthesis in the mutant. The entry of radioactive adenine is not appreciably affected in the mutant by anaerobiosis. It is concluded that coupling factor plays a role in some step(s) of DNA biosynthesis.     

人DJ-1蛋白在NIH3T3细胞中的表达与定位

采用增强型绿色荧光蛋白（EGFP）示踪的方法,研究人DJ-1蛋白在真核细胞中的 定位及其对刺激的反应. 将克隆在pGEX-KG上的DJ-1亚克隆到载体pEGFP-C2上,对 阳性克隆进行PCR、酶切和测序鉴定,用脂质体转染NIH3T3细胞;并用荧光显微镜观察 pEGFP-DJ-1在细胞内的定位以及在血清刺激时的移位;探讨在氧化应激时DJ-1对细胞的保护作用,以及其在细胞内定位的变化. 重组质粒在NIH3T3细胞中得到高效表达,绿色荧光弥散分布于胞质、胞核中,但以胞质居多;血清刺激后,细胞中的绿色荧光从胞质移位到胞核;在200～600 μmol/L H₂O₂刺激下,DJ-1能有效保护细胞抵抗氧化应激,并且也能从胞质移位到胞核.上述研究结果为进一步研究DJ-1蛋白的功能提供了一个重要依据.     

Apoptosis-linked gene 2 binds to the death domain of Fas and dissociates from Fas during Fas-mediated apoptosis in Jurkat cells

Apoptosis-linked gene 2 (ALG-2) is a member of the family of Ca(2+)-binding proteins with penta-EF-hand and is essential for the execution of apoptosis by various signals including Fas activation. We studied the regulation of ALG-2 during Fas-mediated apoptosis in Jurkat cells. The 22-kDa ALG-2 protein is cleaved and becomes a 19-kDa protein after Fas activation. The appearance of 19-kDa ALG-2 protein increases for 4 h after treatment with 200 ng/ml of anti-Fas Ab treatment and gradually degrades afterward. Confocal microscopic analysis showed that ALG-2 translocated from the plasma membrane to the cytosol during Fas-mediated apoptosis. Therefore, we examined if ALG-2 interacts with Fas. The protein-protein interaction of ALG-2 with Fas was demonstrated using yeast two-hybrid assays as well as in vitro GST pull-down assay. Endogenous ALG-2 was immunoprecipitated with anti-Fas Ab in Jurkat cells without Fas activation. However, the endogenous ALG-2 was no longer immunoprecipitated with anti-Fas Ab 2 h after anti-Fas Ab treatment. This study, for the first time, presents a direct molecular connection of ALG-2 to apoptosis by its direct interaction with Fas, and enlists ALG-2 as a new member of posttranslationally modified proteins during Fas-mediated apoptotic process.     

Energy requirement for biosynthesis of DNA in Escherichia coli: Role of membrane-bound energy-transducing ATPase (coupling factor)

A mutant of Escherichia coli missing energy-transducing ATPase and known to be defective in a variety of membrane functions from earlier studies (Yamamoto, T. H., Mével-Ninio, M. and Valentine, R. C. (1973) Biochim. Biophys. Acta 314, 267–275; Thipayathasana, P. and Valentine, R. C. (1974) Biochim. Biophys. Acta 347, 464–468; Mével-Ninio, M. and Yamamoto, T. (1974) Biochim. Biophys. Acta 357, 63–66) has been found to be blocked for anaerobic DNA synthesis. The rate of anaerobic DNA synthesis in the mutant, measured as radioactive adenine incorporation into the alkali-resistant fraction of whole cells, is about 1/6 the rate of DNA synthesis in the wild type culture under similar conditions. Addition of NO₃^- or O₂ restores DNA biosynthesis in the mutant. The entry of radioactive adenine is not appreciably affected in the mutant by anaerobiosis. It is concluded that coupling factor plays a role in some step(s) of DNA biosynthesis.     

核定位信号分析示踪载体——pGST-EGFP的构建及功能鉴定

目的 构建谷胱甘肽转硫酶（GST）与EGFP相融合的新型蛋白质示踪载体--pGST-EGFP,以用于蛋白质细胞亚定位信号序列的深入分析.方法 以质粒pEGFP-N1为骨架,融合从pGEX-2TK载体中扩增的GST编码序列,构建成pGST-EGFP融合表达质粒;再插入人工合成的已知核定位蛋白SV40的核定位序列（NLS）,构建成pGST-EGFP-SV40 NLS作为阳性对照;另外,构建小分子量蛋白TNNI2在pGST-EGFP的融合表达质粒.将对照pEGFP-N1和各重组质粒分别用脂质体介导,瞬时转染HeLa细胞,荧光显微镜下观察蛋白的核定位情况.结果 单独表达的EGFP呈全细胞分布,而GST-EGFP融合蛋白只存在于细胞浆;SV40 NLS能将GST-EGFP融合蛋白带进细胞核.虽然TNNI2-EGFP融合蛋白的细胞亚定位呈现核内丰度更高的特点,但TNNI2-GST-EGFP融合蛋白仅限定于胞浆分布,提示TNNI2不能主动定位到细胞核中.结论 成功构建了蛋白质细胞亚定位示踪载体--pGST-EGFP.作为核定位信号分析系统,其对小分子蛋白细胞亚定位的示踪效果优于传统的pEGFP载体,更适用于科研工作中小分子量蛋白质核定位信号序列的研究.     

Human RBM28 protein is a specific nucleolar component of the spliceosomal snRNPs

The biogenesis of spliceosomal small nuclear RNAs (snRNAs) involves organized translocations between the cytoplasm and certain nuclear domains, such as Cajal bodies and nucleoli. Here we identify human RBM28 protein as a novel snRNP component, based on affinity selection of U6 small nuclear ribonucleoprotein (snRNP). As shown by immunofluorescence, RBM28 is a nucleolar protein. Anti-RBM28 immunoprecipitation from HeLa cell lysates revealed that this protein specifically associates with U1, U2, U4, U5, and U6 snRNAs. Our data provide the first evidence that RBM28 is a common nucleolar component of the spliceosomal ribonucleoprotein complexes, possibly coordinating their transition through the nucleolus.     

One signal is enough: Stepwise transport of two distinct passenger proteins by the Tat pathway across the thylakoid membrane

The twin-arginine translocation (Tat) pathway, one of four protein transport pathways operating at the thylakoid membrane of chloroplasts, shows remarkable substrate flexibility. Here, we have analyzed the thylakoid transport of chimeric tandem substrates that are composed of two different passenger proteins fused to a single Tat transport signal. The chimera 23/23-EGFP in which the reporter protein EGFP is connected to the C-terminus of the OEC23 precursor shows that a single Tat transport signal is sufficient to mediate transport of two distinct passenger proteins in a row. Replacing the transit peptide of OEC23 in 23/23-EGFP by its homolog from OEC16 yields the chimera 16/23-EGFP, which can likewise be fully translocated by the Tat pathway across the thylakoid membrane. However, transport of 16/23-EGFP is retarded at specific steps in the transport process leading to the temporary and consecutive accumulation of three translocation intermediates with distinct membrane topology. They are associated with two oligomeric membrane complexes presumably representing TatBC-receptor complexes. The composition of the translocation intermediates as determined by immunoprecipitation experiments suggests that the two passenger proteins are translocated in a stepwise manner across the membrane.     

Monte Carlo studies of phospholipid lamellae. Effects of proteins, cholesterol, bilayer curvature, and lateral mobility on order parameters

In this paper we present the results of a Monte Carlo study of the effects of protein, cholesterol, bilayer curvature, and mobility on the chain order parameters of a lipid layer. The Monte Carlo method used is identical to the version developed earlier (Scott, Jr., H.L. (1977) Biochim. Biophys. Acta 469, 264–271). Simulations of protein and cholesterol effects are accomplished by insertion of a rigid stationary cylinder into the lipid matrix. The protein studies show the presence of boundary lipid (Jost, P., Griffith, O.H., Capaldi, R.H. and Vanderkooi, G. (1973) Biochim. Biophys. Acta 311, 141–152). The effect of cholesterol is dependent upon the length of the lipid hydrocarbon chains relative to the cholesterol depth of penetration. Our computer studies of bilayer curvature show the manner in which this curvature disrupts chain packing and are consistent with experimental results (Chrzeszczyk, A., Wishnia, A. and Springer, C.S. (1977) Biochim. Biophys. Acta, 470, 161–171). We also find that restricting lateral motion in chains, the simplest manner in which head group interactions can affect hydrocarbon chain order, does not measurably alter the order parameters. We argue that this provides some support for an earlier hypothesis by Scott (Scott, Jr., H.L. (1975) Biochim. Biophys. Acta 406, 329–346) regarding head group-chain interaction in monolayer experiments.     

Two mRNAs exist for the Hs PROS-30 gene encoding a component of human prosomes.

Screening of a λgt11 cDNA expression library of the HeLa cell genome with a monoclonal antibody that specifically recognizes prosomal 30-33-kDa proteins, allowed isolation of a 1264-nucleotide (nt) recombinant cDNA containing a 327-nt untranslated 5'-end. The amino acid (aa) sequence deduced from this cDNA revealed a protein of 269 aa (M_r of 30 227) that includes a consensus box characteristic for Tyr phosphorylation, also observed in other prosomal proteins. Comparison with another prosomal 27-kDa protein, cloned in our laboratory, indicated the presence of three prosomespecific homology boxes observed in these proteins from archaebacteria to man. Interestingly, except for the untranslated 5'-end, as well as the sequence coding for the N-terminal six aa, this cDNA is identical to two recently published cDNAs encoding subunit C2 of human liver proteasome [Tamura et al., Biochim. Biophys. Acta 1089 (1991) 95–102] and subunit NU of human erythrocyte macropain [DeMartino et al., Biochim. Biophys. Acta 1079 (1991) 29–38]. Primer extension and Northern blot analysis using two specific 18-mer oligodeoxyribonucleotides indicated the presence of two mRNAs that have divergent 5'-ends. These results, as confirmed by the polymerase chain reaction, establish the existence of two distinct Hs PROS-30 mRNAs, differing in their 5'-noncoding regions and in the N-terminal six aa of their protein products.     

Phospholipid hydroperoxide glutathione peroxidase is the 18-kDa selenoprotein expressed in human tumor cell lines

Human tumor cell lines cultured in 75Se-containing media demonstrate four major 75Se-labeled cellular proteins (57, 22, 18, and 12 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Among these selenoproteins, an enzymatic activity is known only for the 22-kDa protein, since this protein has been identified as the monomer of glutathione peroxidase. However, all tested cell lines also contained a peroxidase activity with phospholipid hydroperoxides that is completely accounted for by the other selenoenzyme, phospholipid hydroperoxide glutathione peroxidase (PHGPX) (Ursini, F., Maiorino, M., and Gregolin, C. (1985) Biochim. Biophys. Acta 839, 62-70). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of 75Se-labeled proteins separated by gel permeation chromatography supported the identification of PHGPX as the monomeric protein matching the 18 kDa band. This paper is the first report on the identification of PHGPX in human cells.     

Binding of phospholipase A2 to zwitterionic bilayers is promoted by lateral segregation of anionic amphiphiles

Catalytic action of phospholipase A2 is appreciably influenced by the organization and dynamics of bilayers of glycerophosphocholines (Apitz-Castro et al. (1988) Biochim. Biophys. Acta 688, 341-348). However, such effects of the quality of the interface are not observed with bilayers of glycerophosphoryl methanol and other anionic phospholipids (Jain et al. (1986) Biochim. Biophys. Acta 860, 435-447). Such differences between the catalytic susceptibility of zwitterionic versus anionic bilayers are due to a large difference in the affinity of the enzyme for these interfaces. Binding to phospholipase A2 to zwitterionic interfaces can be promoted in the presence of certain anionic additives. For example in the pre-steady-state phase of hydrolysis, segregation of the nacently produced products of hydrolysis could promote binding of phospholipase A2 to regions of higher anionic charge density in the zwitterionic interface. In this paper we show that the dynamics of segregation of the nacently produced products of hydrolysis in zwitterionic bilayers can be readily followed by monitoring the fluorescence intensity of the cationic dye NK-529 (Yu and Jain (1989) Biochim. Biophys. Acta 980, 15-22). The fluorescence emission characteristics of NK-529 change appreciably due to self-quenching of the bound dye molecules as the fatty acid molecules segregate in the bilayer. The kinetics of segregation of fatty acids during the course of hydrolysis of bilayers of zwitterionic phospholipids by phospholipase A2 exhibits an unequivocal correlation with a variety of phenomena that are observed during the transition from the pre-steady-state phase to the steady-state phase of hydrolysis in the reaction progress curves as a function of temperature and in the presence of lipophilic additives.     

Atomic solvation parameters applied to molecular dynamics of proteins in solution.

A solvation energy function for use in the molecular simulation of proteins is proposed. It is based on the accessible surface areas of atoms in the protein and on atomic solvation parameters derived from empirical vapor-to-water free energies of transfer of amino acid side-chain analogs. The energy function and its derivatives were added to the CHARMM molecular simulation program (Brooks, B.R., Bruccoleri, R.E., Olafson, B.D., States, D.J., Swaminathan, S., & Karplus, M., 1983, J. Comput. Chem. 4(2), 187-217). The effect of the added energy term was evaluated by 110 ps of molecular dynamics on the 26-residue protein melittin. The melittin monomer and tetramer were studied both with and without the added term. With the added energy term the monomer partially unfolded, while the secondary structure of the tetramer was preserved, in agreement with reported experiments (Brown, L.R., Lauterwein, J., & Wuethrich, K., 1980, Biochim. Biophys. Acta 622(2), 231-244; Lauterwein, J., Brown, L.R., & Wuethrich, K., 1980, Biochim. Biophys. Acta 622(2), 219-230).     

The use of folding patterns in the prediction of protein topologies

We proved previously that the distribution of formation energies which may be associated with the predicted secondary structures (or nuclei) is specific of the folding process (Busetta, B. 1986, Biochim. Biophys. Acta 870, 327-338). We developed a new predictive algorithm for protein topologies, based on the search of standard 'folding patterns'. In another manner, the strongest predicted nuclei are used to propose a fast sequence-alignment process which is efficient for distantly related proteins.     

EAAT2 density at the astrocyte plasma membrane and Ca(2 + )-regulated exocytosis

We studied whether regulated exocytosis affects the glutamate transporter density in cultured astrocytes, in which the expression of a fluorescently labeled excitatory amino acid transporter 2 (EAAT2-EGFP) predominantly labeled the plasma membrane. The addition of ionomycin that elevates cytosolic Ca(2+) strongly increased the fluorescence of FM 4-64 membrane area dye, confirming the presence of regulated exocytosis in transfected astrocytes. However, concomitant with Ca(2+)-dependent FM 4-64 fluorescence increase, ionomycin induced a significant steady-state decrease in EAAT2-EGFP fluorescence. This is likely due to a secondary inner filter effect since,(i) in the absence of FM 4-64, ionomycin stimulation was ineffective in changing the EAAT2-EGFP fluorescence, and (ii) fluorescence changes in FM 4-64 and EAAT2-EGFP were inversely correlated. To test whether subcellular EAAT2-EGFP structures are translocated from the cytoplasm to the plasma membrane during ionomycin stimulation, EAAT2-EGFP fluorescence was monitored locally at the plasma membrane and a few microns away in the adjacent cytoplasm. Measurements revealed sites with an increase in EAAT2-EGFP plasma membrane fluorescence correlated with a fluorescence decrease beneath the plasma membrane, and sites with plasma membrane fluorescence decrease correlated with fluorescence increase within the adjacent cytoplasm. The sites of rapid translocation/retrieval of EAAT2-EGFP structures to/from the plasma membrane appeared to be distributed in a punctuate pattern around the cell perimeter. The density of EAAT2-EGFP was regulated in a Ca(2+)-dependent manner, since in the absence of extracellular Ca(2+) local translocation/retrieval events were absent, revealing rapid surface density regulation of EAAT2 in astrocytes by regulated exo/endocytosis.     

Isolation of a phosphatidylserine transfer protein from yeast cytosol.

A phospholipid transfer protein with a broad substrate specificity was isolated from yeast cytosol. The rate of transfer catalyzed by this protein in vitro is highest for phosphatidylserine; phosphatidylethanolamine, cardiolipin, phosphatidic acid and ergosterol are transported at a lower rate. In contrast to the yeast phosphatidylinositol transfer protein (Daum, G. and Paltauf, F. (1984) Biochim. Biophys. Acta 794, 385-391) the phosphatidylserine transfer protein does not catalyze the translocation of phosphatidylinositol or phosphatidylcholine. Using chromatographic methods the phosphatidylserine transfer protein was enriched approximately 3000-fold over yeast cytosol. The protein is inactivated by heat, detergents and proteinases. Divalent cations strongly inhibit the transfer of phosphatidylserine in vitro, and EDTA at low concentrations has a stimulatory effect.