PNRC1核定位信号序列的鉴定

为鉴定富含脯氨酸核受体辅调节蛋白1(PNRC1)分子的核定位信号序列（nuclear localization signal sequence, NLS）,在生物信息学方法预测的基础上,先构建野生型PNRC1及删除预测NLS的PNRC1突变体的绿色荧光蛋白（GFP）重组表达载体,转染细胞后通过激光共聚焦显微镜观察PNRC1分子在删除预测NLS后细胞内的定位变化.然后,将预测的NLS编码序列直接连到GFP表达载体上,以及将预测的NLS加到胞浆蛋白上构建其GFP重组表达载体,转染细胞,观察预测的NLS能否把构建的重组体都带到细胞核内.结果显示,删除PNRC1中预测的NLS后,其定位从细胞核中变为主要定位在细胞浆中,而预测的NLS能把GFP或胞浆中的蛋白带到细胞核中.研究表明,预测的NLS为PNRC1分子真正的NLS.     

Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants

A series of versatile cloning vectors has been constructed that facilitate the expression of protein fusions to the Aequorea victoria green fluorescent protein (GFP) in plant cells. Amino-terminal- and carboxy-terminal protein fusions have been created and visualized by epifluorescence microscopy, both in transgenic Arabidopsis thaliana and after transient expression in onion epidermal cells. Using tandem dimers and other protein fusions to GFP, we found that the previously described localization of wild-type GFP to the cell nucleus is most likely due to diffusion of GFP across the nuclear envelope rather than to a cryptic nuclear localization signal. A fluorescence-based, quantitative assay for nuclear localization signals is described. In addition, we have employed the previously characterized mutants GFP–S65T and GFP–Y66H in order to allow for the expression of red-shifted and blue fluorescent proteins, respectively, which are suitable for double-labeling studies. Expression of GFP-fusions was controlled by a cauliflower mosaic virus 35S promoter. Using the Arabidopsis COP1 protein as a model, we confirmed a close similarity in the subcellular localization of native COP1 and the GFP-tagged COP1 protein. We demonstrated that COP1 was localized to discrete subnuclear particles and further confirmed that fusion to GFP did not compromise the activity of the wild-type COP1 protein.     

Nuclear localization of enhanced green fluorescent protein homomultimers

The green fluorescent protein (GFP) and its variants are used in many studies to determine the subcellular localization of other proteins by analyzing fusion proteins. The main problem for nuclear localization studies is the fact that, to some extent, GFP translocates to the nucleus on its own. Because the nuclear import could be due to unspecific diffusion of the relatively small GFP through the nuclear pores, we analyzed the localization of multimers of a GFP variant, the enhanced GFP (EGFP). By detecting the fluorescence of the expressed proteins in gels after nonreducing SDS-PAGE, we demonstrate the integrity of the expressed proteins. Nevertheless, even EGFP homotetramers and homohexamers are found in the nuclei of the five analyzed mammalian cell lines. The use of fusion constructs of small proteins with multimeric EGFP alone, therefore, is not adequate to prove nuclear import processes. Fusion to tetrameric EGFP in combination with a careful quantification of the fluorescence intensities in the nucleus and cytoplasm might be sufficient in many cases to identify a significant difference between the fusion protein and tetrameric EGFP alone to deduce a nuclear localization signal.     

Membrane localization of Arabidopsis acyl-CoA binding protein ACBP2

Cytosolic acyl-CoA binding proteins bind long-chain acyl-CoAs and act as intracellular acyl-CoA transporters and pool formers. Recently, we have characterized Arabidopsis thaliana cDNAs encoding novel forms of ACBP, designated ACBP1 and ACBP2, that contain a hydrophobic domain at the N-terminus and show conservation at the acyl-CoA binding domain to cytosolic ACBPs. We have previously demonstrated that ACBP1 is membrane-associated in Arabidopsis. Here, western blot analysis of anti-ACBP2 antibodies on A. thaliana protein showed that ACBP2 is located in the microsome-containing membrane fraction and in the subcellular fraction containing large particles (mitochondria, chloroplasts and peroxisomes), resembling the subcellular localization of ACBP1. To further investigate the subcellular localization of ACBP2, we fused ACBP2 translationally in-frame to GFP. By means of particle gene bombardment, ACBP2-GFP and ACBP1-GFP fusion proteins were observed transiently expressed at the plasma membrane and at the endoplasmic reticulum in onion epidermal cells. GFP fusions with deletion derivatives of ACBP1 or ACBP2 lacking the transmembrane domain were impaired in membrane targeting. Our investigations also showed that when the transmembrane domain of ACBP1 or that of ACBP2 was fused with GFP, the fusion protein was targeted to the plasma membrane, thereby establishing their role in membrane targeting. The localization of ACBP1-GFP is consistent with our previous observations using immunoelectron microscopy whereby ACBP1 was localized to the plasma membrane and vesicles. We conclude that ACBP2, like ACBP1, is a membrane protein that likely functions in membrane-associated acyl-CoA transfer/metabolism.     

Localization of peroxisomal matrix proteins by photobleaching

The distribution of some enzymes between peroxisomes and cytosol, or a dual localization in both these compartments, can be difficult to reconcile. We have used photobleaching in live cells expressing green fluorescent protein (GFP)-fusion proteins to show that imported bona fide peroxisomal matrix proteins are retained in the peroxisome. The high mobility of the GFP-fusion proteins in the cytosol and absence of peroxisomal escape makes it possible to eliminate the cytosolic fluorescence by photobleaching, to distinguish between exclusively cytosolic proteins and proteins that are also present at low levels in peroxisomes. Using this technique we found that GFP tagged bile acid-CoA:amino acid N-acyltransferase (BAAT) was exclusively localized in the cytosol in HeLa cells. We conclude that the cytosolic localization was due to its carboxyterminal non-consensus peroxisomal targeting signal (-SQL) since mutation of the -SQL to -SKL resulted in BAAT being efficiently imported into peroxisomes.     

Identification of amino acid sequence in the hinge region of human vitamin D receptor that transfers a cytosolic protein to the nucleus

The localization of human vitamin D receptor (VDR) in the absence of its ligand 1,25-dihydroxyvitamin D(3) was investigated using chimera proteins fused to green fluorescent protein (GFP) at either the N or C terminus, and the nuclear localization signal (NLS) was identified. Plasmids carrying the fusion proteins were transiently or stably introduced into COS7 cells, and the subcellular distribution of the fusion proteins was examined. GFP-tagged wild-type VDRs were located predominantly in nuclei but with a significant cytoplasmic presence, while GFP alone was equally distributed throughout the cells. 10(-8) M 1,25-dihydroxyvitamin D(3) promoted the nuclear import of VDR in a few hours. To identify the NLS, we constructed several mutated VDRs fused to GFP. Mutant VDRs that did not bind to DNA were also localized predominantly in nuclei, while the deletion of the hinge region resulted in the loss of preference for nucleus. A short segment of 20 amino acids in the hinge region enabled cytoplasmic GFP-tagged alkaline phosphatase to translocate to nuclei. These results indicate that 1) VDR is located predominantly in nuclei with a significant presence in cytoplasm without the ligand and 2) an NLS consisting of 20 amino acids in the hinge region facilitates the transfer of VDR to the nucleus.     

A plastid protein crucial for Ca2+-regulated stomatal responses

* Guard cell movements are regulated by environmental cues including, for example, elevations in extracellular Ca(2+) concentration. Here, the subcellular localization and physiological function of the Ca(2+)-sensing receptor (CAS) protein was investigated. * CAS protein localization was ascertained by microscopic analyses of green fluorescent protein (GFP) fusion proteins and biochemical fractionation assays. Comparative guard cell movement investigations were performed in wild-type and cas loss-of-function mutant lines of Arabidopsis thaliana. Cytoplasmic Ca(2+) dynamics were addressed in plants expressing the yellow cameleon reporter protein YC3.6. * This study identified CAS as a chloroplast-localized protein that is crucial for proper stomatal regulation in response to elevations of external Ca(2+). CAS fulfils this role through modulation of the cytoplasmic Ca(2+) concentration. * This work reveals a novel role of the chloroplast in cellular Ca(2+) signal transduction.     

Green Fluorescent Protein (GFP)-tagged Cysteine-rich Domains from Protein Kinase C as Fluorescent Indicators for Diacylglycerol Signaling in Living Cells

Cysteine-rich domains (Cys-domains) are ~50–amino acid–long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-γ (Cys1–GFP). Strikingly, stimulation of G-protein or tyrosine kinase–coupled receptors induced a transient translocation of cytosolic Cys1–GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1–GFP in the membrane, whereas DiC8 left Cys1–GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1–GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-γ also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2–GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester–mediated translocation of proteins to selective lipid membranes.     

Searching for new cell wall protein genes in Arabidopsis

The objective of this study was to test an approach that combines bioinformatic and subcellular localization analysis to identify novel cell wall protein genes in Arabidopsis. Proteins with unknown function in the Arabidopsis genome were first identified and scanned for the presence of N-terminal signal peptides. The signal peptide-containing function-unknown proteins were further analyzed to eliminate the ones containing other sequences, such as endoplasmic reticulum and vacuole retention signals, that may prevent a protein from secretion into cell walls. The top ten genes passing the bioinformatic analysis were selected for protein subcellular localization using green fluorescence protein (GFP) as a reporter. A vector was constructed for high throughput gene-GFP fusion protein generation and overexpression in Arabidopsis for gene function analysis. Transformants of six genes showed reasonable expression of GFP fusion protein. However, none of the transformants showed GFP localization in cell walls. The low rate of new cell wall protein discovery suggests that the number of unidentified cell wall proteins in the Arabidopsis genome may be small.     

Green fluorescent protein tagging Drosophila proteins at their native genomic loci with small P elements

We describe a technique to tag Drosophila proteins with GFP at their native genomic loci. This technique uses a new, small P transposable element (the Wee-P) that is composed primarily of the green fluorescent protein (GFP) sequence flanked by consensus splice acceptor and splice donor sequences. We demonstrate that insertion of the Wee-P can generate GFP fusions with native proteins. We further demonstrate that GFP-tagged proteins have correct subcellular localization and can be expressed at near-normal levels. We have used the Wee-P to tag genes with a wide variety of functions, including transmembrane proteins. A genetic analysis of 12 representative fusion lines demonstrates that loss-of-function phenotypes are not caused by the Wee-P insertion. This technology allows the generation of GFP-tagged reagents on a genome-wide scale with diverse potential applications.     

Arabidopsis group Ie formins localize to specific cell membrane domains, interact with actin-binding proteins and cause defects in cell expansion upon aberrant expression

The closely related proteins AtFH4 and AtFH8 represent the group Ie clade of Arabidopsis formin homologues. The subcellular localization of these proteins and their ability to affect the actin cytoskeleton were examined. AtFH4 protein activity was identified using fluorimetric techniques. Interactions between Arabidopsis profilin isoforms and AtFH4 were assayed in vitro and in vivo using pull-down assays and yeast-2-hybrid. The subcellular localization of group Ie formins was observed with indirect immunofluorescence (AtFH4) and an ethanol-inducible green fluorescent protein (GFP) fusion construct (AtFH8). AtFH4 protein affected actin dynamics in vitro, and yeast-2-hybrid assays suggested isoform-specific interactions with the actin-binding protein profilin in vivo. Indirect immunofluorescence showed that AtFH4 localized specifically to the cell membrane at borders between adjoining cells. Expression of an AtFH8 fusion protein resulted in GFP localization to cell membrane zones, similar to AtFH4. Furthermore, aberrant expression of AtFH8 resulted in the inhibition of root hair elongation. Taken together, these data suggest that the group Ie formins act with profilin to regulate actin polymerization at specific sites associated with the cell membrane.     

Combined localization and real-time functional studies using a GFP-tagged ABCG2 multidrug transporter

ABCG2 is a half-transporter which causes multidrug resistance when overexpressed in tumor cells. Availability of combined localization and functional assays would greatly improve cell biology and drug modulation studies for this transporter. Here we demonstrate that an N-terminally GFP-tagged version of the protein (GFP-G2) can be used to directly monitor ABCG2 expression, dimerization, localization and function in living cells. GFP-G2 is fully functional when tested for drug-stimulated ATPase activity, vesicular transport assay, subcellular localization or cell surface epitope conformational changes. By measuring both GFP and Hoechst 33342 dye fluorescence in HEK-293 cells, we provide evidence that a real-time transport assay can be reliably applied to identify ABCG2 substrates, transport modulators, as well as to monitor the cellular functions of this multidrug transporter protein. This approach also avoids the need of cloning, drug selection or other further separation or characterization of the transgene-expressing cells.     

Centriolin anchoring of exocyst and SNARE complexes at the midbody is required for secretory-vesicle-mediated abscission

The terminal step in cytokinesis, called abscission, requires resolution of the membrane connection between two prospective daughter cells. Our previous studies demonstrated that the coiled-coil protein centriolin localized to the midbody during cytokinesis and was required for abscission. Here we show that centriolin interacts with proteins of vesicle-targeting exocyst complexes and vesicle-fusion SNARE complexes. These complexes require centriolin for localization to a unique midbody-ring structure, and disruption of either complex inhibits abscission. Exocyst disruption induces accumulation of v-SNARE-containing vesicles at the midbody ring. In control cells, these v-SNARE vesicles colocalize with a GFP-tagged secreted polypeptide. The vesicles move to the midbody ring asymmetrically from one prospective daughter cell; the GFP signal is rapidly lost, suggesting membrane fusion; and subsequently the cell cleaves at the site of vesicle delivery/fusion. We propose that centriolin anchors protein complexes required for vesicle targeting and fusion and integrates membrane-vesicle fusion with abscission.     

Differential subcellular localization of functionally divergent survivin splice variants

Survivin is an inhibitor of apoptosis protein (IAP) that is markedly overexpressed in most cancers. We identified two novel functionally divergent splice variants, i.e. non-antiapoptotic survivin-2B and antiapoptotic survivin-deltaEx3. Because survivin-2B might be a naturally occurring antagonist of antiapoptotic survivin variants, we analyzed the subcellular distribution of these proteins. PSORT II analysis predicted a preferential cytoplasmic localization of survivin and survivin-2B, but a preferential nuclear localization of survivin-deltaEx3. GFP-tagged survivin variants confirmed the predicted subcellular localization and additionally revealed a cell cycle-dependent nuclear accumulation of survivin-deltaEx3. Moreover, a bipartite nuclear localization signal found exclusively in survivin-deltaEx3 may support cytoplasmic clearance of survivin-deltaEx3. In contrast to the known association between survivin and microtubules or centromeres during mitosis, no corresponding co-localization became evident for survivin-deltaEx3 or survivin-2B. In conclusion, our study provided data on a differential subcellular localization of functionally divergent survivin variants, suggesting that survivin isoforms may perform different functions in distinct subcellular compartments and distinct phases of the cell cycle.     

The carboxy-terminal sequence of the pestivirus glycoprotein E(rns) represents an unusual type of membrane anchor

The E(rns) protein is a structural glycoprotein of pestiviruses that lacks a typical membrane anchor sequence and is known to be secreted from the infected cell. However, major amounts of the protein are retained within the cell and attached to the virion by a so far unknown mechanism. Transient-expression studies with cDNA constructs showed that in a steady-state situation, 16% of the protein is found in the supernatant of the transfected cells while 84% appears as intracellular protein. We show here that E(rns) represents a membrane-bound protein. Membrane binding occurs via the carboxy-terminal region of E(rns). By fusion of this sequence to the carboxy terminus of green fluorescent protein (GFP), the subcellular localization of the reporter protein switched from cytosolic to membrane bound. A core sequence of 11 amino acids necessary for membrane binding was elicited in truncation experiments with GFP constructs. However, this peptide is not sufficient to confer membrane anchoring but needs either upstream or downstream accessory sequences. Analyses with different extraction procedures showed that E(rns) is neither easily stripped from the membrane, like a peripheral membrane protein, nor as tightly membrane bound as a transmembrane protein.     

Expression of Green Fluorescent Protein Fused to Magnetosome Proteins in Microaerophilic Magnetotactic Bacteria

The magnetosomes of magnetotactic bacteria are prokaryotic organelles consisting of a magnetite crystal bounded by a phospholipid bilayer that contains a distinct set of proteins with various functions. Because of their unique magnetic and crystalline properties, magnetosome particles are potentially useful as magnetic nanoparticles in a number of applications, which in many cases requires the coupling of functional moieties to the magnetosome membrane. In this work, we studied the use of green fluorescent protein (GFP) as a reporter for the magnetosomal localization and expression of fusion proteins in the microaerophilic Magnetospirillum gryphiswaldense by flow cytometry, fluorescence microscopy, and biochemical analysis. Although optimum conditions for high fluorescence and magnetite synthesis were mutually exclusive, we established oxygen-limited growth conditions, which supported growth, magnetite biomineralization, and GFP fluorophore formation at reasonable rates. Under these optimized conditions, we studied the subcellular localization and expression of the GFP-tagged magnetosome proteins MamC, MamF, and MamG by fluorescence microscopy and immunoblotting. While all fusions specifically localized at the magnetosome membrane, MamC-GFP displayed the strongest expression and fluorescence. MamC-GFP-tagged magnetosomes purified from cells displayed strong fluorescence, which was sensitive to detergents but stable under a wide range of temperature and salt concentrations. In summary, our data demonstrate the use of GFP as a reporter for protein localization under magnetite-forming conditions and the utility of MamC as an anchor for magnetosome-specific display of heterologous gene fusions.     

Exercise does not alter subcellular localization, but increases phosphorylation of insulin-signaling proteins in human skeletal muscle

The subcellular localization of insulin signaling proteins is altered by various stimuli such as insulin, insulin-like growth factor I, and oxidative stress and is thought to be an important mechanism that can influence intracellular signal transduction and cellular function. This study examined the possibility that exercise may also alter the subcellular localization of insulin signaling proteins in human skeletal muscle. Nine untrained males performed 60 min of cycling exercise (approximately 67% peak pulmonary O2 uptake). Muscle biopsies were sampled at rest, immediately after exercise, and 3 h postexercise. Muscle was fractionated by centrifugation into the following crude fractions: cytosolic, nuclear, and a high-speed pellet containing membrane and cytoskeletal components. Fractions were analyzed for protein content of insulin receptor, insulin receptor substrate (IRS)-1 and -2, p85 subunit of phosphatidylinositol 3-kinase, Akt, and glycogen synthase kinase-3 (GSK-3). There was no significant change in the protein content of the insulin signaling proteins in any of the crude fractions after exercise or 3 h postexercise. Exercise had no significant effect on the phosphorylation of IRS-1 Tyr612 in any of the fractions. In contrast, exercise increased (P < 0.05) the phosphorylation of Akt Ser473 and GSK-3alpha/beta Ser9/21 in the cytosolic fraction only. In conclusion, exercise can increase phosphorylation of downstream insulin signaling proteins specifically in the cytosolic fraction but does not result in changes in the subcellular localization of insulin signaling proteins in human skeletal muscle. Change in the subcellular protein localization is therefore an unlikely mechanism to influence signal transduction pathways and cellular function in skeletal muscle after exercise.     

Keratin-containing inclusions affect cell morphology and distribution of cytosolic cellular components

Many neurodegenerative diseases are characterized by the presence of protein aggregates bundled with intermediate filaments (IFs) and similar structures, known as Mallory bodies (MBs), are observed in various liver diseases. IFs are anchored at desmosomes and hemidesmosomes, however, interactions with other intercellular junctions have not been determined. We investigated the effect of IF inclusions on junction-associated and cytosolic proteins in various cultured cells. We performed gene transfection of the green fluorescent protein (GFP)-tagged cytokeratin (CK) 18 mutant arg89cys (GFP-CK18 R89C) in cultured cells and observed CK aggregations as well as loss of IF networks. Among various junction-associated proteins, zonula occludens-1 and beta-catenin were colocalized with CK aggregates on immunofluorescent analyses. Similar results were obtained on immunostaining for cytosolic proteins, 14-3-3 zeta protein, glucose-6-phosphate dehydrogenase and DsRed. E-cadherin, a basolateral membrane protein in polarized epithelia, was present on both the apical and basolateral domains in GFP-CK18 R89C-transfected cells. Furthermore, cells containing CK aggregates were significantly larger than GFP-tagged wild type CK18 (GFP-WT CK18)-transfected or non-transfected cells (P < 0.01) and sometimes their morphology was significantly altered. Our data indicate that CK aggregates affect not only cell morphology but also the localization of various cytosolic components, which may affect the cellular function.