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.     

The DNA binding activity of p53 displays reaction-diffusion kinetics

The tumor suppressor protein p53 plays a key role in maintaining the genomic stability of mammalian cells and preventing malignant transformation. In this study, we investigated the intracellular diffusion of a p53-GFP fusion protein using confocal fluorescence recovery after photobleaching. We show that the diffusion of p53-GFP within the nucleus is well described by a mathematical model for diffusion of particles that bind temporarily to a spatially homogeneous immobile structure with binding and release rates k1 and k2, respectively. The diffusion constant of p53-GFP was estimated to be Dp53-GFP=15.4 microm2 s-1, significantly slower than that of GFP alone, DGFP=41.6 microm2 s-1. The reaction rates of the binding and unbinding of p53-GFP were estimated as k1=0.3 s-1 and k2=0.4 s-1, respectively, values suggestive of nonspecific binding. Consistent with this finding, the diffusional mobilities of tumor-derived sequence-specific DNA binding mutants of p53 were indistinguishable from that of the wild-type protein. These data are consistent with a model in which, under steady-state conditions, p53 is latent and continuously scans DNA, requiring activation for sequence-specific DNA binding.     

GFP as a tool to analyze the organization, dynamics and function of nuclei and microtubules in Neurospora crassa

We report the construction of a versatile GFP expression plasmid and demonstrate its utility in Neurospora crassa. To visualize nuclei and microtubules, we generated carboxy-terminal fusions of sgfp to Neurospora histone H1 (hH1) and beta-tubulin (Bml). Strong expression of GFP fusion proteins was achieved with the inducible Neurospora ccg-1 promoter. Nuclear and microtubule organization and dynamics were observed in live vegetative hyphae, developing asci, and ascospores by conventional and confocal laser scanning fluorescence microscopy. Observations of GFP fusion proteins in live cells largely confirmed previous results obtained by examination of fixed cells with various microscopic techniques. H1-GFP revealed dynamic nuclear shapes. Microtubules were mostly aligned parallel to the growth axis in apical compartments but more randomly arranged in sub-apical compartments. Time-lapse imaging of beta-tubulin-GFP in germinating macroconidia revealed polymerization and depolymerization of microtubules. In heterozygous crosses, H1-GFP and beta-tubulin-GFP expression was silenced, presumably by meiotic silencing. H1-GFP was translated in the vicinity of hH1+-sgfp+ nuclei in the common cytoplasm of giant Banana ascospores, but it diffused into all nuclei, another illustration of the utility of GFP fusion proteins.     

An Apo-14 promoter-driven transgenic zebrafish that marks liver organogenesis

Several transgenic zebrafish lines for liver development studies had been obtained in the first decade of this century, but not any transgenic GFP zebrafish lines that mark the through liver development and organogenesis were reported. In this study, we analyzed expression pattern of endogenous Apo-14 in zebrafish embryogenesis by whole-mount in situ hybridization, and revealed its expression in liver primordium and in the following liver development. Subsequently, we isolated zebrafish Apo-14 promoter of 1763 bp 5'-flanking sequence, and developed an Apo-14 promoter-driven transgenic zebrafish Tg(Apo14: GFP). And, maternal expression and post-fertilization translocation of Apo-14 promoter-driven GFP were observed in the transgenic zebrafish line. Moreover, we traced onset expression of Apo-14 promoter-driven GFP and developmental behavior of the expressed cells in early heterozygous embryos by out-crossing the Tg(Apo14: GFP) male to the wild type female. Significantly, the Apo-14 promoter-driven GFP is initially expressed around YSL beneath the embryo body at 10 hpf when the embryos develop to tail bud prominence. In about 14-somite embryos at 16-17 hpf, a typical "salt-and-pepper" expression pattern is clearly observed in YSL around the yolk sac. Then, a green fluorescence dot begins to appear between the notochord and the yolk sac adjacent to otic vesicle at about 20 hpf, which is later demonstrated to be liver primordium that gives rise to liver. Furthermore, we investigated dynamic progression of liver organogenesis in the Tg(Apo14: GFP) zebrafish, because the Apo-14 promoter-driven GFP is sustainably expressed from hepatoblasts and liver progenitor cells in liver primordium to hepatocytes in the larval and adult liver. Additionally, we observed similar morphology between the liver progenitor cells and the GFP-positive nuclei on the YSL, suggesting that they might originate from the same progenitor cells in early embryos. Overall, the current study provides a transgenic zebrafish line that marks the through liver organogenesis.     

Identification of a C-terminal region that is required for the nuclear translocation of ERK2 by passive diffusion

Extracellular signal-regulated kinase 2 (ERK2) is located in the cytoplasm of resting cells and translocates into the nucleus upon extracellular stimuli by active transport of a dimer. Passive transport of an ERK2 monomer through the nuclear pore is also reported to coexist. We attempted to characterize the cytoplasmic retention and nuclear translocation of fusion proteins between deletion and site-directed mutants of ERK2 and green fluorescent protein (GFP). The overexpressed ERK2-GFP fusion protein is usually localized to both the cytoplasm and the nucleus unless a cytoplasmic anchoring protein is coexpressed. Deletion of 45 residues, but not 43 residues, from the C terminus of ERK2 prevented the nuclear distribution of the ERK2-GFP fusion protein. Substitution of a part of residues 299-313 to alanine residues also prevented the nuclear distribution of the ERK2-GFP fusion protein without abrogation of its nuclear active transport. These observations may indicate that the passive diffusion of ERK2 into the nucleus is not simple diffusion but includes a specific interaction process between residues 299-313 and the nuclear pore complex and that this interaction is not required for the active transport. We also showed that substitution of Tyr(314) to alanine residue abrogated the cytoplasmic retention of the ERK2-GFP fusion protein by PTP-SL but not by MEK1.     

Subcellular localization and targeting of glucocorticoid receptor protein fusions expressed in transgenic Arabidopsis thaliana

An animal system of inducible activation of protein fusions with the binding domain of glucocorticoid receptor (BDGR) was tested in Arabidopsis thaliana by monitoring dexamethasone (DEX)-induced nuclear targeting of reporter constructs. Two constructs containing green fluorescent protein (GFP), human homeobox protein Hanf-1 and Xenopus laevis BDGR were used, GFP/Hanf-1/BDGR and GFP/BDGR. The control construct contained GFP alone. In the absence of DEX both fusion proteins were uniformly distributed in the cytoplasm of root cells, but showed strong association with plastids in plant aerial parts. DEX treatment of roots prompted a strong and reversible nuclear accumulation of GFP/Hanf-1/BDGR, but not GFP/BDGR. Thus, in roots, the specific nuclear translocation of GFP/Hanf-1/BDGR was driven by Hanf-1 and tightly regulated by BDGR. However, in plant aerial parts treated with DEX, nuclear translocation of GFP/Hanf-1/BDGR was observed only in a few cases, and most part of the fusion protein was incorrectly and irreversibly targeted to plastids. Protease X digestion of isolated chloroplasts showed that BDGR fusion proteins were translocated into the chloroplast envelope and bound to envelope membranes, probably due to association with the chloroplast import apparatus. Thus, for efficient use of the glucocorticoid-inducible system in plants, it will be necessary to modify BDGR structure to prevent incorrect targeting of fusion proteins.     

Establishment of insect cell lines expressing green fluorescent protein on cell surface based on AcMNPV GP64 membrane fusion characteristic

Displaying a protein on the surface of cells has been provided a very successful strategy to function research of exogenous proteins. Based on the membrane fusion characteristic of Autographa californica multiple nucleopolyhedrovirus envelope protein GP64, we amplified and cloned N-terminal signal peptide and C-terminal transmembrane domain as well as cytoplasmic tail domain of gp64 gene into vector pIZ/V5-His with multi-cloning sites to construct the cell surface expression vector pIZ/V5-gp64. To verify that the vector can be used to express proteins on the membrane of insect cells, a recombinant plasmid pIZ/V5-gp64-GFP was constructed by introducing the PCR amplified green fluorescent protein (GFP) gene and transfected into insect cell lines Sf9 and H5. The transected cells were screened with zeocin and cell cloning. PCR verification results showed that the GFP gene was successfully integrated into these cells. Green fluorescence in Sf9-GFP and H5-GFP cells was observed by using confocal laser scanning microscopy and immunofluorescence detection indicated that GFP protein was located on the cell membrane. Western blot results showed that a fusion protein GP64-GFP of about 40 kDa was expressed on the membrane of Sf9-GFP and H5-GFP cells. The expression system constructed in this paper can be used for localization and continuous expression of exogenous proteins on insect cell membrane.     

Subcellular localization of the homocitrate synthase in<Emphasis Type="Italic"> Penicillium chrysogenum</Emphasis>

There are conflicting reports regarding the cellular localization in Saccharomyces cerevisiae and filamentous fungi of homocitrate synthase, the first enzyme in the lysine biosynthetic pathway. The homocitrate synthase (HS) gene (lys1) of Penicillium chrysogenum was disrupted in three transformants (HS(-)) of the Wis 54-1255 pyrG strain. The three mutants named HS1(-), HS2(-) and HS3(-) all lacked homocitrate synthase activity and showed lysine auxotrophy, indicating that there is a single gene for homocitrate synthase in P. chrysogenum. The lys1 ORF was fused in frame to the gene for the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria. Homocitrate synthase-deficient mutants transformed with a plasmid containing the lys1-GFP fusion recovered prototrophy and showed similar levels of homocitrate synthase activity to the parental strain Wis 54-1255, indicating that the hybrid protein retains the biological function of wild-type homocitrate synthase. Immunoblotting analysis revealed that the HS-GFP fusion protein is maintained intact and does not release the GFP moiety. Fluorescence microscopy analysis of the transformants showed that homocitrate synthase was mainly located in the cytoplasm in P. chrysogenum; in S. cerevisiae the enzyme is targeted to the nucleus. The control nuclear protein StuA was properly targeted to the nucleus when the StuA (targeting domain)-GFP hybrid protein was expressed in P. chrysogenum. The difference in localization of homocitrate synthase between P. chrysogenum and S. cerevisiae suggests that this protein may play a regulatory function, in addition to its catalytic function, in S. cerevisiae but not in P. chrysogenum.     

Impediments to secretion of green fluorescent protein and its fusion from Saccharomyces cerevisiae

While it has been demonstrated that GFP-tagged proteins were transported to their correct cellular compartments in most cells, attempts to secrete GFP/GFP-fusion through the default secretory pathway have not been as successful. In an attempt to induce secretion of GFP and Hexokinase (HXK)-GFP fusion in Saccharomycescerevisiae, we have cloned constructs that employed four different yeast secretion signal sequences, i.e., INU1, SUC2, PHO5, and MEL1. The expression is under the control of the galactose-inducible GAL1 promoter. Our results showed that all eight constructs entered the secretory pathway successfully, and the signal peptides were all cleaved off. However, none of the eight constructs were able to lead to secretion into the culture media or the periplasmic space. The expression levels of the eight constructs differ dramatically, depending on both the signal peptide and whether GFP was fused with HXK. Confocal microscopy studies revealed that the eight constructs also led to very different localization patterns. Among them, two constructs targeted GFP to the vacuole partially or exclusively, whereas others were mostly retained in the ER/Golgi compartments. Our efforts, together with those of others, seem to suggest that the signal peptide itself is not enough to lead to secretion of GFP from S. cerevisiae, although it has been successful in some other organisms. Nonetheless, the advantage of GFP's in vivo detection makes it a powerful tool for investigating protein localization events.     

Molecular Control of Nuclear and Subnuclear Targeting of the Plant CDK Inhibitor ICK1 and ICK1-Mediated Nuclear Transport of CDKA

ICK1 is the first member of a family of plant cyclin-dependent kinase (CDK) inhibitors. It has been shown that ICK1 is localized in the nuclei of transgenic Arabidopsis plants. Since cellular localization is important for the functions of cell cycle regulators, a comprehensive analysis was undertaken to identify specific sequences regulating the cellular localization of ICK1. Deletion and site-specific mutants fused to the green fluorescent protein (GFP) were used in transgenic Arabidopsis plants and transfected tobacco cells. Surprisingly, three separate sequences in the N-terminal, central and C-terminal regions of ICK1 could independently confer nuclear localization of the GFP fusion proteins. The central nuclear localization signal NLS^ICK1 could transport the much larger GUS (β-glucuronidase)-GFP fusion protein into nuclei, while the other two sequences were unable to. These results suggest that NLS^ICK1 is a strong NLS that actively transports the fusion protein into nuclei, while the other two sequences are either a weaker NLS or confer the nuclear localization of GFP indirectly. It was further observed that the N-terminal sequence specifies a punctate pattern of subnuclear localization, while the C-terminal sequence suppresses it. Furthermore, co-expression of ICK1 and Arabidopsis CDKA, tagged with different GFP variants, showed that ICK1 could mediate the transport of CDKA into nuclei while a mutant ICK1^1–162 that does not interact with CDKA lost this ability. These results illustrate how the nuclear localization of ICK1 is regulated and also suggest a possible role of ICK1 in regulating the cellular distribution of CDKA.     

Lamina-associated polypeptide 2beta (LAP2beta) is contained in a protein complex together with A- and B-type lamins

Lamina-associated polypeptide 2beta (LAP2beta) of vertebrates is an integral membrane protein of the inner nuclear membrane that is generated by alternative splicing from the LAP2 gene. In the majority of Xenopus somatic cells including cultured kidney epithelial cells (A6 cells) there is only one major LAP2 isoform expressed that has the highest similarities with the mammalian LAP2beta whereas isoforms corresponding in size to the mammalian LAP2gamma and alpha are not detectable. We selected A6 cells and A6 cells stably expressing GFP fusion proteins of Xenopus LAP2beta (XLAP2Pbeta) as a model system to study interactions between LAP2beta and lamins. In vitro binding experiments with GST-XLAP2beta fusion proteins and immunoprecipitations with antibodies to GFP revealed that XLAP2beta is part of a complex that contains A- and B-type lamins. For the targeting to the nuclear envelope and the in vivo formation of this complex, GFP fusion proteins were sufficient comprising only the carboxyterminal 135 amino acids of XLAP2beta or the comparable region of zebrafish LAP2beta. A highly conserved 36 amino acids long sequence is located in this region of LAP2beta that is part of the lamina-binding domain previously identified in rat LAP2beta. GFP-LAP2beta fusion proteins of Xenopus, zebrafish, and rat that contained this sequence do compete with endogenous LAP2 in transfected cells for the same binding sites in the lamina. Our data indicate that the lamina-binding site of LAP2beta has been highly conserved during vertebrate evolution and suggests that this region of LAP2beta mediates the interactions between polymers of A- and B-type lamins.     

Eci1p uses a PTS1 to enter peroxisomes: either its own or that of a partner, Dci1p

Saccharomyces cerevisiae delta3,delta2-enoyl-CoA isomerase (Eci1p), encoded by ECI1, is an essential enzyme for the betaoxidation of unsaturated fatty acids. It has been reported, as well as confirmed in this study, to be a peroxisomal protein. Unlike many other peroxisomal proteins, Ecilp possesses both a peroxisome targeting signal type 1 (PTS1)-like signal at its carboxy-terminus (-HRL) and a PTS2-like signal at its amino-terminus (RIEGPFFIIHL). We have found that peroxisomal targeting of a fusion protein consisting of Eci1p in front of green fluorescent protein (GFP) is not dependent on Pex7p (the PTS2 receptor), ruling out a PTS2 mechanism, but is dependent on Pex5p (the PTS1 receptor). This Pex5p-dependence was unexpected, since the putative PTS1 of Ecilp is not at the C-terminus of the fusion protein; indeed, deletion of this signal (-HRL-) from the fusion did not affect the Pex5p-dependent targeting. Consistent with this, Pex5p interacted in two-hybrid assays with both Eci1p and Eci1PdeltaHRL. Ecilp-GFP targeting and Eci1pdeltaHRL interaction were abolished by replacement of Pex5p with Pex5p(N495K), a point-mutated Pex5p that specifically abolishes the PTS1 protein import pathway. Thus, Eci1p peroxisomal targeting does require the Pex5p-dependent PTS1 pathway, but does not require a PTS1 of its own. By disruption of ECI1 and DCI1, we found that Dci1p, a peroxisomal PTS1 protein that shares 50% identity with Eci1p, is necessary for Eci1p-GFP targeting. This suggests that the Pex5p-dependent import of Eci1p-GFP is due to interaction and co-import with Dci1p. Despite the dispensability of the C-terminal HRL for import in wild-type cells, we have also shown that this tripeptide can function as a PTS1, albeit rather weakly, and is essential for targeting in the absence of Dci1p. Thus, Eci1p can be targeted to peroxisomes by its own PTS1 or as a hetero-oligomer with Dcilp. These data demonstrate a novel, redundant targeting pathway for Eci1p.     

The Golgi-localized Arabidopsis endomembrane protein12 contains both endoplasmic reticulum export and Golgi retention signals at its C terminus

Endomembrane proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and mammalian cells, are characterized by the presence of a large lumenal N terminus, nine transmembrane domains, and a short cytoplasmic tail. The Arabidopsis thaliana genome contains 12 EMP members (EMP1 to EMP12), but little is known about their protein subcellular localization and function. Here, we studied the subcellular localization and targeting mechanism of EMP12 in Arabidopsis and demonstrated that (1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; (2) GFP fusion at the C terminus of EMP12 caused mislocalization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in Arabidopsis cells; (3) the EMP12 cytoplasmic tail contained dual sorting signals (i.e., an endoplasmic reticulum export motif and a Golgi retention signal that interacted with COPII and COPI subunits, respectively); and (4) the Golgi retention motif of EMP12 retained several post-Golgi membrane proteins within the Golgi apparatus in gain-of-function analysis. These sorting signals are highly conserved in all plant EMP isoforms and, thus, likely represent a general mechanism for EMP targeting in plant cells.     

Assembly and sorting of the tonoplast potassium channel AtTPK1 and its turnover by internalization into the vacuole

The assembly, sorting signals, and turnover of the tonoplast potassium channel AtTPK1 of Arabidopsis (Arabidopsis thaliana) were studied. We used transgenic Arabidopsis expressing a TPK1-green fluorescent protein (GFP) fusion or protoplasts transiently transformed with chimeric constructs based on domain exchange between TPK1 and TPK4, the only TPK family member not located at the tonoplast. The results show that TPK1-GFP is a dimer and that the newly synthesized polypeptides transiently interact with a thus-far unidentified 20-kD polypeptide. A subset of the TPK1-TPK4 chimeras were unable to assemble correctly and these remained located in the endoplasmic reticulum where they interacted with the binding protein chaperone. Therefore, TPK1 must assemble correctly to pass endoplasmic reticulum quality control. Substitution of the cytosolic C terminus of TPK4 with the corresponding domain of TPK1 was sufficient to allow tonoplast delivery, indicating that this domain contains tonoplast sorting information. Pulse-chase labeling indicated that TPK1-GFP has a half-life of at least 24 h. Turnover of the fusion protein involves internalization into the vacuole where the GFP domain is released. This indicates a possible mechanism for the turnover of tonoplast proteins.     

Targeting of green fluorescent protein expression to the cell surface.

We have previously reported on GPI-anchored fusion proteins that bind radioactive isotopes. We targeted their expression to the cell surface to obtain a marker protein detectable by nuclear and optical imaging (1, 2). Here we suggest a novel approach for targeting a model protein (GFP) to the exoplasmic surface of the plasma membrane. An expression vector (pcPEP-GFP) was constructed containing GFP cDNA fused with the fragment encoding the N-terminal cytoplasmic domain and signal peptide/membrane anchoring domain of the rabbit neutral endopeptidase (PEP-GFP). Flow cytometry showed green fluorescence in 45% of cells transfected with GFP and in 34% of cells transfected with PEP-GFP (24 h after transfection). Fluorescence microscopy of fixed cells stained with rhodaminated anti-GFP antibodies showed positive reaction only in the case of PEP-GFP-transfected cells indicating cell-surface expression. The PEP-GFP fusion protein was identified as a component of the light microsomal and Golgi fractions by immunoblotting.     

Subcompartment localization of the side chain xyloglucan-synthesizing enzymes within Golgi stacks of tobacco suspension-cultured cells

Xyloglucan is the dominant hemicellulosic polysaccharide of the primary cell wall of dicotyledonous plants that plays a key role in plant development. It is well established that xyloglucan is assembled within Golgi stacks and transported in Golgi-derived vesicles to the cell wall. It is also known that the biosynthesis of xyloglucan requires the action of glycosyltransferases including α-1,6-xylosyltransferase, β-1,2-galactosyltransferase and α-1,2-fucosyltransferase activities responsible for the addition of xylose, galactose and fucose residues to the side chains. There is, however, a lack of knowledge on how these enzymes are distributed within subcompartments of Golgi stacks. We have undertaken a study aiming at mapping these glycosyltransferases within Golgi stacks using immunogold-electron microscopy. To this end, we generated transgenic lines of tobacco (Nicotiana tabacum) BY-2 suspension-cultured cells expressing either the α-1,6-xylosyltransferase, AtXT1, the β-1,2-galactosyltransferase, AtMUR3, or the α-1,2-fucosyltransferase AtFUT1 of Arabidopsis thaliana fused to green-fluorescent protein (GFP). Localization of the fusion proteins within the endomembrane system was assessed using confocal microscopy. Additionally, tobacco cells were high pressure-frozen/freeze-substituted and subjected to quantitative immunogold labelling using anti-GFP antibodies to determine the localization patterns of the enzymes within subtypes of Golgi cisternae. The data demonstrate that: (i) all fusion proteins, AtXT1-GFP, AtMUR3-GFP and AtFUT1-GFP are specifically targeted to the Golgi apparatus; and (ii) AtXT1-GFP is mainly located in the cis and medial cisternae, AtMUR3-GFP is predominantly associated with medial cisternae and AtFUT1-GFP mostly detected over trans cisternae suggesting that initiation of xyloglucan side chains occurs in early Golgi compartments in tobacco cells.