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.     

Cytoplasmic, nuclear, and golgi localization of RGS proteins. Evidence for N-terminal and RGS domain sequences as intracellular targeting motifs

RGS proteins comprise a family of proteins named for their ability to negatively regulate heterotrimeric G protein signaling. Biochemical studies suggest that members of this protein family act as GTPase-activating proteins for certain Galpha subunits, thereby accelerating the turn-off mechanism of Galpha and terminating signaling by both Galpha and Gbetagamma subunits. In the present study, we used confocal microscopy to examine the intracellular distribution of several RGS proteins in COS-7 cells expressing RGS-green fluorescent protein (GFP) fusion proteins and in cells expressing RGS proteins endogenously. RGS2 and RGS10 accumulated in the nucleus of COS-7 cells transfected with GFP constructs of these proteins. In contrast, RGS4 and RGS16 accumulated in the cytoplasm of COS-7 transfectants. As observed in COS-7 cells, RGS4 exhibited cytoplasmic localization in mouse neuroblastoma cells, and RGS10 exhibited nuclear localization in human glioma cells. Deletion or alanine substitution of an N-terminal leucine repeat motif present in both RGS4 and RGS16, a domain identified as a nuclear export sequence in HIV Rev and other proteins, promoted nuclear localization of these proteins in COS-7 cells. In agreement with this observation, treatment of mouse neuroblastoma cells with leptomycin B to inhibit nuclear protein export by exportin1 resulted in accumulation of RGS4 in the nucleus of these cells. GFP fusions of RGS domains of RGS proteins localized in the nucleus, suggesting that nuclear localization of RGS proteins results from nuclear targeting via RGS domain sequences. RGSZ, which shares with RGS-GAIP a cysteine-rich string in its N-terminal region, localized to the Golgi complex in COS-7 cells. Deletion of the N-terminal domain of RGSZ that includes the cysteine motif promoted nuclear localization of RGSZ. None of the RGS proteins examined were localized at the plasma membrane. These results demonstrate that RGS proteins localize in the nucleus, the cytoplasm, or shuttle between the nucleus and cytoplasm as nucleo-cytoplasmic shuttle proteins. RGS proteins localize differentially within cells as a result of structural differences among these proteins that do not appear to be important determinants for their G protein-regulating activities. These findings suggest involvement of RGS proteins in more complex cellular functions than currently envisioned.     

Nubp1 Is Required for Lung Branching Morphogenesis and Distal Progenitor Cell Survival in Mice

The lung is a complex system in biology and medicine alike. Whereas there is a good understanding of the anatomy and histology of the embryonic and adult lung, less is known about the molecular details and the cellular pathways that ultimately orchestrate lung formation and affect its health. From a forward genetic approach to identify novel genes involved in lung formation, we identified a mutated Nubp1 gene, which leads to syndactyly, eye cataract and lung hypoplasia. In the lung, Nubp1 is expressed in progenitor cells of the distal epithelium. Nubp1(m1Nisw) mutants show increased apoptosis accompanied by a loss of the distal progenitor markers Sftpc, Sox9 and Foxp2. In addition, Nubp1 mutation disrupts localization of the polarity protein Par3 and the mitosis relevant protein Numb. Using knock-down studies in lung epithelial cells, we also demonstrate a function of Nubp1 in regulating centrosome dynamics and microtubule organization. Together, Nubp1 represents an essential protein for lung progenitor survival by coordinating vital cellular processes including cell polarity and centrosomal dynamics.     

The N-terminus of porcine circovirus type 2 replication protein is required for nuclear localization and ori binding activities

Porcine circovirus type 2 possesses a circular, single-stranded DNA genome that requires the replication protein (Rep) for virus replication. To characterize the DNA binding potential and the significant region that confers the nuclear localization of the Rep protein, the defined coding regions of rep gene were cloned and expressed. All of the recombinant proteins except for the N-terminal 110 residues deletion mutant could bind to the double-stranded minimal binding site of replication origin (ori). In addition, the N-terminal deletion mutant lacking 110 residues exhibited mainly cytoplasmic staining in the transfected cells in contrast to the others, which localized dominantly in the nucleus, suggesting that this N-terminal domain is essential for nuclear localization. Furthermore, a series of green fluorescence proteins (GFP) containing potential nuclear localization signal (NLS) sequences were tested for their cellular distribution. The ability of the utmost 20 residues of the N-terminal region to target the GFP to the nucleus confirmed its role as a functional NLS.     

Cellular distribution of mammalian DNA topoisomerase II is determined by its catalytically dispensable C-terminal domain.

Mammalian cells express two genetically distinct isoforms of DNA topoisomerase II, designated topoisomerase IIalphaand topoisomerase IIbeta. We have recently shown that mouse topoisomerase IIalpha can substitute for the yeast topoisomerase II enzyme and complement yeast top2 mutations. This functional complementation allowed functional analysis of the C-terminal domain (CTD) of mammalian topoisomerase II, where the amino acid sequences are divergent and species-specific, in contrast to the highly conserved N-terminal and central domains. Several C-terminal deletion mutants of mouse topoisomerase IIalpha were constructed and expressed in yeast top2 cells. We found that the CTD of topoisomerase IIalphais dispensable for enzymatic activity in vitro but is required for nuclear localization in vivo. Interestingly, the CTD of topoisomerase IIbetawas also able to function as a signal for nuclear targeting. We therefore examined whether the CTD alone is sufficient for nuclear localization in vivo . The C-terminal region was fused to GFP (green fluorescent protein) and expressed under the GAL1 promoter in yeast cells. As expected, GFP signal was exclusively detected in the nucleus, irrespective of the CTD derived from either topoisomerase IIalphaor IIbeta. Surprisingly, when the upstream sequence of each CTD was added nuclear localization of the GFP signal was found to be cell cycle dependent: topoisomerase IIalpha-GFP was seen in the mitotic nucleus but was absent from the interphase nucleus, while topoisomerase IIbeta-GFP was detected predominantly in the interphase nucleus and less in the mitotic nucleus. Our results suggest that the catalytically dispensable CTD of topoisomerase II is sufficient as a signal for nuclear localization and that yeast cells can distinguish between the two isoforms of mammalian topoisomerase II, localizing each protein properly.     

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.     

Functional roles for fatty acylated amino-terminal domains in subcellular localization

Several membrane-associating signals, including covalently linked fatty acids, are found in various combinations at the N termini of signaling proteins. The function of these combinations was investigated by appending fatty acylated N-terminal sequences to green fluorescent protein (GFP). Myristoylated plus mono/dipalmitoylated GFP chimeras and a GFP chimera containing a myristoylated plus a polybasic domain were localized similarly to the plasma membrane and endosomal vesicles, but not to the nucleus. Myristoylated, nonpalmitoylated mutant chimeric GFPs were localized to intracellular membranes, including endosomes and the endoplasmic reticulum, and were absent from the plasma membrane, the Golgi, and the nucleus. Dually palmitoylated GFP was localized to the plasma membrane and the Golgi region, but it was not detected in endosomes. Nonacylated GFP chimeras, as well as GFP, showed cytosolic and nuclear distribution. Our results demonstrate that myristoylation is sufficient to exclude GFP from the nucleus and associate with intracellular membranes, but plasma membrane localization requires a second signal, namely palmitoylation or a polybasic domain. The similarity in localization conferred by the various myristoylated and palmitoylated/polybasic sequences suggests that biophysical properties of acylated sequences and biological membranes are key determinants in proper membrane selection. However, dual palmitoylation in the absence of myristoylation conferred significant differences in localization, suggesting that multiple palmitoylation sites and/or enzymes may exist.     

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.     

Nuclear translocation of Acinetobacter baumannii transposase induces DNA methylation of CpG regions in the promoters of E-cadherin gene

Nuclear targeting of bacterial proteins has emerged as a pathogenic mechanism whereby bacterial proteins induce host cell pathology. In this study, we examined nuclear targeting of Acinetobacter baumannii transposase (Tnp) and subsequent epigenetic changes in host cells. Tnp of A. baumannii ATCC 17978 possesses nuclear localization signals (NLSs), (225)RKRKRK(230). Transient expression of A. baumannii Tnp fused with green fluorescent protein (GFP) resulted in the nuclear localization of these proteins in COS-7 cells, whereas the truncated Tnp without NLSs fused with GFP were exclusively localized in the cytoplasm. A. baumannii Tnp was found in outer membrane vesicles, which delivered this protein to the nucleus of host cells. Nuclear expression of A. baumannii Tnp fused with GFP in A549 cells induced DNA methylation of CpG regions in the promoters of E-cadherin (CDH1) gene, whereas the cytoplasmic localization of the truncated Tnp without NLSs fused with GFP did not induce DNA methylation. DNA methylation in the promoters of E-cadherin gene induced by nuclear targeting of A. baumannii Tnp resulted in down-regulation of gene expression. In conclusion, our data show that nuclear traffic of A. baumannii Tnp induces DNA methylation of CpG regions in the promoters of E-cadherin gene, which subsequently down-regulates gene expression. This study provides a new insight into the epigenetic control of host genes by bacterial proteins.     

Two novel mouse genes--Nubp2, mapped to the t-complex on chromosome 17, and Nubp1, mapped to chromosome 16--establish a new gene family of nucleotide-binding proteins in eukaryotes.

Two novel mouse genes and one novel human gene that define distinctive eukaryotic nucleotide-binding proteins (NUBP) and are related to the mrp gene of prokaryotes are characterized. Phylogenetic analyses of the genes, encoding a short form (Nubp2) and a long form (Nubp1) of NUBP, clearly establish them as a new NUBP/MRP gene family that is well conserved throughout phylogeny. In addition to conserved ATP/GTP-binding motifs A (P-loop) and A', members of this family share at least two highly conserved sequence motifs, NUBP/MRP motifs alpha and beta. Only one type of NUBP/MRP gene has been observed thus far in prokaryotes, but there are two types in eukaryotes. One group includes mouse Nubp1, human NBP, yeast NBP35, and Caenorhabditis elegans F10G8.6 and is characterized by a unique N-terminal sequence with four cysteine residues that is lacking in the other group, which includes mouse Nubp2, human NUBP2, and yeast YIA3w. Northern blot analyses of the two mouse genes show distinctive patterns consistent with this classification. Mouse Nubp2 is mapped to the t-complex region of mouse Chromosome 17, whereas Nubp1 is mapped to the proximal region of mouse Chromosome 16. Interestingly, both regions are syntenic with human chromosome 16p13.1-p13.3, suggesting that a chromosomal breakage between Nubp2 and Nubp1 probably occurred during the evolution of mouse chromosomes.     

Molecular cloning of one isotype of human lamina-associated polypeptide 1s and a topological analysis using its deletion mutants

LAP1s (lamina-associated polypeptide 1s) are type 2 integral membrane proteins with a single membrane-spanning region of the inner nuclear membrane. We report here on the cloning of the full-length cDNA of human LAP1B (huLAP1B) that encodes 584 amino acids. The sequence homology between the predicted rat LAP1B and huLAP1B was found to be 73.6%. A topological analysis was carried out by transiently expressing N-terminal GFP fused deletion mutants of huLAP1B in cells. The transmembrane (TM) domain (aa 346-368) is required for the localization of the nuclear and endoplasmic reticulum membrane and that the TM domain and the C-terminal half of the nucleoplasmic domain (aa 190-331) are sufficient for the proper localization of LAP1B. In contrast, the well-conserved lumenal domain of the nuclear membrane is not required for its topological function. Biochemical analysis showed that huLAP1B is retained within the nucleus via interactions of the nucleoplasmic portion with nuclear components.     

DDM1 binds Arabidopsis methyl-CpG binding domain proteins and affects their subnuclear localization

Methyl-CpG binding domain (MBD) proteins in Arabidopsis thaliana bind in vitro methylated CpG sites. Here, we aimed to characterize the binding properties of AtMBDs to chromatin in Arabidopsis nuclei. By expressing in wild-type cells AtMBDs fused to green fluorescent protein (GFP), we showed that AtMBD7 was evenly distributed at all chromocenters, whereas AtMBD5 and 6 showed preference for two perinucleolar chromocenters adjacent to nucleolar organizing regions. AtMBD2, previously shown to be incapable of binding in vitro-methylated CpG, was dispersed within the nucleus, excluding chromocenters and the nucleolus. Recruitment of AtMBD5, 6, and 7 to chromocenters was disrupted in ddm1 and met1 mutant cells, where a significant reduction in cytosine methylation occurs. In these mutant cells, however, AtMBD2 accumulated at chromocenters. No effect on localization was observed in the chromomethylase3 mutant showing reduced CpNpG methylation or in kyp-2 displaying a reduction in Lys 9 histone H3 methylation. Transient expression of DDM1 fused to GFP showed that DDM1 shares common sites with AtMBD proteins. Glutathione S-transferase pull-down assays demonstrated that AtMBDs bind DDM1; the MBD motif was sufficient for this interaction. Our results suggest that the subnuclear localization of AtMBD is not solely dependent on CpG methylation; DDM1 may facilitate localization of AtMBDs at specific nuclear domains.     

Nuclear localization of angiotensinogen in astrocytes

In the brain, angiotensinogen (AGT) is primarily expressed in astrocytes; brain ANG II derived from locally produced AGT has been shown to influence blood pressure. To better understand the molecular basis of AGT expression in the brain, we identified a human astrocytoma cell line, CCF-STTG1, that expresses endogenous AGT mRNA and produces AGT protein. Studies examining CCF-STTG1 cell AGT after N- and O-glycosidase suggest that AGT may not be posttranslationally modified by glycosylation in these cells as it is in plasma. Small amounts of AGT (5% of HepG2) were detected in the culture medium, suggesting a low rate of AGT secretion. Immunocytochemical examination of AGT in CCF-STTG1 cells revealed mainly nuclear localization. Although this has not been previously reported, it is consistent with nuclear localization of other serpin family members. To examine this further, we generated a fusion protein consisting of green fluorescent protein (GFP) and human AGT and examined subcellular localization by confocal microscopy after confirming expression of the fusion protein by Western blot. In CCF-STTG1 cells, a control GFP construct lacking AGT was mainly localized in the cytoplasm, whereas the GFP-AGT fusion protein was primarily localized in the nucleus. To map the location of a potential nuclear localization signal, overlapping 500-bp fragments of human AGT cDNA were fused in frame downstream of GFP. Although four of the fusion proteins exhibited either perinuclear or cytoplasmic localization, one fusion protein encoding the COOH terminus of AGT was localized in the nucleus. Importantly, nuclear localization of human AGT was confirmed in primary cultures of glial cells isolated from transgenic mice expressing the human AGT under the control of its own endogenous promoter. Our results suggest that AGT may have a novel intracellular role in the brain apart from its predicted endocrine function.     

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.     

TransientAgrobacterium-mediated gene expression in theArabidopsis hydroponics root system for subcellular localization studies

To a great extent, the cellular compartmentalization and molecular interactions are indicative of the function of a protein. The development of simple and efficient tools for testing the subcellular location of proteins is indispensable to elucidate the function of genes in plants. In this report, we assessed the feasibility ofAgrobacterium-mediated transformation of hydroponically grown roots to follow intracellular targeting of proteins fused to green fluorescent protein (GFP). We developed a simple in planta assay for subcellular localization of proteins inArabidopsis roots via transient transformation and tested this method by expressing a GFP fusion of a known nuclear protein, IQD1. Visualization of transiently expressed GFP fusion proteins in roots by means of confocal microscopy is superior to the analysis of green tissues because the roots are virtually transparent and free of chlorophyll autofluorescence.