Immunofluorescent localization of the proteins of nuclear ribonucleoprotein complexes

Antibodies were raised in chickens against heterogeneous nuclear RNA (hnRNA)-binding proteins from 30S ribonucleoprotein (RNP) complexes of mouse Taper hepatoma ascites cell nuclei. The antibody preparations were characterized for immunological specificity and purity by double- diffusion gels, binding to specific bands in SDS polyacrylamide gels, and crossed immunoelectrophoresis. Antibodies raised against either whole 30S RNP complexes or purified RNP core proteins had a strong selective affinity for the four 34,000- to 40,000-dalton polypeptides which comprise the major structural proteins of hnRNP. The intracellular distribution of 30S RNP antigens in mouse ascites cells was determined by indirect immunofluorescence microsacopy. In interphase cells immunofluorescent sites were restricted to the nucleus, and nucleoli were free of fluorescence. The chicken anti-mouse- RNP antibodies were also able to react with cells from many different vertebrate species, showing a similar nucleus-restricted localization of the reacting sites. The antibodies also bound chick 30S RNP-proteins and reacted with the nuclei of chick cells. An exception to this was the failure of the antibody to bind to adult chick erythrocytes, suggesting that these major hnRNA binding proteins may be found only in nuclei capable of RNA synthesis.     

Functional characterization of nuclear localization signals in yeast Sm proteins

In mammals, nuclear localization of U-snRNP particles requires the snRNA hypermethylated cap structure and the Sm core complex. The nature of the signal located within the Sm core proteins is still unknown, both in humans and yeast. Close examination of the sequences of the yeast SmB, SmD1, and SmD3 carboxyl-terminal domains reveals the presence of basic regions that are reminiscent of nuclear localization signals (NLSs). Fluorescence microscopy studies using green fluorescent protein (GFP)-fusion proteins indicate that both yeast SmB and SmD1 basic amino acid stretches exhibit nuclear localization properties. Accordingly, deletions or mutations in the NLS-like motifs of SmB and SmD1 dramatically reduce nuclear fluorescence of the GFP-Sm mutant fusion alleles. Phenotypic analyses indicate that the NLS-like motifs of SmB and SmD1 are functionally redundant: each NLS-like motif can be deleted without affecting yeast viability whereas a simultaneous deletion of both NLS-like motifs is lethal. Taken together, these findings suggest that, in the doughnut-like structure formed by the Sm core complex, the carboxyl-terminal extensions of Sm proteins may form an evolutionarily conserved basic amino acid-rich protuberance that functions as a nuclear localization determinant.     

Proteasome-dependent processing of nuclear proteins is correlated with their subnuclear localization

Although proteasomes are abundant in the nucleoplasm little is known of proteasome-dependent proteolysis within the nucleus. Thus, we monitored the subcellular distribution of nuclear proteins in correlation with proteasomes. The proteasomal pathway clears away endogenous proteins, regulates numerous cellular processes, and delivers immunocompetent peptides to the antigen presenting machinery. Confocal laser scanning microscopy revealed that histones, splicing factor SC35, spliceosomal components, such as U1-70k or SmB/B('), and PML partially colocalize with 20S proteasomes in nucleoplasmic substructures, whereas the centromeric and nucleolar proteins topoisomerase I, fibrillarin, and UBF did not overlap with proteasomes. The specific inhibition of proteasomal processing with lactacystin induced accumulation of histone protein H2A, SC35, spliceosomal components, and PML, suggesting that these proteins are normally degraded by proteasomes. In contrast, concentrations of centromeric proteins CENP-B and -C and nucleolar proteins remained constant during inhibition of proteasomes. Quantification of fluorescence intensities corroborated that nuclear proteins which colocalize with proteasomes are degraded by proteasome-dependent proteolysis within the nucleoplasm. These data provide evidence that the proteasome proteolytic pathway is involved in processing of nuclear components, and thus may play an important role in the regulation of nuclear structure and function.     

Interactions and three-dimensional localization of a group of nuclear pore complex proteins

We have used antibodies directed against a number of nuclear pore complex (NPC) proteins to determine their mutual interactions and location within the three-dimensional structure of the NPC. A monoclonal antibody, termed QE5, recognized three NPC polypeptides, p250, NUP153, and p62 on Western blots, and labeled the nuclear envelope of several cultured cell lines by immunofluorescence microscopy. These three polypeptides contained O-linked N- acetylglucosamine residues and were released from the NPC by detergent/high-salt treatment as discrete high molecular weight complexes. p250 was found in association with a novel 75 kD protein, NUP153 was released as a homo-oligomer of about 1 megadalton, and p62 was associated with polypeptides of 58 and 54 kD (previously reported by Finlay, D. R., E. Meier, P. Bradley, J. Horecka, and D. J. Forbes. 1991. J. Cell Biol. 114:169-183). p75, p58, and p54 were not galactosylated in vitro. Xenopus oocyte NEs were labeled with gold- conjugated QE5 and prepared for electron microscopy by quick freezing/freeze drying/rotary metal shadowing. This EM preparation method enabled us to more precisely localize the epitopes of this antibody to the cytoplasmic filaments and the nuclear basket of the NPC. Since QE5 recognizes three O-linked NPC glycoproteins, its labeling was compared with that of the lectin wheat germ agglutinin which recognizes O-linked N-acetylglucosamine moieties. The two probes were found to yield similar, although not identical, distributions of label. To identify the individual proteins with particular NPC components, we have used an anti-peptide antibody against NUP153 and a monospecific anti-p250 polyclonal antibody. Labeling with these two antibodies has documented that NUP153 is a constituent of the nuclear basket with at least one of its epitopes residing in its terminal ring, whereas p250 is a constituent of the cytoplasmic filaments.     

Nuclear and nuclear envelope binding proteins of the glucocorticoid receptor nuclear localization peptide identified by crosslinking

The molecular mechanisms underlying the nuclear entry of steroid receptors and possible regulation of steroid hormone action during receptor passage across the nuclear envelope have not been elucidated. A nuclear localization signal has been identified in the hinge region of the glucocorticoid receptor. A synthetic peptide corresponding to this sequence was radio-iodinated and incubated with high salt- and detergent-extracted rat liver nuclei or nuclear envelope in the presence of crosslinker. After SDS-PAGE, two nuclear polypeptides of 60 and 76 kDa which had been specifically crosslinked were identified by autoradiography. A 60 kDa polypeptide was also crosslinked in the nuclear envelope fraction. ATP and elevated temperatures enhanced the crosslinking of both nuclear peptides. Finally, we showed that the pattern of crosslinking of the simian virus 40 large tumour antigen nuclear localization signal was identical to that of the glucocorticoid receptor signal to the nuclear polypeptides. The crosslinked peptides are good candidates for nuclear importers of the glucocorticoid receptor. In addition, the data suggest that these binding sites may be part of a general mechanism for nuclear entry of proteins.     

Charge versus sequence for nuclear/nucleolar localization of plant ribosomal proteins

Ribosomal subunit assembly in the nucleolus is dependent on efficient targeting of ribosomal proteins (RPs) from the cytoplasm into the nucleus and nucleolus. Nuclear/nucleolar localization of a protein is generally mediated by one or more specific stretches of basic amino acids—nuclear/nucleolar localization signals (NLSs/NoLSs). Arabidopsis thaliana RPL23aA has eight putative NLSs/NoLSs (pNLSs/NoLSs). Here we mutated all eight NLS/NoLSs individually and in groups and showed, via transient expression in tobacco cells that nucleolar localization of RPL23aA was disrupted by mutation of various combinations of five or more pNLSs/NoLSs. Mutation of all eight pNLSs/NoLSs, a 50 % reduction in total basic charge of RPL23aA, resulted in a complete disruption of nucleolar localization, however, the protein can still localize to the nucleus. As no individual or specific combination of NoLSs was absolutely required for nucleolar localization, we suggest that nucleolar localization/retention of RPL23aA is dependent on the overall basic charge. In addition to the optimal basic charge conferred by these NoLSs, nucleolar localization/retention of RPL23aA also required a C-terminal putative 26S rRNA binding site. In contrast, in the RPs RPS8A and RPL15A, mutation of just two and three N-terminal pNLSs, respectively, disrupted both nuclear and nucleolar localization of these two RPs, indicating differential signal requirements for nuclear and nucleolar localization of the three Arabidopsis RPs RPL23aA, RPL15A and RPS8A.     

Prediction of bacterial proteins carrying a nuclear localization signal and nuclear targeting of HsdM from Klebsiella pneumoniae

Nuclear targeting of bacterial proteins is an emerging pathogenic mechanism whereby bacterial proteins can interact with nuclear molecules and alter the physiology of host cells. The fully sequenced bacterial genome can predict proteins that target the nuclei of host cells based on the presence of nuclear localization signal (NLS). In the present study, we predicted bacterial proteins with the NLS sequences from Klebsiella pneumoniae by bioinformatic analysis, and 13 proteins were identified as carrying putative NLS sequences. Among them, HsdM, a subunit of KpnAl that is a type I restriction-modification system found in K. pneumoniae, was selected for the experimental proof of nuclear targeting in host cells. HsdM carried the NLS sequences, ₇KKAKAKK₁₃, in the N-terminus. A transient expression of HsdM-EGFP in COS-1 cells exhibited exclusively a nuclear localization of the fusion proteins, whereas the fusion proteins of HsdM with substitutions in residues lysine to alanine in the NLS sequences, ₇AAAKAAA₁₃, were localized in the cytoplasm. HsdM was co-localized with importin o in the nuclei of host cells. Recombinant HsdM alone methylated the eukaryotic DNA in vitro assay. Although HsdM tested in this study has not been considered to be a virulence factor, the prediction of NLS motifs from the full sequenced genome of bacteria extends our knowledge of functional genomics to understand subcellular targeting of bacterial proteins.     

Antibodies to nuclear thyroid hormone-binding proteins. Antibody characterization and immunofluorescent localization

To further investigate the mechanism by which thyroid hormones regulate target cell function, we have prepared and partially characterized antibodies to highly purified nuclear thyroid hormone-binding proteins (NTBP). NTBPs were prepared from bovine liver nuclear extracts by bio-specific elution from an affinity gel containing immobilized 3,5,3'-triiodo-L-thyronine (T3). Antibodies (Ab) raised to NTBP in BALB/c mice were assayed for Ab-NTBP complex formation on HPLC TSK SW3000 molecular exclusion gels and found to be species-specific and non-cross-reactive with serum thyroid hormone-binding proteins. Most of the antibody activity was directed against two fractions of molecular weight (MW) 89 000 and 53 000, which were associated with thyroxine (T4)-binding activity. The 89 000 D T4-binding activity was shifted to a higher MW complex when incubated with specific antibody. Indirect immunofluorescence showed antibody activity against discrete, clumped chromatin sites, nuclear envelope and plasma membrane in hepatocytes. Intense fluorescence was also observed in the cells lining the hepatic sinusoids and in the cytoskeleton of bovine aortic endothelial cells in culture. The data suggest that thyroid hormone target cells contain extranuclear loci that share antigenic sites with NTBP and may also represent specific NTBP-like sites of thyroid hormone binding.     

Involvement of 14-3-3 proteins in nuclear localization of telomerase

Maintenance of telomeres is implicated in chromosome stabilization and cell immortalization. Telomerase, which catalyzes de novo synthesis of telomeres, is activated in germ cells and most cancers. Telomerase activity is regulated by gene expression for its catalytic subunit, TERT, whereas several lines of evidence have suggested a post-translational regulation of telomerase activity. Here we identify the 14-3-3 signaling proteins as human TERT (hTERT)-binding partners. A dominant-negative 14-3-3 redistributed hTERT, which was normally predominant in the nucleus, into the cytoplasm. Consistent with this observation, hTERT-3A, a mutant that could not bind 14-3-3, was localized into the cytoplasm. Leptomycin B, an inhibitor of CRM1/exportin 1-mediated nuclear export, or disruption of a nuclear export signal (NES)-like motif located just upstream of the 14-3-3 binding site in hTERT impaired the cytoplasmic localization of hTERT. Compared with wild-type hTERT, hTERT-3A increased its association with CRM1. 14-3-3 binding was not required for telomerase activity either in vitro or in cell extracts. These observations suggest that 14-3-3 enhances nuclear localization of TERT by inhibiting the CRM1 binding to the TERT NES-like motif.     

Predicting nuclear localization

Nuclear localization of proteins is a crucial element in the dynamic life of the cell. It is complicated by the massive diversity of targeting signals and the existence of proteins that shuttle between the nucleus and cytoplasm. Nevertheless, a majority of subcellular localization tools that predict nuclear proteins have been developed without involving dual localized proteins in the data sets. Hence, in general, the existing models are focused on predicting statically nuclear proteins, rather than nuclear localization itself. We present an independent analysis of existing nuclear localization predictors, using a nonredundant data set extracted from Swiss-Prot R50.0. We demonstrate that accuracy on truly novel proteins is lower than that of previous estimations, and that existing models generalize poorly to dual localized proteins. We have developed a model trained to identify nuclear proteins including dual localized proteins. The results suggest that using more recent data and including dual localized proteins improves the overall prediction. The final predictor NUCLEO operates with a realistic success rate of 0.70 and a correlation coefficient of 0.38, as established on the independent test set. (NUCLEO is available at: http://pprowler.itee.uq.edu.au.).     

A switching system regulating subcellular localization of nuclear proteins using a viral protease

We explored a novel approach to the functional regulation of nuclear proteins; altering their subcellular localization. To anchor a nuclear protein, beta-galactosidase with the nuclear localization signal of SV40 (nbeta-gal), within the cytoplasm, nbeta-gal was fused to the transmembrane domain of granulocyte colony-stimulating factor receptor (G-CSFR), a membrane protein. To liberate the nbeta-gal portion from the fusion protein, we used a protease derived from a plant virus, whose recognition sequence was inserted between the G-CSFR and nbeta-gal. Western analysis showed that the chimeric protein was cleaved in the presence of the protease in 293 cells and that the fusion protein without the recognition sequence remained intact. This chimeric protein was localized exclusively in the cytoplasm as visualized by X-gal staining and immunofluorescence microscopy. In contrast, when expressed together with the protease, beta-gal was predominantly detected in the nuclei. Moreover, we isolated 293-cell clones constitutively expressing the protease, indicating that this protease is not cytotoxic. These results suggest that the viral protease-mediated alteration of subcellular localization can potentially regulate the function of nuclear proteins.     

Systematic deletion and mitotic localization of the nuclear pore complex proteins of Aspergillus nidulans

To define the extent of the modification of the nuclear pore complex (NPC) during Aspergillus nidulans closed mitosis, a systematic analysis of nuclear transport genes has been completed. Thirty genes have been deleted defining 12 nonessential and 18 essential genes. Several of the nonessential deletions caused conditional phenotypes and self-sterility, whereas deletion of some essential genes caused defects in nuclear structure. Live cell imaging of endogenously tagged NPC proteins (Nups) revealed that during mitosis 14 predicted peripheral Nups, including all FG repeat Nups, disperse throughout the cell. A core mitotic NPC structure consisting of membrane Nups, all components of the An-Nup84 subcomplex, An-Nup170, and surprisingly, An-Gle1 remained throughout mitosis. We propose this minimal mitotic NPC core provides a conduit across the nuclear envelope and acts as a scaffold to which dispersed Nups return during mitotic exit. Further, unlike other dispersed Nups, An-Nup2 locates exclusively to mitotic chromatin, suggesting it may have a novel mitotic role in addition to its nuclear transport functions. Importantly, its deletion causes lethality and defects in DNA segregation. This work defines the dramatic changes in NPC composition during A. nidulans mitosis and provides insight into how NPC disassembly may be integrated with mitosis.     

Ultrastructural localization of nuclear matrix proteins in HeLa cells using silver-enhanced ultra-small gold probes

We describe a method for immunogold staining of nuclear matrix proteins using ultra-small gold particles. The nuclear matrix of HeLa cells is obtained by two fractionation steps: (a) cell permeabilization with Triton X-100 to isolate the cytoskeleton, and (b) nuclease digestion followed by an incubation in 0.25 M ammonium sulfate to isolate the nuclear matrix. To prevent redistribution of internal matrix proteins during nuclear matrix preparation, pre-fixation with 0.1% acrolein was performed. Under this condition up to 80% of protein and 90% of DNA and RNA could be removed on nuclear matrix isolation, without redistribution of internal nuclear matrix proteins. For immunogold labeling, 1-nm gold probes appeared to be required to obtain optimal penetration into the nucleus. These particles can be visualized after silver enhancement. After gold labeling the matrices are stained, embedded in Epon, and ultra-thin sections are prepared for examination in the electron microscope. The applicability of this method is examplified by the localization of a 125 KD internal nuclear matrix protein and the lamins A and C in nuclear matrix preparations of HeLa cells.