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.     

NucPred--predicting nuclear localization of proteins

NucPred analyzes patterns in eukaryotic protein sequences and predicts if a protein spends at least some time in the nucleus or no time at all. Subcellular location of proteins represents functional information, which is important for understanding protein interactions, for the diagnosis of human diseases and for drug discovery. NucPred is a novel web tool based on regular expression matching and multiple program classifiers induced by genetic programming. A likelihood score is derived from the programs for each input sequence and each residue position. Different forms of visualization are provided to assist the detection of nuclear localization signals (NLSs). The NucPred server also provides access to additional sources of biological information (real and predicted) for a better validation and interpretation of results. AVAILABILITY: The web interface to the NucPred tool is provided at http://www.sbc.su.se/~maccallr/nucpred. In addition, the Perl code is made freely available under the GNU Public Licence (GPL) for simple incorporation into other tools and web servers.     

Subcellular localization of cytoplasmic lattice-associated proteins is dependent upon fixation and processing procedures

We and others have recently demonstrated by immuno-EM and mutation analysis that two oocyte-restricted maternal effect genes, PADI6 and MATER, localize, in part, to the oocyte cytoplasmic lattices (CPLs). During these ongoing studies, however, we found that the localization of these factors by confocal immunofluorescence (IF) analysis can vary dramatically depending upon how the oocytes and embryos are processed, with the localization pattern sometimes appearing more uniformly cytoplasmic while at other times appearing to be primarily cortical. We set out to better understand this differential staining pattern by testing a range of IF protocol parameters, changing mainly time and temperature conditions of the primary antibody solution incubation, as well as fixation methods. We found by confocal IF whole mount analysis that PADI6 and MATER localization in germinal vesicle stage oocytes is mainly cytoplasmic when the oocytes are fixed and then incubated with primary antibodies at room temperature for 1 hour, while the localization of these factors is largely limited to the cortex when the oocytes are fixed and incubated in primary antibody at 4 °C overnight. We then probed sections of fixed/embedded ovaries and isolated two-cell embryos with specific antibodies and found that, under these conditions, PADI6 and MATER were again primarily cytoplasmically localized, although the staining for these factors is slightly more cortical at the two-cell stage. Taken together, our results suggest that the localization of CPL-associated proteins by confocal IF is particularly affected by processing conditions. Further, based on our current observations, it appears that PADI6 and MATER are primarily distributed throughout the cytoplasm as opposed to the oocyte subcortex.     

Cytoplasmic microtubule-associated proteins: Phosphorylation at novel sites is correlated with their incorporation into assembled microtubules

We have analyzed the detailed structure and cytoplasmic distribution of cytoplasmic microtubule-associated proteins. The procedure used to identify these proteins, based on preparation of detergent-extracted cytoskeletons, permits separation of fractions containing assembled and unassembled microtubule proteins. We show that two of these proteins, 69 and 80 kd, are closely related to one another and that each protein is present as a set of structurally related polypeptides with differing isoelectric points. In both neuroblastoma and pheochromocytoma cells, several of the isoelectric variants are greatly enriched in the fraction containing assembled microtubule components. Their differential distribution is correlated with phosphorylation at novel sites on the protein. These results support the possibility that covalent modification of a cytoskeletal component may specify its functional state.     

Yeast proteins that recognize nuclear localization sequences

A variety of peptides can mediate the localization of proteins to the nucleus. We have identified yeast proteins of 70 and 59 kD that bind to nuclear localization peptides of SV-40 T antigen, Xenopus nucleoplasmin, and the yeast proteins Ga14 and histone H2B. These proteins are assayed by the binding of peptide-albumin conjugates to proteins immobilized on nitrocellulose filters. These binding proteins fractionate with nuclei and are extractable with salt but not detergent. Radiolabeled peptide-albumin conjugates also bind to isolated nuclei; the binding is saturable and can be extracted with salt. Different nuclear localization peptides compete with each other, implying that a single class of proteins is responsible for their recognition. The 70- and 59-kD proteins have the properties expected for a receptor that would act to direct proteins to the nucleus.     

The subnuclear localization of tRNA ligase in yeast

Yeast tRNA ligase is an enzyme required for tRNA splicing. A study by indirect immune fluorescence shows that this enzyme is localized in the cell nucleus. At higher resolution, studies using indirect immune electron microscopy show this nuclear location to be primarily at the inner membrane of the nuclear envelope, most likely at the nuclear pore. There is a more diffuse, secondary location of ligase in a region of the nucleoplasm within 300 nm of the nuclear envelope. When the amount of ligase in the cell is increased, nuclear staining increases but staining of the nuclear envelope remains constant. This experiment indicates that there are a limited number of ligase sites at the nuclear envelope. Since the other tRNA splicing component, the endonuclease, has the characteristics of an integral membrane protein, we hypothesize that it constitutes the site for the interaction of ligase with the nuclear envelope.     

ATM regulates proteasome-dependent subnuclear localization of TRF1, which is important for telomere maintenance

Ataxia telangiectasia mutated (ATM), a PI-3 kinase essential for maintaining genomic stability, has been shown to regulate TRF1, a negative mediator of telomerase-dependent telomere extension. However, little is known about ATM-mediated TRF1 phosphorylation site(s) in vivo. Here, we report that ATM phosphorylates S367 of TRF1 and that this phosphorylation renders TRF1 free of chromatin. We show that phosphorylated (pS367)TRF1 forms distinct non-telomeric subnuclear foci and that these foci occur predominantly in S and G2 phases, implying that their formation is cell cycle regulated. We show that phosphorylated (pS367)TRF1-containing foci are sensitive to proteasome inhibition. We find that a phosphomimic mutation of S367D abrogates TRF1 binding to telomeric DNA and renders TRF1 susceptible to protein degradation. In addition, we demonstrate that overexpressed TRF1-S367D accumulates in the subnuclear domains containing phosphorylated (pS367)TRF1 and that these subnuclear domains overlap with nuclear proteasome centers. Taken together, these results suggest that phosphorylated (pS367)TRF1-containing foci may represent nuclear sites for TRF1 proteolysis. Furthermore, we show that TRF1 carrying the S367D mutation is unable to inhibit telomerase-dependent telomere lengthening or to suppress the formation of telomere doublets and telomere loss in TRF1-depleted cells, suggesting that S367 phosphorylation by ATM is important for the regulation of telomere length and stability.     

Proteasome-dependent degradation of oxidized proteins in MRC-5 fibroblasts

Fibroblasts were exposed to various concentrations of hydrogen peroxide and the removal of oxidized proteins was followed by determining protein-bound carbonyls. Fibroblasts are able to increase the turnover of metabolically radiolabeled proteins after treatment with hydrogen peroxide. It was demonstrated for the first time, that the increased protein turnover was accompanied by a removal of protein-bound carbonyl groups. The proteasome-specific inhibitor lactacystin was able to inhibit the elimination of protein-bound carbonyl groups. Therefore, the key role of the proteasome in the degradation of oxidized proteins in fibroblasts could be demonstrated.     

Identification and nuclear localization of yeast pre-messenger RNA processing components: RNA2 and RNA3 proteins

Temperature-sensitive mutations in the RNA2 through RNA11 genes of yeast prevent the processing of nuclear pre-mRNAs. We have raised antisera that detect the RNA2 and RNA3 proteins in immunoblots of extracts of yeast containing high copy number RNA2 and RNA3 plasmids. Subcellular fractionation of yeast cells that overproduce the RNA2 and RNA3 proteins has revealed that these proteins are enriched in nuclear fractions. Indirect immunofluorescence results have indicated that these proteins are localized in yeast nuclei. These localization results are consistent with the fact that these genes have a role in processing yeast pre-mRNA.     

DNA binding is not sufficient for nuclear localization of regulatory proteins in Saccharomyces cerevisiae.

We showed by immunofluorescence that the procaryotic DNA-binding protein LexA and a chimeric protein that contains the DNA-binding portion of LexA (amino acids 1 to 87) and a large portion (amino acids 74 to 881) of the Saccharomyces cerevisiae positive regulatory GAL4 protein (GAL4 gene product) are not preferentially localized in the nucleus in S. cerevisiae.