Changes in nuclear proteins of rat testis cells separated by velocity sedimentation.

The technique of velocity sedimentation at unit gravity has been used to separate rat testis cell suspensions into fractions enriched in particular cell types. Changes in the nuclear proteins from the various fractions have been characterized by polyacrylamide gel electrophoresis, and correlated with the changing morphology of the nucleus during spermatogenesis. The most striking alterations in both protein composition and nuclear morphology occur during spermatid maturation as both histone and non-histone proteins are replaced by highly basic, low molecular weight, spermatidal proteins. This replacement process is accompanied by a quantitative reduction in both histone and non-histone proteins. The synthesis of at least three basic proteins has been identified with late stage spermatids. One of these proteins is a highly basic sperm-specific protein containing high levels of cyst(e)ine and arginine. A second protein synthesized in late stage spermatids is lysine rich, while the third protein contains cyst(e)ine and co-migrates with histone F2a1 on acid-urea polyacrylamide gels. The changes in protein composition of rat testis nuclei after irradiation or hypophysectomy reflect the resulting changes in the cellular composition of the testis. After selective elimination of the germinal cells by irradiation, the electrophoretic pattern of acid-soluble proteins from the testis is very similar to that of somatic tissue. Thus, the cellular specificity of nuclear proteins demonstrated here using cell separation techniques is also apparent following treatments which selectively alter the cellular composition of the testis.     

Purification and properties of the Xenopus Hat1 acetyltransferase: association with the 14-3-3 proteins in the oocyte nucleus.

We have purified the Xenopus histone acetyltransferase Hat1 holoenzyme from oocytes. The holoenzyme contains the catalytic subunit Hat1, the retinoblastoma associated protein RbAp48, and members of the phosphoserine binding family of 14-3-3 proteins. We have determined that the Hat1 holoenzyme specifically acetylates free histone H4 but not nucleosomal histones. RbAp48 is a phosphoprotein that contains a consensus recognition motif for the 14-3-3 proteins. The 14-3-3 proteins provide a regulatory function for the activity of many phosphoproteins. We find that the hugely abundant Hat1 holoenzyme is present in 10 000-fold excess over somatic cell levels. The holoenzyme is localized in the oocyte nucleus where acetylated histones are stored. The oocyte form of the Xenopus Hat1 holoenzyme may represent a specialized storage form of histone acetyltransferase. Following oocyte maturation and subsequent embryogenesis, the Hat1 enzyme is redistributed to the cytoplasm, where new histones are synthesized.     

Interaction of the tobacco lectin with histone proteins

The tobacco (Nicotiana tabacum) agglutinin or Nictaba is a member of a novel class of plant lectins residing in the nucleus and the cytoplasm of tobacco cells. Since tobacco lectin expression is only observed after the plant has been subjected to stress situations such as jasmonate treatment or insect attack, Nictaba is believed to act as a signaling protein involved in the stress physiology of the plant. In this paper, a nuclear proteomics approach was followed to identify the binding partners for Nictaba in the nucleus and the cytoplasm of tobacco cv Xanthi cells. Using lectin affinity chromatography and pull-down assays, it was shown that Nictaba interacts primarily with histone proteins. Binding of Nictaba with histone H2B was confirmed in vitro using affinity chromatography of purified calf thymus histone proteins on a Nictaba column. Elution of Nictaba-interacting histone proteins was achieved with 1 m N-acetylglucosamine (GlcNAc). Moreover, mass spectrometry analyses indicated that the Nictaba-interacting histone proteins are modified by O-GlcNAc. Since the lectin-histone interaction was shown to be carbohydrate dependent, it is proposed that Nictaba might fulfill a signaling role in response to stress by interacting with O-GlcNAcylated proteins in the plant cell nucleus.     

Nuclear proteins. V. Studies of histone-binding proteins from mouse liver by affinity chromatography

We examined four extracts of mouse liver for histone-binding proteins using histone affinity chromatography and positively charged resins. The extracts used were cytoplasm and washes from isolated nuclei with buffers containing 0.05 M Tris, 0.15 M NaCl or 0.35 M NaCl. Proteins from the nuclear washes showed greater binding to the columns than proteins from the cytoplasm. The binding fractions were heterogeneous in gel electrophoresis systems. Proteins bound to affinity columns of individual histones were similar to those bound to columns of whole histone, polylysine and DEAE. A 25,000 dalton polypeptide (J2), found only in nuclear washes was a prominent histone-binding protein. It could be competitively eluted from DEAE with histones, suggesting polypeptide J2 may show a specific affinity for histones. Polypeptide J2 has an acidic to basic amino acid ratio of 1.58, and its amino acid composition is not similar to that of the high mobility group 1 protein. Polypeptide J2 binds to hydrophobic columns and may play a role in modifying histone-histone and histone-DNA interactions.     

Identification of two HSP70-related Xenopus oocyte proteins that are capable of recycling across the nuclear envelope

Two 70-kD polypeptides, B3 and B4, are present in equivalent concentrations in the nucleus and cytoplasm of Xenopus oocytes. The objectives of this study were to determine if they (a) are members of the 70-kD family of heat shock proteins, and (b) recycle between the nuclear and cytoplasmic compartments. Evidence based on high-affinity binding to ATP, cross-reactivity of B3/B4-specific antibodies with rat hsc70, and a comparison of cyanogen bromide cleavage peptide maps with hsc70, verified that B3 and B4 are members of the 70-kD family of heat- shock proteins. Nuclear uptake studies were performed by microinjecting 125I-labeled B3/B4, rat hsc70, and BSA into the cytoplasm of oocytes, and examining their subsequent intracellular distributions. By 6 h postinjection, the nuclear concentration of B3/B4 and hsc70 were approximately 24-fold greater than BSA controls. It was also found that B3/B4-coated gold particles as large as 120A in diameter were able to enter the nucleus by passing through the pores. Nuclear efflux was analyzed by microinjecting the iodinated proteins directly into the oocyte nuclei. 2 h after nuclear injection, at least 46% of the B3/B4 and 60% of the hsc70 were found in the cytoplasmic fractions, compared with less than 10% for the BSA controls. Cell fusion experiments, in which labeled, anucleate oocyte vegetal hemispheres were fused, under oil, with nucleate unlabeled animal hemispheres, demonstrated that cytoplasmic B3 and B4 could enter the nucleus after equilibration was reached, arguing against the existence of separate nuclear and cytoplasmic populations. Collectively, these results show that B3, B4, and rat hsc70 are transported across the nuclear envelope and recycle between the nucleus and cytoplasm.     

Interacting proteins and differences in nuclear transport reveal specific functions for the NAP1 family proteins in plants

Nucleosome assembly protein 1 (NAP1) is conserved from yeast to human and facilitates the in vitro assembly of nucleosomes as a histone chaperone. Inconsistent with their proposed function in the nucleus, however, many NAP1 proteins had been reported to localize in the cytoplasm. We investigated the subcellular localization of tobacco (Nicotiana tabacum) and rice (Oryza sativa) NAP1 family proteins first by identification of interacting partners and by direct examination of the localization of green fluorescent protein-tagged proteins. Through treatment of tobacco cells with leptomycin B and mutagenesis of nuclear export signal, we demonstrated that Nicta;NAP1;1 and Orysa;NAP1;1 shuttle between the cytoplasm and the nucleus. Together with the demonstration that tobacco NAP1 proteins bind histone H2A and H2B, our results support the current model and provide additional evidence that function of NAP1 as histone chaperones appears to be conserved in plants. In addition, we show that tobacco NAP1 proteins interact with tubulin and the mitotic cyclin Nicta;CYCB1;1, suggesting a role for NAP1 in microtubule dynamics. Interestingly, in spite of their high homology with the above NAP1 proteins, the other three tobacco proteins and Orysa;NAP1;2 did not show nucleocytoplasmic shuttling and were localized only in the cytoplasm. Moreover, Orysa;NAP1;3 that lacks a typical nuclear localization signal sequence was localized in both the cytoplasm and the nucleus. Finally, we show that only Orysa;NAP1;3 could be phosphorylated by casein kinase 2alpha in vitro. However, this phosphorylation was not responsible for nuclear import of Orysa;NAP1;3 as being demonstrated through mutagenesis studies. Together, our results provide an important step toward elucidating the molecular mechanism of function of the NAP1 family proteins in plants.     

Analysis of herpesvirus saimiri structural proteins with monoclonal antibodies.

Herpesvirus saimiri is a lymphotrophic virus isolated from squirrel monkeys that causes highly malignant lymphomas in owl monkeys, marmosets, and rabbits, but not in its natural host. We have been interested in exploring immunological and biological aspects of this phenomenon and describe in this report the isolation of 27 monoclonal antibodies (MCAs) to herpesvirus saimiri which were grouped into 11 distinct sets based on the proteins they immunoprecipitated. In total, these 11 groups of MCAs identify the majority of the proteins present in the virion. Immunofluorescence was used to study how the viral antigens are compartmentalized in infected cells. One antibody produced intense nuclear staining and immunoprecipitated both the largest protein seen in gels (150,000 daltons) and one of the smallest (13,000 daltons). All of the other groups of MCAs principally stained the cytoplasm of infected cells. One of the unexpected results of this study was the observation that a majority of the MCAs precipitated more than one protein (15 of 27 antibodies, 7 of 11 groups). Whereas one group of MCAs, which normally precipitated four proteins, identified only single polypeptides after dithiothreitol pretreatment, the other sets of antibodies continued to recognize two or more viral antigens under reducing conditions. Immunoprecipitation of viral proteins with polyvalent sera obtained from virus-infected rabbits and monkeys was carried out to verify and extend previously published reports on the proteins of herpesvirus saimiri.