The chimeric eukaryote: Origin of the nucleus from the karyomastigont in amitochondriate protists

We present a testable model for the origin of the nucleus, the membrane-bounded organelle that defines eukaryotes. A chimeric cell evolved via symbiogenesis by syntrophic merger between an archaebacterium and a eubacterium. The archaebacterium, a thermoacidophil resembling extant Thermoplasma, generated hydrogen sulfide to protect the eubacterium, a heterotrophic swimmer comparable to Spirochaeta or Hollandina that oxidized sulfide to sulfur. Selection pressure for speed swimming and oxygen avoidance led to an ancient analogue of the extant cosmopolitan bacterial consortium “Thiodendron latens.” By eubacterial-archaebacterial genetic integration, the chimera, an amitochondriate heterotroph, evolved. This “earliest branching protist” that formed by permanent DNA recombination generated the nucleus as a component of the karyomastigont, an intracellular complex that assured genetic continuity of the former symbionts. The karyomastigont organellar system, common in extant amitochondriate protists as well as in presumed mitochondriate ancestors, minimally consists of a single nucleus, a single kinetosome and their protein connector. As predecessor of standard mitosis, the karyomastigont preceded free (unattached) nuclei. The nucleus evolved in karyomastigont ancestors by detachment at least five times (archamoebae, calonymphids, chlorophyte green algae, ciliates, foraminifera). This specific model of syntrophic chimeric fusion can be proved by sequence comparison of functional domains of motility proteins isolated from candidate taxa.

Kinetics and mechanism of DNA uptake into the cell nucleus

Gene transfer to eukaryotic cells requires the uptake of exogenous DNA into the cell nucleus. Except during mitosis, molecular access to the nuclear interior is limited to passage through the nuclear pores. Here we demonstrate the nuclear uptake of extended linear DNA molecules by a combination of fluorescence microscopy and single-molecule manipulation techniques, using the latter to follow uptake kinetics of individual molecules in real time. The assays were carried out on nuclei reconstituted in vitro from extracts of Xenopus eggs, which provide both a complete complement of biochemical factors involved in nuclear protein import, and unobstructed access to the nuclear pores. We find that uptake of DNA is independent of ATP or GTP hydrolysis, but is blocked by wheat germ agglutinin. The kinetics are much slower than would be expected from hydrodynamic considerations. A fit of the data to a simple model suggests femto-Newton forces and a large friction relevant to the uptake process.

RNA Polymerase Subunits Encoded by the Plastid rpo Genes Are Not Shared with the Nucleus-Encoded Plastid Enzyme1

Plastid genes in photosynthetic higher plants are transcribed by at least two RNA polymerases. The plastid rpoA, rpoB, rpoC1, and rpoC2 genes encode subunits of the plastid-encoded plastid RNA polymerase (PEP), an Escherichia coli-like core enzyme. The second enzyme is referred to as the nucleus-encoded plastid RNA polymerase (NEP), since its subunits are assumed to be encoded in the nucleus. Promoters for NEP have been previously characterized in tobacco plants lacking PEP due to targeted deletion of rpoB (encoding the β-subunit) from the plastid genome. To determine if NEP and PEP share any essential subunits, the rpoA, rpoC1, and rpoC2 genes encoding the PEP α-, β′-, and β"-subunits were removed by targeted gene deletion from the plastid genome. We report here that deletion of each of these genes yielded photosynthetically defective plants that lack PEP activity while maintaining transcription specificity from NEP promoters. Therefore, rpoA, rpoB, rpoC1, and rpoC2 encode PEP subunits that are not essential components of the NEP transcription machinery. Furthermore, our data indicate that no functional copy of rpoA, rpoB, rpoC1, or rpoC2 that could complement the deleted plastid rpo genes exists outside the plastids.

Dual Targeting of Osh1p, a Yeast Homologue of Oxysterol-binding Protein, to both the Golgi and the Nucleus-Vacuole Junction

Oxysterol binding protein (OSBP) is the only protein known to bind specifically to the group of oxysterols with potent effects on cholesterol homeostasis. Although the function of OSBP is currently unknown, an important role is implicated by the existence of multiple homologues in all eukaryotes so far examined. OSBP and a subset of homologues contain pleckstrin homology (PH) domains. Such domains are responsible for the targeting of a wide range of proteins to the plasma membrane. In contrast, OSBP is a peripheral protein of Golgi membranes, and its PH domain targets to the trans-Golgi network of mammalian cells. In this article, we have characterized Osh1p, Osh2p, and Osh3p, the three homologues of OSBP in Saccharomyces cerevisiae that contain PH domains. Examination of a green fluorescent protein (GFP) fusion to Osh1p revealed a striking dual localization with the protein present on both the late Golgi, and in the recently described nucleus-vacuole (NV) junction. Deletion mapping revealed that the PH domain of Osh1p specified targeting to the late Golgi, and an ankyrin repeat domain targeting to the NV junction, the first such targeting domain identified for this structure. GFP fusions to Osh2p and Osh3p showed intracellular distributions distinct from that of Osh1p, and their PH domains appear to contribute to their differing localizations.

Perturbation of the Nucleus: A Novel Hog1p-independent, Pkc1p-dependent Consequence of Hypertonic Shock in Yeast

Hypertonic shock of Saccharomyces cerevisiae activates the Hog1p MAP kinase cascade. In contrast, protein kinase C (Pkc1p) and the “cell integrity” MAP kinase cascade are critical for the response to hypotonic shock. We observed that hypertonic shock transiently relocated many, but not all, nuclear and nucleolar proteins to the cytoplasm. We hypothesized that the relocation of nuclear proteins was due to activation of the Hog1p kinase cascade, yet, surprisingly, Hog1p was not required for these effects. In contrast, Pkc1p kinase activity was required, although the Pkc1p MAP kinase cascade and several factors known to lie upstream and downstream of Pkc1p were not. Moreover, sudden induction of a hyperactive form of Pkc1p was sufficient to relocate nuclear proteins. Taken together, these observations show that the scope of involvement of Pkc1p in the organization of the nucleus considerably exceeds what has been characterized previously. The relocation of nuclear proteins is likely to account for the profound inhibition of RNA synthesis that was observed during hypertonic shock.

Occurrence of two somatostatin variants in the frog brain: characterization of the cDNAs, distribution of the mRNAs, and receptor-binding affinities of the peptides.

In tetrapods, only one gene encoding a somatostatin precursor has been identified so far. The present study reports the characterization of the cDNA clones that encode two distinct somatostatin precursors in the brain of the frog Rana ridibunda. The cDNAs were isolated by using degenerate oligonucleotides based on the sequence of the central region of somatostatin to screen a frog brain cDNA library. One of the cDNAs encodes a 115-amino acid protein (prepro-somatostatin-14; PSS1) that exhibits a high degree of structural similarity with the mammalian somatostatin precursor. The other cDNA encodes a 103-amino acid protein (prepro-[Pro2, Met13]somatostatin-14; PSS2) that contains the sequence of the somatostatin analog (peptide SS2) at its C terminus, but does not exhibit appreciable sequence similarity with PSS1 in the remaining region. In situ hybridization studies indicate differential expression of the PSS1 and PSS2 genes in the septum, the lateral part of the pallium, the amygdaloid complex, the posterior nuclei of the thalamus, the ventral hypothalamic nucleus, the torus semicircularis and the optic tectum. The somatostatin variant SS2 was significantly more potent (4-6 fold) than somatostatin itself in displacing [125I-Tyr0, D-Trp8] somatostatin-14 from its specific binding sites. The present study indicates that the two somatostatin variants could exert different functions in the frog brain and pituitary. These data also suggest that distinct genes encoding somatostatin variants may be expressed in the brain of other tetrapods.

The junction-associated protein, zonula occludens-1, localizes to the nucleus before the maturation and during the remodeling of cell-cell contacts.

The junction-associated protein zonula occludens-1 (ZO-1) is a member of a family of membrane-associated guanylate kinase homologues thought to be important in signal transduction at sites of cell-cell contact. We present evidence that under certain conditions of cell growth, ZO-1 can be detected in the nucleus. Two different antibodies against distinct portions of the ZO-1 polypeptide reveal nuclear staining in subconfluent, but not confluent, cell cultures. An exogenously expressed, epitope-tagged ZO-1 can also be detected in the nuclei of transfected cells. Nuclear accumulation can be stimulated at sites of wounding in cultured epithelial cells, and immunoperoxidase detection of ZO-1 in tissue sections of intestinal epithelial cells reveals nuclear labeling only along the outer tip of the villus. These results suggest that the nuclear localization of ZO-1 is inversely related to the extent and/or maturity of cell contact. Since cell-cell contacts are specialized sites for signaling pathways implicated in growth and differentiation, we suggest that the nuclear accumulation of ZO-1 may be relevant for its suggested role in membrane-associated guanylate kinase homologue signal transduction.

A 104-kDa diacylglycerol kinase containing ankyrin-like repeats localizes in the cell nucleus.

The cDNA corresponding to a fourth species of diacylglycerol (DG) kinase (EC 2.7.1.107) was isolated from cDNA libraries of rat retina and brain. This cDNA encoded a 929-aa, 104-kDa polypeptide termed DGK-IV. DGK-IV was different from previously identified mammalian DG kinase species, DGK-I, DGK-II, and DGK-III, in that it contained no EF-hand motifs but did contain four ankyrin-like repeats at the carboxyl terminus. These structural features of DGK-IV closely resemble the recently cloned, eye-specific DG kinase of Drosophila that is encoded by the retinal degeneration A (rdgA) gene. However, DGK-IV was expressed primarily in the thymus and brain with relatively low expression in the eye and intestine. Furthermore, the primary structure of the DGK-IV included a nuclear targeting motif, and immunocytochemical analysis revealed DGK-IV to localize in the nucleus of COS-7 cells transfected with the epitope-tagged cDNA, suggesting an involvement of DGK-IV in intranuclear processes.

Induction of a physiological memory in the cerebral cortex by stimulation of the nucleus basalis.

Auditory cortical receptive field plasticity produced during behavioral learning may be considered to constitute "physiological memory" because it has major characteristics of behavioral memory: associativity, specificity, rapid acquisition, and long-term retention. To investigate basal forebrain mechanisms in receptive field plasticity, we paired a tone with stimulation of the nucleus basalis, the main subcortical source of cortical acetylcholine, in the adult guinea pig. Nucleus basalis stimulation produced electroencephalogram desynchronization that was blocked by systemic and cortical atropine. Paired tone/nucleus basalis stimulation, but not unpaired stimulation, induced receptive field plasticity similar to that produced by behavioral learning. Thus paired activation of the nucleus basalis is sufficient to induce receptive field plasticity, possibly via cholinergic actions in the cortex.

Translocation of PKN from the cytosol to the nucleus induced by stresses.

Effects of environmental stresses on the subcellular localization of PKN were investigated in NIH 3T3, BALB/c 3T3, and Rat-1 cells. The immunofluorescence of PKN resided prominently in the cytoplasmic region in nonstressed cells. When these cells were treated at 42 degrees C, there was a time-dependent decrease of the immunofluorescence of PKN in the cytoplasmic region that correlated with an increase within the nucleus as observed by confocal microscope. After incubation at 37 degrees C following beat shock, the immunofluorescence of PKN returned to the perinuclear and cytoplasmic regions from the nucleus. The nuclear translocation of PKN by heat shock was supported by the biochemical subcellular fractionation and immunoblotting. The nuclear localization of PKN was also observed when the cells were exposed to other stresses such as sodium arsenite and serum starvation. These results raise the possibility that there is a pathway mediating stress signals from the cytosol to the nucleus through PKN.

Ethanol causes translocation of cAMP-dependent protein kinase catalytic subunit to the nucleus.

Short- and long-term ethanol exposures have been shown to alter cellular levels of cAMP, but little is known about the effects of ethanol on cAMP-dependent protein kinase (PKA). When cAMP levels increase, the catalytic subunit of PKA (C alpha) is released from the regulatory subunit, phosphorylates nearby proteins, and then translocates to the nucleus, where it regulates gene expression. Altered localization of C alpha would have profound effects on multiple cellular functions. Therefore, we investigated whether ethanol alters intracellular localization of C alpha. NG108-15 cells were incubated in the presence or absence of ethanol for as long as 48 h, and localization of PKA subunits was determined by immunocytochemistry. We found that ethanol exposure produced a significant translocation of C alpha from the Golgi area to the nucleus. C alpha remained in the nucleus as long as ethanol was present. There was no effect of ethanol on localization of the type I regulatory subunit of PKA. Ethanol also caused a 43% decrease in the amount of type I regulatory subunit but had no effect on the amount of C alpha as determined by Western blot. These data suggest that ethanol-induced translocation of C alpha to the nucleus may account, in part, for diverse changes in cellular function and gene expression produced by alcohol.

Migration of vesicular stomatitis virus glycoprotein to the nucleus of infected cells.

A new means of direct visualization of the early events of viral infection by selective fluorescence labeling of viral proteins coupled with digital imaging microscopy is reported. The early phases of viral infection have great importance for understanding viral replication and pathogenesis. Vesicular stomatitis virus, the best-studied rhabdovirus, is composed of an RNA genome of negative sense, five viral proteins, and membrane lipids derived from the host cell. The glycoprotein of vesicular stomatitis virus was labeled with fluorescein isothiocyanate, and the labeled virus was incubated with baby hamster kidney cells. After initiation of infection, the fluorescence of the labeled glycoprotein was first seen inside the cells in endocytic vesicles. The fluorescence progressively migrated to the nucleus of infected cells. After 1 h of infection, the virus glycoprotein was concentrated in the nucleus and could be recovered intact in a preparation of purified nuclei. These results suggest that uncoating of the viral RNA occurs close to the nuclear membrane, which would precede transcription of the leader RNA that enters the nucleus to shut off cellular RNA synthesis and DNA replication.

Ultraviolet-induced movement of the human DNA repair protein, Xeroderma pigmentosum type G, in the nucleus.

Xeroderma pigmentosum type G (XPG) is a human genetic disease exhibiting extreme sensitivity to sunlight. XPG patients are defective XPG endonuclease, which is an enzyme essential for DNA repair of the major kinds of solar ultraviolet (UV)-induced DNA damages. Here we describe a novel dynamics of this protein within the cell nucleus after UV irradiation of human cells. Using confocal microscopy, we have localized the immunofluorescent, antigenic signal of XPG protein to foci throughout the cell nucleus. Our biochemical studies also established that XPG protein forms a tight association with nuclear structure(s). In human skin fibroblast cells, the number of XPG foci decreased within 2 h after UV irradiation, whereas total nuclear XPG fluorescence intensity remained constant, suggesting redistribution of XPG from a limited number of nuclear foci to the nucleus overall. Within 8 h after UV, most XPG antigenic signal was found as foci. Using beta-galactosidase-XPG fusion constructs (beta-gal-XPG) transfected into HeLa cells, we have identified a single region of XPG that is evidently responsible both for foci formation and for the UV dynamic response. The fusion protein carrying the C terminus of XPG (amino acids 1146-1185) localized beta-gal specific antigenic signal to foci and to the nucleolus regions. After UV irradiation, antigenic beta-gal translocated reversibly from the subnuclear structures to the whole nucleus with kinetics very similar to the movements of XPG protein. These findings lead us to propose a model in which distribution of XPG protein may regulate the rate of DNA repair within transcriptionally active and inactive compartments of the cell nucleus.

Overview of the most prevalent hypothalamus-specific mRNAs, as identified by directional tag PCR subtraction.

We applied the directional tag PCR subtractive hybridization method to construct a rat hypothalamic cDNA library from which cerebellar and hippocampal sequences had been depleted, enriching 20-30-fold for sequences expressed selectively in the hypothalamus. We studied a sample of 94 clones selected for enrichment in the subtracted library. These clones corresponded to 43 distinct mRNA species, about half of which were novel. Thirty-eight of these 43 mRNAs (corresponding to 85 of the clones in the sample) exhibited enrichment in the hypothalamus; 23 were highly enriched. In situ hybridization studies revealed that one novel species was restricted to cells in a small bilaterally symmetric area of the paraventricular hypothalamus. Other novel mRNAs showed substantial enrichment in basal diencephalic structures, particularly the hypothalamus, without restriction to single hypothalamic nuclei. The data suggest that the hypothalamus utilizes at least two distinct strategies for employing its selectively expressed proteins. Secretory neuropeptides utilized for intercellular communication are produced by functionally discrete nuclei, while several other proteins are shared by structures that are unrelated in their physiological roles but may share biochemical systems.