A compact system of small planets around a former red-giant star.

Planets that orbit their parent star at less than about one astronomical unit (1 AU is the Earth-Sun distance) are expected to be engulfed when the star becomes a red giant. Previous observations have revealed the existence of post-red-giant host stars with giant planets orbiting as close as 0.116 AU or with brown dwarf companions in tight orbits, showing that these bodies can survive engulfment. What has remained unclear is whether planets can be dragged deeper into the red-giant envelope without being disrupted and whether the evolution of the parent star itself could be affected. Here we report the presence of two nearly Earth-sized bodies orbiting the post-red-giant, hot B subdwarf star KIC 05807616 at distances of 0.0060 and 0.0076 AU, with orbital periods of 5.7625 and 8.2293 hours, respectively. These bodies probably survived deep immersion in the former red-giant envelope. They may be the dense cores of evaporated giant planets that were transported closer to the star during the engulfment and triggered the mass loss necessary for the formation of the hot B subdwarf, which might also explain how some stars of this type did not form in binary systems.

Genetic analyses involving interactions between the ergosterol biosynthetic enzymes, lanosterol synthase (Erg7p) and 3-ketoreductase (Erg27p), in the yeast Saccharomyces cerevisiae.

Protein-protein interaction studies in the Saccharomyces cerevisiae ergosterol biosynthetic pathway suggest that enzymes in this pathway may act as an integrated multienzyme complex. The yeast sterol 3-ketoreductase (Erg27p) required for C-4 demethylation of sterols has previously been shown to also be required for the function of the upstream oxidosqualene cyclase/lanosterol synthase (Erg7p); thus, erg27 mutants accumulate oxidosqualenes as precursors rather than 3-ketosterones. In the present study, we have created various mutations in the ERG27 gene. These mutations include 5 C-terminal truncations, 6 internal deletions, and 32 point mutants of which 14 were obtained by site-directed mutagenesis and 18 by random mutagenesis. We have characterized these ERG27 mutations by determining the following: Erg27 and Erg7 enzyme activities, presence of Erg27p as determined by western immunoblots, ability to grow on various sterol substrates and GC sterol profiles. Mutations of the predicted catalytic residues, Y202F and K206A, resulted in the endogenous accumulation of 3-ketosterones rather than oxidosqualenes suggesting retention of Erg7 enzyme activity. This novel phenotype demonstrated that the catalytic function of Erg27p can be separated from its Erg7p chaperone ability. Other erg27 mutations resulted in proteins that were present, as determined by western immunoblotting, but unable to interact with the Erg7 protein. We also classify Erg27p as belonging to the SDR (short-chain dehydrogenase/reductase) family of enzymes and demonstrate the possibility of homo- or heterodimerization of the protein. This study provides new insights into the role of Erg27p in sterol biosynthesis.

Protein-protein interactions among C-4 demethylation enzymes involved in yeast sterol biosynthesis.

A Saccharomyces cerevisae microarray expression study indicated that an ORF, YER044C, now designated ERG28, was strongly coregulated with ergosterol biosynthesis. Disruption of the ERG28 gene results in slow growth and accumulation of sterol intermediates similar to those observed in erg26 and erg27 null strains, suggesting that the Erg28p may interact with Erg26p and/or Erg27p. In this study, a peptide from human hemagglutinin protein (HA) epitope tag was added to ERG26 and ERG27 genes, and a Myc tag was added to the ERG28 gene to detect interactions between Erg28p and Erg26p/Erg27p. Differential centrifugation showed that Erg26p, Erg27p, and Erg28p are all membrane-associated proteins. Green fluorescent protein-fusion protein localization studies showed that Erg26p, Erg27p, and Erg28p are all located in the endoplasmic reticulum. Solubilized membrane protein coimmunoprecipitation studies using rabbit anti-Erg25p indicated that Erg25p coimmunoprecipitates with both Erg27p and Erg28p. Erg28p was also shown to reciprocally coimmunoprecipitate with Erg27p. However, no coimmunoprecipitation was observed with Erg26p, most likely because of the poor solubilization of this protein. Sucrose gradient ultracentrifugation studies suggested that Erg25p/Erg26p/Erg27p/Erg28p, along with other proteins in sterol biosynthesis, might form a complex between 66 and 200 kDa. Using an anti-HA column with Erg27p-HA and Erg26p-HA as target proteins, a complex containing Erg25p/Erg26p/Erg27p/Erg28p was identified. Thus, we suggest that Erg28p works as a transmembrane scaffold to tether Erg27p and possibly other C-4 demethylation proteins (Erg25p, Erg26p), forming a demethylation complex in the endoplasmic reticulum.

Dynamic localization of rop GTPases to the tonoplast during vacuole development.

Vacuoles are essential pleomorphic organelles that undergo dynamic changes during cell growth and differentiation in plants. How developmental signals are linked to vacuole biogenesis and development is poorly understood. In this report, we show that a Rop GTPase is localized to developing vacuoles in pea (Pisum sativum cv Extra Early Alaska). Rop belongs to the RHO family of Ras-related small GTP-binding proteins that are key molecular switches in a wide variety of eukaryotic signal transduction pathways. Using indirect immunofluorescence and an anti-Rop antibody, we showed that Rop proteins accumulate to high levels in rapidly growing tapetal cells of pea anthers. In these cells, Rop is localized to an endomembrane system that exists as dynamic pleomorphic networks: a perinuclear fine network decorated with punctate dots, a network composed of small spheres and tubules, and interconnected chambers. Colocalization with a tonoplast annexin VCaB42 shows that these dynamic networks represent the tonoplast. Our results suggest that the dynamic Rop-containing tonoplast networks represent a unique stage of vacuole development. The specific localization of Rop to developing vacuoles supports a role for Rop in signal transduction that mediates vacuole development in plants.

Amplification of padlock probes for DNA diagnostics by cascade rolling circle amplification or the polymerase chain reaction.

CONTEXT: Padlock probes are highly specific reagents for DNA diagnostics that can discriminate gene sequences with single base mutations. When the 3' and 5' terminal regions of the oligonucleotide probes are juxtaposed on a target DNA sequence, they can be circularized by enzymatic ligation and become topologically locked to the target. However, to be useful in solution-based diagnostics, the sensitivity of padlock probes must be markedly enhanced. OBJECTIVE: To describe two methods for geometric amplification of circularized padlock probes. DESIGN: Cascade rolling circle amplification is an isothermal system that uses generic primers and a DNA polymerase with strong strand displacement activity to amplify circularized probes by a mechanism combining rolling circle replication and strand displacement synthesis. One of the primers was designed as an energy transfer-labeled primer, which generates a fluorescence signal only when incorporated into the amplified product, enabling a direct means of detection. RESULTS: Using pUC19 as a model target to circularize an 89-base probe, a 10 billion-fold amplification was achieved with Bst DNA polymerase (large fragment) within 1 hour starting with as few as 10 probe molecules. The polymerase chain reaction was also used to amplify ligated padlock probes in a rare target detection system. In mixing experiments containing both normal and mutant p53 or c-Ki-ras2 gene target sequences, mutant targets were easily detected in the presence of a 500-fold excess of normal target copies. CONCLUSION: These results indicate that padlock probes can be amplified to the high levels required for solution-based DNA diagnostics.

A new class of proteins capable of binding transition metals.

Ion uptake, transport, and sequestration are essential to meet the nutritional requirements for plant growth and development. Furthermore, regulation of these processes is critical for plants to tolerate toxic levels of ions. The examination of isoprenylated proteins encoded by Arabidopsis thaliana and Glycine max cDNAs revealed a unique family of proteins containing putative metal-binding motifs (the core sequence is M/LXCXXC). Here, we describe this new class of proteins, which are capable of being isoprenylated and binding transition metal ions. Members of this family contain consensus isoprenylation (CaaX) sites, which we demonstrate are efficiently isoprenylated in vitro. ATFP3, a representative of the Arabidopsis family, was expressed in Escherichia coli and examined for metal-binding activity in vitro. Analysis of the interaction of ATFP3 with metal-chelating columns (IMAC) suggested that it binds to Cu2+, Ni2+, or Zn2+. To test whether proteins with these characteristics are present in other plant species, tobacco BY2 cells were labeled in vivo with [14C]mevalonate and the resulting mevalonate-labeled proteins were tested for metal-binding activity. Several soluble, isoprenylated proteins which bound copper-IMAC columns were revealed. Consistent with a wide-spread distribution of these proteins in plants, their presence was observed in Arabidopsis, soybean, and tobacco.

A Vacuole-Associated Annexin Protein, VCaB42, Correlates with the Expansion of Tobacco Cells.

A Ca-dependent membrane-binding protein of the annexin family, VCaB42, has previously been shown to associate with vacuolar vesicles at physiological levels of Ca. In this study we used suspension-cultured cells of tobacco (Nicotiana tabacum BY-2) to show that VCaB42 is enriched 4.5-fold in intact vacuoles, whereas evacuolated protoplasts show a 12-fold reduction in VCaB42. VCaB42 distribution is thus comparable to that of the vacuole-associated H+-ATPase but is distinct from the endoplasmic reticulum-localized protein calnexin. Because VCaB42 is a vacuole-associated annexin, and given the putative function of annexins in vesicle fusion, we hypothesize a role for this protein in the vacuolation process of expanding cells. Consistent with this hypothesis, we show that VCaB42 levels correlate with age-associated and hormonally induced changes in cell volume in tobacco suspension cultures. The association of VCaB42 with vacuoles and its correlative pattern of expression relative to the expansion of cells is consistent with a possible role for VCaB42 in the early events of vacuole biogenesis.

Prenylation of oncogenic human PTP(CAAX) protein tyrosine phosphatases.

Many isoprenylated proteins are known to participate in signal transduction, but not all have been identified. Using an in vitro prenylation screen, two human cDNAs (PTP(CAAXI) and PTP(CAAX2)) homologous to the rat PRL-1 and human OV-1 protein tyrosine phosphatase genes were identified. PTP(CAAXI) and PTP(CAAX2) were farnesylated in vitro by mammalian farnesyl:protein transferase, and epitope-tagged PTP(CAAX2) was prenylated in epithelial cells. Overexpression of PTP(CAAXI) and PTP(CAAX2) in epithelial cells caused a transformed phenotype in culture and tumor growth in nude mice. Thus, PTP(CAAXI) and PTP(CAAX2) represent a novel class of isoprenylated, oncogenic protein tyrosine phosphatases.

Identification of a DinB/UmuC homolog in the archeon Sulfolobus solfataricus.

To date, eight closely related homologs of the Escherichia coli UmuC protein have been identified. All of these homologs appear to play critical roles in damage-inducible mutagenesis in enterobacteriaceae. Recently, a distantly related UmuC-homolog, DinB, has also been identified in E. coli. Using the polymerase chain reaction together with degenerate primers designed against conserved regions found in UmuC-like proteins, we have identified a new member of the UmuC-superfamily in the archeon Sulfolobus solfataricus. This new homolog shows high sequence similarity to DinB and a lower level of similarity to UmuC. As a consequence, we have called this new gene dbh (dinB homolog). Analysis of approximately 2.7 kb DNA encompassing the dbh region revealed several open reading frames (orfs). One, encoding a putative ribokinase, was located immediately upstream of dbh. This orf overlaps the dbh gene by 4 bp suggesting that both proteins might be coordinately expressed. Further upstream of the ribokinase-dbh locus was another orf encoding a potential ATPase homologous to two uncharacterized S. cerevisiae proteins (YD9346.02c and SC38KCXVI_20) and another E. coli DNA repair protein, RuvB. While this is the first report of a UmuC-like homolog in an archeon, we detected additional homologs using protein sequence comparisons in Gram-positive bacteria, cyanobacteria, and among potential human EST products, indicating that UmuC-related proteins comprise a ubiquitous superfamily of proteins probably involved in DNA repair and mutagenesis.

Identification and isoprenylation of plant GTP-binding proteins.

To identify isoprenylated plant GTP-binding proteins, Arabidopsis thaliana and Nicotiana tabacum cDNA expression libraries were screened for cDNA-encoded proteins capable of binding [32P]GTP in vitro. ATGB2, an Arabidopsis homologue of the GTP-binding protein Rab2, was found to bind GTP in vitro and to be a substrate for a geranylgeranyl:protein transferase (GGTase) present in plant extracts. The carboxyl terminus of this protein contains a -GCCG sequence, which has not previously been shown to be recognized by any prenyl:protein transferase (PTase), but which most closely resembles that isoprenylated by the type II GGTase (-XXCC, -XCXC, or -CCXX). In vitro geranylgeranylation of an Arabidopsis Rab1 protein containing a carboxyl-terminal-CCGQ sequence confirmed the presence of a type II GGTase-like activity in plant extracts. Several other proteins were also identified by in vitro GTP binding, including Arabidopsis and tobacco homologues of Rab11, ARF (ADP-ribosylation factor) and Sar proteins, as well as a novel 22 kDa Arabidopsis protein (ATG81). This 22 kDa protein had consensus GTP-binding motifs and bound GTP with high specificity, but its structure was not closely related to that of any known GTP-binding protein (it most resembled proteins within the ARF/Sar and G protein alpha-subunit superfamilies).

Identification of cDNAs encoding isoprenylated proteins.

Isoprenylated proteins are involved in signal transduction, control of cell growth and differentiation, organization of the nuclear lamina and cytoskeleton, and vesicle sorting. The isoprenoid moiety facilitates the interaction of these proteins with membranes and/or other proteins. However, many isoprenylated proteins remain unidentified. A method is described for identifying novel and known cDNAs encoding isoprenylated proteins. Sufficient details of the screening procedure are given so that this method may be easily used to identify cDNAs encoding other covalently modified proteins or proteins possessing high affinity ligand binding sites.

Changes in Protein Isoprenylation during the Growth of Suspension-Cultured Tobacco Cells.

Isoprenylation facilitates the association of proteins with intracellular membranes and/or other proteins. In mammalian and yeast cells, isoprenylated proteins are involved in signal transduction, cell division, organization of the cytoskeleton, and vesicular transport. Recently, protein isoprenylation has been demonstrated in higher plants, but little is currently known about the functions of isoprenylated plant proteins. We report that inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (lovastatin) or prenyl:protein transferases (perilly alcohol) severely impair the growth of cultured tobacco (Nicotiana tabacum) cells but only when added within the first 2 d following transfer to fresh medium, before any increase in culture volume is detectable. This "window" of sensitivity to inhibitors of protein isoprenylation correlates temporally with an increase in [14C]mevalonate incorporation into tobacco cell proteins in vitro. We have also observed a marked increase in farnesyl:protein transferase activity at this early time in the growth of tobacco cultures. In contrast, type I geranylgeranyl:protein transferase activity does not change significantly during culture growth. Although these events coincide with the replication of DNA, I [mu]M lovastatin-treated cells are capable of DNA synthesis, suggesting that lovastatin-induced cell growth arrest is not due to inhibition of DNA replication. Together, these data support the hypothesis that protein isoprenylation is necessary for the early stages of growth of tobacco cultures.

A 42-kilodalton annexin-like protein is associated with plant vacuoles.

A 42-kD, calcium-dependent, membrane-binding protein (VCaB42) was associated with partially purified vacuole membrane. Membrane-dissociation assays indicated that VCaB42 binding to vacuole membranes was selective for calcium over other cations and that 50% of VCaB42 remained membrane bound at 61 +/- 11 nM free calcium. A 13-amino acid sequence obtained from VCaB42 showed 85% similarity with the endonexin fold, a sequence found in the annexin family of proteins that is thought to be essential for calcium and lipid binding. The greatest similarity in amino acid sequence was observed with annexin VIII (VAC-beta). The calcium-binding properties and sequence similarities suggest that VCaB42 is a member of the annexin family of calcium-dependent, membrane-binding proteins. Functional assays for VCaB42 on vacuole membrane transport processes indicated that it did not significantly affect the initial rate of calcium uptake into vacuole membrane vesicles. Because VCaB42 is vacuole localized (likely on the cytosolic surface of the vacuole) and is 50% dissociated within the physiological range of cytosolic free calcium, we hypothesize that this protein is a sensor that monitors cytosolic calcium levels and transmits that information to the vacuole.

Novel isoprenylated proteins identified by an expression library screen.

Isoprenylated proteins are involved in eukaryotic cell growth and signal transduction. The protein determinant for prenylation is a short carboxyl-terminal motif containing a cysteine, to which the isoprenoid is covalently attached via thioether linkage. To date, isoprenylated proteins have almost all been identified by demonstrating the attachment of an isoprenoid to previously known proteins. Thus, many isoprenylated proteins probably remain undiscovered. To identify novel isoprenylated proteins for subsequent biochemical study, colony blots of a Glycine max cDNA expression library were [3H]farnesyl-labeled in vitro. Proteins identified by this screen contained several different carboxyl termini that conform to consensus farnesylation motifs. These proteins included known farnesylated proteins (DnaJ homologs) and several novel proteins, two of which contained six or more tandem repeats of a hexapeptide having the consensus sequence (E/G)(G/P)EK(P/K)K. Thus, plants contain a diverse array of genes encoding farnesylated proteins, and our results indicate that fundamental differences in the identities of farnesylated proteins may exist between plants and other eukaryotes. Expression library screening by direct labeling can be adapted to identify isoprenylated proteins from other organisms, as well as proteins with other post-translational modifications.

Mutation of the cyclin-dependent kinase phosphorylation site in simian virus 40 (SV40) large T antigen specifically blocks SV40 origin DNA unwinding.

A mutant simian virus 40 (SV40) large tumor (T) antigen bearing alanine instead of threonine at residue 124 (T124A) failed to replicate SV40 DNA in infected monkey cells (J. Schneider and E. Fanning, J. Virol. 62:1598-1605, 1988). We investigated the biochemical properties of T124A T antigen in greater detail by using purified protein from a baculovirus expression system. Purified T124A is defective in SV40 DNA replication in vitro, but does bind specifically to the viral origin under the conditions normally used for DNA replication. The mutant protein forms double-hexamer complexes at the origin in an ATP-dependent fashion, although the binding reaction requires somewhat higher protein concentrations than the wild-type protein. Binding of T124A protein results in local distortion of the origin DNA similar to that observed with the wild-type protein. These findings indicate that the replication defect of T124A protein is not due to failure to recognize and occupy the origin. Under some conditions T124A is capable of unwinding short origin DNA fragments. However, the mutant protein is almost completely defective in unwinding of circular plasmid DNA molecules containing the SV40 origin. Since the helicase activity of T124A is essentially identical to that of the wild-type protein, we conclude that the mutant is defective in the initial opening of the duplex at the origin, possibly as a result of altered hexamer-hexamer interactions. The phenotype of T124A suggests a possible role for phosphorylation of threonine 124 by cyclin-dependent kinases in controlling the origin unwinding activity of T antigen in infected cells.

Protein isoprenylation in suspension-cultured tobacco cells.

Many mammalian and yeast proteins, including small ras-like GTP binding proteins, heterotrimeric G protein gamma subunits, and nuclear lamins, have been shown to be covalently linked to isoprenoid derivatives of mevalonic acid. Isoprenylation of these proteins is required for their assembly into membranes and, hence, for their biological activity. In this report, it is shown that cultured tobacco cells, when pretreated with an inhibitor of endogenous mevalonic acid synthesis (lovastatin), incorporate radioactivity from 14C-mevalonic acid into proteins. Most of these proteins are membrane associated, and many are similar in mass to mammalian ras-like GTP binding proteins and nuclear lamins. Furthermore, it is shown that tobacco cell extracts catalyze the transfer of radioactivity from 3H-farnesyl pyrophosphate and 3H-geranylgeranyl pyrophosphate to protein substrates in vitro. These studies indicate the presence of at least two distinct prenyl:protein transferases in tobacco extracts: one that utilizes farnesyl pyrophosphate and preferentially modifies a substrate protein with a CAIM carboxy terminus (farnesyl:protein transferase) and one that utilizes geranylgeranyl pyrophosphate and preferentially modifies a substrate protein with a CAIL carboxy terminus (geranylgeranyl:protein transferase type I). This work provides a basis for future work on the role of protein isoprenylation in plant cell growth, signal transduction, and membrane biogenesis.

Characterization of vacuolar calcium-binding proteins.

The vacuole plays a major structural and biochemical role in the higher plant cell. Among the most studied properties of the vacuole have been transport activities. One important aspect of vacuolar function is its participation in the regulation of cytosolic calcium levels. To identify the molecular entities involved in calcium regulation, a study of vacuole-associated, calcium-binding proteins (CaBs) was initiated. A competition assay was used, and it was observed that the majority of the total cellular membrane-associated, calcium-binding activity resided in low-density fractions enriched in vacuole membranes. Much of that calcium-binding activity was inactivated by a 0.5 m KI wash, and of the remaining activity, 77% was estimated to be peripherally associated with vacuolar membranes, whereas 23% was integrally associated with the vacuolar membrane. Calcium-ligand blots were used, and four major CaBs, with apparent molecular masses of 64, 58, 55, and 42 kD, were detected in purified vacuole membrane fractions. Two of these, the 58- and the 55-kD polypeptide, also appear to be present in significant amounts in endoplasmic reticulum-enriched fractions. However, the 64- and the 42-kD polypeptide are found primarily in vacuolar fractions. It is interesting that expression of the 42-kD polypeptide appears to be restricted to the heavily vacuolated cortical tissues (i.e. it is not found in vascular tissues). The localization of CaBs in the vacuole is consistent with the presence of calcium uptake (H(+)/Ca(2+) antiport) and release mechanisms (inositol trisphosphate sensitive) on vacuolar membranes. These vacuole-associated CaBs, which may play a role in calcium buffering, together with the calcium transport systems, could mediate the vacuolar component of cellular calcium homeostasis.

A heat shock protein complex isolated from rabbit reticulocyte lysate can reconstitute a functional glucocorticoid receptor-Hsp90 complex.

When unliganded glucocorticoid receptor that has been stripped free of associated proteins is incubated with rabbit reticulocyte lysate, the receptor becomes associated with the 70- and 90-kDa heat shock proteins (hsp70 and hsp90), and the untransformed state of the receptor is functionally reconstituted [Scherrer, L. C., Dalman, F. C., Massa, E., Meshinchi, S., & Pratt, W. B. (1990) J. Biol. Chem. 265, 21397-21400]. Recently, an hsp70-containing protein complex (200-250 kDa) purified from rabbit reticulocyte lysate was shown to maintain a fusion protein bearing the mitochondrial matrix-targeting signal in a state that is competent for mitochondrial import [Sheffield, W. P., Shore, G. C., & Randall, S. K. (1990) J. Biol. Chem. 265, 11069-11076]. In this work, we show that this partially purified mitochondrial import-competent fraction contains both hsp90 and hsp70. When the purified fraction is immunoadsorbed with a monoclonal antibody specific for hsp90, a significant portion of the hsp70 is co-immunoadsorbed, suggesting that hsp90 and hsp70 are present together as a complex. The partially purified fraction maintains a hybrid precursor protein containing the mitochondrial matrix-targeting signal of rat pre-ornithine carbamyl transferase in an import-competent state. Incubation of immunopurified glucocorticoid receptor with this fraction of reticulocyte lysate results in ATP-dependent association of the receptor with both hsp70 and hsp90, and the resulting complexes are functional as assessed by return of the receptor to the high-affinity steroid binding conformation. The glucocorticoid receptor hetero-complex reconstituting activity of the lysate fraction is low relative to its mitochondrial import activity. Importantly, however, this is the first demonstration of the functional and structural reconstitution of the untransformed state of any steroid receptor utilizing a partially purified system.

The fate of parental nucleosomes during SV40 DNA replication.

The fate of parental nucleosomes during the replication of chromatin templates was studied using a modification of the cell-free SV40 DNA replication system. Plasmid DNA molecules containing the SV40 origin were assembled into chromatin with purified core histones and fractionated assembly factors derived from HeLa cells. When these templates were replicated in vitro, the resulting progeny retained a nucleosomal organization. To determine whether the nucleosomes associated with the progeny molecules resulted from displacement of parental histones during replication followed by reassembly, the replication reactions were performed in the presence of control templates. It was observed that the progeny genomes resulting from the replication of chromatin templates retained a nucleosomal structure, whereas the progeny of the control DNA molecules were not assembled into chromatin. Additional experiments, involving direct addition of histones to the replication reaction mixtures, confirmed that the control templates were not sequestered in some form which made them unavailable for nucleosome assembly. Thus, our data demonstrate that parental nucleosomes remain associated with the replicating molecules and are transferred to the progeny molecules without displacement into solution. We propose a simple model in which nucleosomes ahead of the fork are transferred intact to the newly synthesized daughter duplexes.