Tomato SP-Interacting Proteins Define a Conserved Signaling System That Regulates Shoot Architecture and Flowering

Divergent architecture of shoot models in flowering plants reflects the pattern of production of vegetative and reproductive organs from the apical meristem. The SELF-PRUNING (SP) gene of tomato is a member of a novel CETS family of regulatory genes (CEN, TFL1, and FT) that controls this process. We have identified and describe here several proteins that interact with SP (SIPs) and with its homologs from other species: a NIMA-like kinase (SPAK), a bZIP factor, a novel 10-kD protein, and 14-3-3 isoforms. SPAK, by analogy with Raf1, has two potential binding sites for 14-3-3 proteins, one of which is shared with SP. Surprisingly, overexpression of 14-3-3 proteins partially ameliorates the effect of the sp mutation. Analysis of the binding potential of chosen mutant SP variants, in relation to conformational features known to be conserved in this new family of regulatory proteins, suggests that associations with other proteins are required for the biological function of SP and that ligand binding and protein-protein association domains of SP may be separated. We suggest that CETS genes encode a family of modulator proteins with the potential to interact with a variety of signaling proteins in a manner analogous to that of 14-3-3 proteins.     

Characterization of a Tomato Xyloglucan Endotransglycosylase Gene That Is Down-Regulated by Auxin in Etiolated Hypocotyls

The reorganization of the cellulose-xyloglucan matrix is proposed to serve as an important mechanism in the control of strength and extensibility of the plant primary cell wall. One of the key enzymes associated with xyloglucan metabolism is xyloglucan endotransglycosylase (XET), which catalyzes the endocleavage and religation of xyloglucan molecules. As with other plant species, XETs are encoded by a gene family in tomato (Lycopersicon esculentum cv T5). In a previous study, we demonstrated that the tomato XET gene LeEXT was abundantly expressed in the rapidly expanding region of the etiolated hypocotyl and was induced to higher levels by auxin. Here, we report the identification of a new tomato XET gene, LeXET2, that shows a different spatial expression and diametrically opposite pattern of auxin regulation from LeEXT. LeXET2 was expressed more abundantly in the mature nonelongating regions of the hypocotyl, and its mRNA abundance decreased dramatically following auxin treatment of etiolated hypocotyl segments. Analysis of the effect of several plant hormones on LeXET2 expression revealed that the inhibition of LeXET2 mRNA accumulation also occurred with cytokinin treatment. LeXET2 mRNA levels increased significantly in hypocotyl segments treated with gibberellin, but this increase could be prevented by adding auxin or cytokinin to the incubation media. Recombinant LeXET2 protein obtained by heterologous expression in Pichia pastoris exhibited greater XET activity against xyloglucan from tomato than that from three other species. The opposite patterns of expression and differential auxin regulation of LeXET2 and LeEXT suggest that they encode XETs with distinct roles during plant growth and development.     

The Membrane Protein Alkaline Phosphatase Is Delivered to the Vacuole by a Route That Is Distinct from the VPS-dependent Pathway

Membrane trafficking intermediates involved in the transport of proteins between the TGN and the lysosome-like vacuole in the yeast Saccharomyces cerevisiae can be accumulated in various vps mutants. Loss of function of Vps45p, an Sec1p-like protein required for the fusion of Golgi-derived transport vesicles with the prevacuolar/endosomal compartment (PVC), results in an accumulation of post-Golgi transport vesicles. Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment.

The vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as defined by mutations in VPS27. In this study we demonstrate that, whereas VPS45 and VPS27 are required for the vacuolar delivery of several membrane proteins, the vacuolar membrane protein alkaline phosphatase (ALP) reaches its final destination without the function of these two genes. Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole.

Using a combination of immunofluorescence localization and pulse/chase immunoprecipitation analysis, we demonstrate that, in addition to ALP, the vacuolar syntaxin Vam3p also follows this VPS45/27-independent pathway to the vacuole. In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.

     

Dmc1 Functions in a Saccharomyces Cerevisiae Meiotic Pathway That Is Largely Independent of the Rad51 Pathway

Meiotic recombination in the yeast Saccharomyces cerevisiae requires two similar recA-like proteins, Dmc1p and Rad51p. A screen for dominant meiotic mutants provided DMC1-G126D, a dominant allele mutated in the conserved ATP-binding site (specifically, the A-loop motif) that confers a null phenotype. A recessive null allele, dmc1-K69E, was isolated as an intragenic suppressor of DMC1-G126D. Dmc1-K69Ep, unlike Dmc1p, does not interact homotypically in a two-hybrid assay, although it does interact with other fusion proteins identified by two-hybrid screen with Dmc1p. Dmc1p, unlike Rad51p, does not interact in the two-hybrid assay with Rad52p or Rad54p. However, Dmc1p does interact with Tid1p, a Rad54p homologue, with Tid4p, a Rad16p homologue, and with other fusion proteins that do not interact with Rad51p, suggesting that Dmc1p and Rad51p function in separate, though possibly overlapping, recombinational repair complexes. Epistasis analysis suggests that DMC1 and RAD51 function in separate pathways responsible for meiotic recombination. Taken together, our results are consistent with a requirement for DMC1 for meiosis-specific entry of DNA double-strand break ends into chromatin. Interestingly, the pattern on CHEF gels of chromosome fragments that result from meiotic DNA double-strand break formation is different in DMC1 mutant strains from that seen in rad50S strains.     

Corolla Is a Novel Protein That Contributes to the Architecture of the Synaptonemal Complex of Drosophila

In most organisms the synaptonemal complex (SC) connects paired homologs along their entire length during much of meiotic prophase. To better understand the structure of the SC, we aim to identify its components and to determine how each of these components contributes to SC function. Here, we report the identification of a novel SC component in Drosophila melanogaster female oocytes, which we have named Corolla. Using structured illumination microscopy, we demonstrate that Corolla is a component of the central region of the SC. Consistent with its localization, we show by yeast two-hybrid analysis that Corolla strongly interacts with Cona, a central element protein, demonstrating the first direct interaction between two inner-synaptonemal complex proteins in Drosophila. These observations help provide a more complete model of SC structure and function in Drosophila females.     

The Identification of a Novel Gene,MAPO2, That Is Involved in the Induction of Apoptosis Triggered by O6-Methylguanine

O⁶-Methylguanine, one of alkylated DNA bases, is especially mutagenic. Cells containing this lesion are eliminated by induction of apoptosis, associated with the function of mismatch repair (MMR) proteins. A retrovirus-mediated gene-trap mutagenesis was used to isolate new genes related to the induction of apoptosis, triggered by the treatment with an alkylating agent, N-methyl-N-nitrosourea (MNU). This report describes the identification of a novel gene, MAPO2 (O⁶-methylguanine-induced apoptosis 2), which is originally annotated as C1orf201. The MAPO2 gene is conserved among a wide variety of multicellular organisms and encodes a protein containing characteristic PxPxxY repeats. To elucidate the function of the gene product in the apoptosis pathway, a human cell line derived from HeLa MR cells, in which the MAPO2 gene was stably knocked down by expressing specific miRNA, was constructed. The knockdown cells grew at the same rate as HeLa MR, thus indicating that MAPO2 played no role in the cellular growth. After exposure to MNU, HeLa MR cells and the knockdown cells underwent cell cycle arrest at G₂/M phase, however, the production of the sub-G₁ population in the knockdown cells was significantly suppressed in comparison to that in HeLa MR cells. Moreover, the activation of BAK and caspase-3, and depolarization of mitochondrial membrane, hallmarks for the induction of apoptosis, were also suppressed in the knockdown cells. These results suggest that the MAPO2 gene product might positively contribute to the induction of apoptosis triggered by O⁶-methylguanine.     

Evidence That a Precursor Glycoprotein Is Cleaved to Yield the Major Glycoprotein of Avian Tumor Virus

A glycoprotein designated pr90, which is recognized by anti-gp85 serum, is present in lysates of pulse-labeled transformed cells. Under chase conditions, a reduction in the level of labeled pr90 is observed concomitant with the appearance of labeled, cell-associated viral glycoprotein.     

Molybdenum-Containing Nicotine Hydroxylase Genes in a Nicotine Degradation Pathway That Is a Variant of the Pyridine and Pyrrolidine Pathways

Ochrobactrum sp. strain SJY1 utilizes nicotine as a sole source of carbon, nitrogen, and energy via a variant of the pyridine and pyrrolidine pathways (the VPP pathway). Several strains and genes involved in the VPP pathway have recently been reported; however, the first catalyzing step for enzymatic turnover of nicotine is still unclear. In this study, a nicotine hydroxylase for the initial hydroxylation step of nicotine degradation was identified and characterized. The nicotine hydroxylase (VppA), which converts nicotine to 6-hydroxynicotine in the strain SJY1, is encoded by two open reading frames (vppA_S and vppA_L [subunits S and L, respectively]). The vppA genes were heterologously expressed in the non-nicotine-degrading strains Escherichia coli DH5α and Pseudomonas putida KT2440; only the Pseudomonas strain acquired the ability to degrade nicotine. The small subunit of VppA contained a [2Fe-2S] cluster-binding domain, and the large subunit of VppA contained a molybdenum cofactor-binding domain; however, an FAD-binding domain was not found in VppA. Resting cells cultivated in a molybdenum-deficient medium had low nicotine transformation activity, and excess molybdenum was detected in the purified VppA by inductively coupled plasma-mass spectrometry analysis. Thus, it is demonstrated that VppA is a two-component molybdenum-containing hydroxylase.     

Evidence for a Shared Nuclear Pore Complex Architecture That Is Conserved from the Last Common Eukaryotic Ancestor

The nuclear pore complex (NPC) is a macromolecular assembly embedded within the nuclear envelope that mediates bidirectional exchange of material between the nucleus and cytoplasm. Our recent work on the yeast NPC has revealed a simple modularity in its architecture and suggested a common evolutionary origin of the NPC and vesicle coating complexes in a progenitor protocoatomer. However, detailed compositional and structural information is currently only available for vertebrate and yeast NPCs, which are evolutionarily closely related. Hence our understanding of NPC composition in a full evolutionary context is sparse. Moreover despite the ubiquitous nature of the NPC, sequence searches in distant taxa have identified surprisingly few NPC components, suggesting that much of the NPC may not be conserved. Thus, to gain a broad perspective on the origins and evolution of the NPC, we performed proteomics analyses of NPC-containing fractions from a divergent eukaryote (Trypanosoma brucei) and obtained a comprehensive inventory of its nucleoporins. Strikingly trypanosome nucleoporins clearly share with metazoa and yeast their fold type, domain organization, composition, and modularity. Overall these data provide conclusive evidence that the majority of NPC architecture is indeed conserved throughout the Eukaryota and was already established in the last common eukaryotic ancestor. These findings strongly support the hypothesis that NPCs share a common ancestry with vesicle coating complexes and that both were established very early in eukaryotic evolution.Nearly all eukaryotic cells possess an extensive endomembrane system that is principally responsible for protein targeting and modification (1). The nucleus, the defining eukaryotic feature, is separated from the cytoplasm by a double bilayered nuclear envelope (NE)¹ that is contiguous with the rest of this endomembrane system via connections to the endoplasmic reticulum. Nuclear pore complexes (NPCs) fenestrate the NE, serving as the exclusive sites mediating exchange between the nucleoplasmic and cytoplasmic compartments. Macromolecules are chaperoned through the NPC by numerous transport factors. It has been proposed that the endomembrane system and nucleus have an autogenous origin (i.e. evolving from invaginations of an ancestral plasma membrane) and were established early in eukaryotic evolution (2).The composition of the NPC has been cataloged at ∼30 distinct nucleoporins (Nups) (3) for the yeast Saccharomyces cerevisiae (4) and vertebrates (5), two members of the Opisthokonta (animals, fungi, and closely related protists). Ultrastructural studies have identified objects morphologically similar (at a first approximation) to opisthokont NPCs in the other major eukaryote supergroups (6–8). However, very few data are available concerning the detailed NPC molecular composition and architecture for nearly all eukaryotic lineages, leaving a relatively narrow view of the “typical” NPC and its origins. A few examples of potential Nup orthologs beyond the opisthokonts have been reported, leading to the suggestion that substantial portions of the NPC may have an ancient, pre-last common eukaryotic ancestor (LCEA) origin (9). However, a more extensive study has concluded that LCEA possessed a primitive ancestral NPC that passed few components to its modern descendants (10).In yeast and vertebrates, the NPC consists of an eight-spoked core surrounding a central tube that serves as the conduit for macromolecular exchange. Each spoke can be divided into two similar nucleoplasmic and cytoplasmic halves. The eight spokes connect to form several coaxial rings: the membrane rings, the two outer rings at the nucleoplasmic and cytoplasmic periphery, and the two adjacent inner rings (11). Groups of Nups that we term “linker Nups” are attached between both sets of outer and inner rings. Another group of related proteins, collectively termed phenylalanine-glycine (FG) Nups, are largely exposed on the inner surface of the spokes and anchored either to the inner rings or to the linker Nups (11).Opisthokont Nups can be grouped into three structural classes (11, 12). The first class comprises membrane-bound proteins that anchor the NPC into the NE. The second class is the core scaffold Nups; these proteins constitute the bulk of the NPC mass, form the central tube, and provide the scaffold for the deployment of the third class of Nups across both faces of the NPC. The core scaffold Nups are remarkably restricted at the structural level and contain only three distinct arrangements of 2-fold types: proteins dominated by an α-solenoid fold (also termed a helix-turn-helix repeat domain), proteins consisting of a β-propeller fold, and finally proteins composed of an amino-terminal β-propeller fold followed by a carboxyl-terminal α-solenoid fold (which we here term a β-α structure) (12). FG Nups comprise the third class. These Nups carry multiply repeated degenerate “Phe-Gly” motifs (FG repeats) separated by hydrophilic or charged residues that form large unstructured domains. Each FG Nup also contains a small structured domain (often a coiled coil motif) that serves as the anchor site for interaction with the remainder of the NPC.Many transport factors belong to a structurally related protein family collectively termed karyopherins (Kaps) (13, 14). Transport across the NPC depends on the interactions between Kaps, cargo molecules, and the disordered repeat domains of FG Nups; the latter are thought to form the selective barrier for nucleocytoplasmic transport, guiding the Kap·cargo complexes (and other transport factors) through the central tube while excluding other macromolecules (for reviews, see Refs. 3 and 15–22).Significantly we have previously noted that the fold composition and arrangement of many of the core scaffold Nups are shared with proteins that form coating structures that participate in the generation and transport of vesicles between different endomembrane compartments; significantly many vesicle coating complex proteins and NPC scaffold Nups share an α-solenoid fold, β-propeller fold, or β-α structure (12, 23–28). These similarities gave rise to the “protocoatomer hypothesis,” which suggests a common ancestry for the NPC and these vesicle coat complexes. However, it is unclear how many, if any, of these particular core scaffold Nups are widely conserved, and hence it is unclear how general this potential relationship is throughout the Eukaryota. Thus, two scenarios are possible. The first is that the coatomer-like proteins are only found in a subset of the eukaryotes (including the opisthokonts), indicating that they are a relatively recent acquisition of only some eukaryotes and are not a general feature of all NPCs. The second is that the coatomer-like proteins are conserved in all eukaryotes, providing strong support to the protocoatomer hypothesis. To directly address this issue we characterized the NPC of Trypanosoma brucei, a highly divergent but experimentally tractable organism, using proteomics. The resulting data indicate an ancient origin for the majority of the NPC components and shed light on the origin of LCEA itself.     

Systemic Induction of Photosynthesis via Illumination of the Shoot Apex Is Mediated Sequentially by Phytochrome B,Auxin and Hydrogen Peroxide in Tomato

Systemic signaling of upper leaves promotes the induction of photosynthesis in lower leaves, allowing more efficient use of light flecks. However, the nature of the systemic signals has remained elusive. Here, we show that preillumination of the tomato (Solanum lycopersicum) shoot apex alone can accelerate photosynthetic induction in distal leaves and that this process is light quality dependent, where red light promotes and far-red light delays photosynthetic induction. Grafting the wild-type rootstock with a phytochome B (phyB) mutant scion compromised light-induced photosynthetic induction as well as auxin biosynthesis in the shoot apex, auxin signaling, and RESPIRATORY BURST OXIDASE HOMOLOG1 (RBOH1)-dependent hydrogen peroxide (H₂O₂) production in the systemic leaves. Light-induced systemic H₂O₂ production in the leaves of the rootstock also was absent in plants grafted with an auxin-resistant diageotropica (dgt) mutant scion. Cyclic electron flow around photosystem I and associated ATP production were increased in the systemic leaves by exposure of the apex to red light. This enhancement was compromised in the systemic leaves of the wild-type rootstock with phyB and dgt mutant scions and also in RBOH1-RNA interference leaves with the wild type as scion. Silencing of ORANGE RIPENING, which encodes NAD(P)H dehydrogenase, compromised the systemic induction of photosynthesis. Taken together, these results demonstrate that exposure to red light triggers phyB-mediated auxin synthesis in the apex, leading to H₂O₂ generation in systemic leaves. Enhanced H₂O₂ levels in turn activate cyclic electron flow and ATP production, leading to a faster induction of photosynthetic CO₂ assimilation in the systemic leaves, allowing plants better adaptation to the changing light environment.As a consequence of their sessile lifestyle, plants have evolved a high capacity for the regulation of physiology, growth, and development that facilitates survival in a constantly changing environment. Environmental stimuli perceived within an organ not only influence morphogenetic and physiological changes within that organ but also generate systemic effects in other organs that are remote from the site of signal perception. This crucial phenomenon is called systemic signaling or systemic regulation. Systemic signaling prepares other tissues of a plant for future challenges that may initially only be sensed by a few local tissues or cells. Several types of systemic responses are known. These include systemic acquired resistance, which is typically activated by pathogens such as viruses, bacteria, and fungi (Fu and Dong, 2013), induced systemic resistance, which is triggered by beneficial soil microorganisms or others (Pieterse and Dicke, 2007), and systemic acquired acclimation, which is initiated by abiotic stresses such as high light, UV radiation, heat, cold, and salinity (Mittler and Blumwald, 2015).The light utilization efficiency of photosynthesis is important for the survival of understory plants and plants growing in canopies. In particular, the efficient use of the energy contained in light (sun) flecks is important because light flecks contribute up to 60% to 80% of photosynthetically active radiation received by understory plants (Pearcy and Seemann, 1990; Leakey et al., 2003, 2005). Earlier studies have shown the existence of systemic regulation of stomatal development and of photosynthesis in developing leaves in response to environmental signals perceived by mature leaves, such as changing irradiance and atmospheric CO₂ conditions (Lake et al., 2002; Coupe et al., 2006; Araya et al., 2008). Phytochome B (phyB) is important in the transmission of the systemic signals that modulate stomatal development in young leaves of Arabidopsis (Arabidopsis thaliana; Casson and Hetherington, 2014). In tomato (Solanum lycopersicum), there are two forms of phyB, phyB1 and phyB2, that work together to mediate red (R) light-induced responses, such as hypocotyl elongation and greening in seedlings (Hauser et al., 1995; Weller et al., 2000).Photosynthesis is completely switched off in the dark, specifically to prevent futile cycling of metabolites through the reductive and oxidative pentose phosphate pathways. Hence, leaves need time to reactivate the enzymes of carbon assimilation after a period of darkness. The time taken to reach maximum net rates of photosynthesis upon illumination is called photosynthetic induction (Walker, 1973). Systemic signaling also has been observed for the regulation of photosynthesis in relation to leaf ontology in understory plants (Montgomery and Givnish, 2008). The uppermost leaves, which are generally the first to receive sunlight, display faster photosynthetic induction times than understory leaves (Bai et al., 2008). Photosynthetic induction in understory leaves is enhanced by the preillumination of upper leaves but not lower leaves, suggesting a directional signal transfer (Hou et al., 2015). While this process allows plants to use the light energy in sun flecks more efficiently, the nature of the systemic signals and their transmission pathways remain largely unresolved. Although systemic signaling between different leaf ranks has been suggested to occur through the xylem (Thorpe et al., 2007) and also via electrical signals (Zimmermann et al., 2009), it is likely that systemic signals also pass through the phloem (Turgeon and Wolf, 2009; Hou et al., 2015). In addition, the phytohormone auxin is produced in the shoot apex and redistributed throughout the shoot by rapid nonpolar phloem transport (Ljung et al., 2001). Changes in the light environment can dramatically alter auxin homeostasis, which is regulated in a light quality- and photoreceptor-dependent manner (Halliday et al., 2009).The photosynthetic electron transport chain exhibits enormous flexibility in the relative rates of NADPH and ATP production in order to accommodate the varying requirements of metabolism (Foyer et al., 2012). Noncyclic, pseudocyclic, and cyclic electron flow (CEF) pathways operate in the photosynthetic electron transport chain to drive the proton gradient across the thylakoid membrane (Allen, 2003). Photosynthetic induction is not only associated with the activation of the light- and thiol-dependent activation of carbon assimilation enzymes but also dependent on a high rate of CEF to drive ATP synthesis (Foyer et al., 1992). Considerable overreduction of the electron transport acceptors occurs during the photosynthetic induction period, and this continues until carbon assimilation can be activated. CEF around PSI, an essential component of photosynthesis, drives the proton gradient in a situation when NADP reduction has reached its highest capacity and this essential electron acceptor is no longer available (Yamori et al., 2015; Yamori and Shikanai, 2016). CEF is particularly sensitive to the reduction-oxidation (redox) status of the chloroplast, which in turn is responsive to cellular redox homeostasis. Oxidants such as hydrogen peroxide (H₂O₂), which are produced by pseudocyclic electron flow in the chloroplasts, play a crucial role in the activation of CEF through modulation of the activity of the NADPH-plastoquinone reductase complex (Strand et al., 2015). Hormone-mediated generation of H₂O₂ also can stimulate CO₂ assimilation (Jiang et al., 2012).Auxins such as indole-3-acetic acid (IAA) generate H₂O₂ (Ivanchenko et al., 2013; Peer et al., 2013) and can regulate CO₂ assimilation (Bidwell and Turner, 1966; Hayat et al., 2009; Peng et al., 2013). Therefore, we used tomato plants to test the hypothesis that the systemic signaling that regulates photosynthetic induction in understory leaves arises from light-induced changes in auxin and H₂O₂ homeostasis involving the modulation of CEF in systemic leaves. We present evidence showing that R light perceived in the shoot apex by a phyB-dependent pathway alters IAA signaling in a systemic manner. IAA signals from the apex, perceived in distal leaves, trigger systemic H₂O₂ production that accelerates photosynthetic induction by increasing CEF-dependent ATP production in the systemic leaves. These findings provide new insights into the elaborate plant regulatory network that allows light adaptation in different organs.     

The Mitochondrial 2-Oxoglutarate Carrier Is Part of a Metabolic Pathway That Mediates Glucose- and Glutamine-stimulated Insulin Secretion

Glucose-stimulated insulin secretion from pancreatic islet β-cells is dependent in part on pyruvate cycling through the pyruvate/isocitrate pathway, which generates cytosolic α-ketoglutarate, also known as 2-oxoglutarate (2OG). Here, we have investigated if mitochondrial transport of 2OG through the 2-oxoglutarate carrier (OGC) participates in control of nutrient-stimulated insulin secretion. Suppression of OGC in clonal pancreatic β-cells (832/13 cells) and isolated rat islets by adenovirus-mediated delivery of small interfering RNA significantly decreased glucose-stimulated insulin secretion. OGC suppression also reduced insulin secretion in response to glutamine plus the glutamate dehydrogenase activator 2-amino-2-norbornane carboxylic acid. Nutrient-stimulated increases in glucose usage, glucose oxidation, glutamine oxidation, or ATP:ADP ratio were not affected by OGC knockdown, whereas suppression of OGC resulted in a significant decrease in the NADPH:NADP⁺ ratio during stimulation with glucose but not glutamine + 2-amino-2-norbornane carboxylic acid. Finally, OGC suppression reduced insulin secretion in response to a membrane-permeant 2OG analog, dimethyl-2OG. These data reveal that the OGC is part of a mechanism of fuel-stimulated insulin secretion that is common to glucose, amino acid, and organic acid secretagogues, involving flux through the pyruvate/isocitrate cycling pathway. Although the components of this pathway must remain intact for appropriate stimulus-secretion coupling, production of NADPH does not appear to be the universal second messenger signal generated by these reactions.     

Entry into Mitosis in Vertebrate Somatic Cells Is Guarded by a Chromosome Damage Checkpoint That Reverses the Cell Cycle When Triggered during Early but Not Late Prophase

When vertebrate somatic cells are selectively irradiated in the nucleus during late prophase (<30 min before nuclear envelope breakdown) they progress normally through mitosis even if they contain broken chromosomes. However, if early prophase nuclei are similarly irradiated, chromosome condensation is reversed and the cells return to interphase. Thus, the G₂ checkpoint that prevents entry into mitosis in response to nuclear damage ceases to function in late prophase. If one nucleus in a cell containing two early prophase nuclei is selectively irradiated, both return to interphase, and prophase cells that have been induced to returned to interphase retain a normal cytoplasmic microtubule complex. Thus, damage to an early prophase nucleus is converted into a signal that not only reverses the nuclear events of prophase, but this signal also enters the cytoplasm where it inhibits e.g., centrosome maturation and the formation of asters. Immunofluorescent analyses reveal that the irradiation-induced reversion of prophase is correlated with the dephosphorylation of histone H1, histone H3, and the MPM2 epitopes. Together, these data reveal that a checkpoint control exists in early but not late prophase in vertebrate cells that, when triggered, reverses the cell cycle by apparently downregulating existing cyclin-dependent kinase (CDK1) activity.     

PqqD Is a Novel Peptide Chaperone That Forms a Ternary Complex with the Radical S-Adenosylmethionine Protein PqqE in the Pyrroloquinoline Quinone Biosynthetic Pathway

Pyrroloquinoline quinone (PQQ) is a product of a ribosomally synthesized and post-translationally modified pathway consisting of five conserved genes, pqqA-E. PqqE is a radical S-adenosylmethionine (RS) protein with a C-terminal SPASM domain, and is proposed to catalyze the formation of a carbon-carbon bond between the glutamate and tyrosine side chains of the peptide substrate PqqA. PqqD is a 10-kDa protein with an unknown function, but is essential for PQQ production. Recently, in Klebsiella pneumoniae (Kp), PqqD and PqqE were shown to interact; however, the stoichiometry and K_D were not obtained. Here, we show that the PqqE and PqqD interaction transcends species, also occurring in Methylobacterium extorquens AM1 (Me). The stoichiometry of the MePqqD and MePqqE interaction is 1:1 and the K_D, determined by surface plasmon resonance spectroscopy (SPR), was found to be ∼12 μm. Moreover, using SPR and isothermal calorimetry techniques, we establish for the first time that MePqqD binds MePqqA tightly (K_D ∼200 nm). The formation of a ternary MePqqA-D-E complex was captured by native mass spectrometry and the K_D for the MePqqAD-MePqqE interaction was found to be ∼5 μm. Finally, using a bioinformatic analysis, we found that PqqD orthologues are associated with the RS-SPASM family of proteins (subtilosin, pyrroloquinoline quinone, anaerobic sulfatase maturating enzyme, and mycofactocin), all of which modify either peptides or proteins. In conclusion, we propose that PqqD is a novel peptide chaperone and that PqqD orthologues may play a similar role in peptide modification pathways that use an RS-SPASM protein.     

An Apical-Type Trafficking Pathway Is Present in Cultured Oligodendrocytes but the Sphingolipid-enriched Myelin Membrane Is the Target of a Basolateral-Type Pathway

Myelin sheets originate from distinct areas at the oligodendrocyte (OLG) plasma membrane and, as opposed to the latter, myelin membranes are relatively enriched in glycosphingolipids and cholesterol. The OLG plasma membrane can therefore be considered to consist of different membrane domains, as in polarized cells; the myelin sheet is reminiscent of an apical membrane domain and the OLG plasma membrane resembles the basolateral membrane. To reveal the potentially polarized membrane nature of OLG, the trafficking and sorting of two typical markers for apical and basolateral membranes, the viral proteins influenza virus–hemagglutinin (HA) and vesicular stomatitis virus–G protein (VSVG), respectively, were examined. We demonstrate that in OLG, HA and VSVG are differently sorted, which presumably occurs upon their trafficking through the Golgi. HA can be recovered in a Triton X-100-insoluble fraction, indicating an apical raft type of trafficking, whereas VSVG was only present in a Triton X-100-soluble fraction, consistent with its basolateral sorting. Hence, both an apical and a basolateral sorting mechanism appear to operate in OLG. Surprisingly, however, VSVG was found within the myelin sheets surrounding the cells, whereas HA was excluded from this domain. Therefore, despite its raft-like transport, HA does not reach a membrane that shows features typical of an apical membrane. This finding indicates either the uniqueness of the myelin membrane or the requirement of additional regulatory factors, absent in OLG, for apical delivery. These remarkable results emphasize that polarity and regulation of membrane transport in cultured OLG display features that are quite different from those in polarized cells.     

Reduced Expression of the Retinoblastoma Protein Shows That the Related Signaling Pathway Is Essential for Mediating the Antineoplastic Activity of Erufosine

Erufosine is a new antineoplastic agent of the group of alkylphosphocholines, which interferes with signal transduction and induces apoptosis in various leukemic and tumor cell lines. The present study was designed to examine for the first time the mechanism of resistance to erufosine in malignant cells with permanently reduced expression of the retinoblastoma (Rb) protein. Bearing in mind the high number of malignancies with reduced level of this tumor-suppressor, this investigation was deemed important for using erufosine, alone or in combination, in patients with compromised RB1 gene expression. For this purpose, clones of the leukemic T-cell line SKW-3 were used, which had been engineered to constantly express differently low Rb levels. The alkylphosphocholine induced apoptosis, stimulated the expression of the cyclin dependent kinase inhibitor p27^Kip1 and inhibited the synthesis of cyclin D3, thereby causing a G₂ phase cell cycle arrest and death of cells with wild type Rb expression. In contrast, Rb-deficiency impeded the changes induced by eru-fosine in the expression of these proteins and abrogated the induction of G₂ arrest, which was correlated with reduced antiproliferative and anticlonogenic activities of the compound. In conclusion, analysis of our results showed for the first time that the Rb signaling pathway is essential for mediating the antineoplastic activity of erufosine and its efficacy in patients with malignant diseases may be predicted by determining the Rb status.     

Activation of the Protein Kinase C1 Pathway upon Continuous Heat Stress in Saccharomyces cerevisiae Is Triggered by an Intracellular Increase in Osmolarity due to Trehalose Accumulation

This paper reports on physiological and molecular responses of Saccharomyces cerevisiae to heat stress conditions. We observed that within a very narrow range of culture temperatures, a shift from exponential growth to growth arrest and ultimately to cell death occurred. A detailed analysis was carried out of the accumulation of trehalose and the activation of the protein kinase C1 (PKC1) (cell integrity) pathway in both glucose- and ethanol-grown cells upon temperature upshifts within this narrow range of growth temperatures. It was observed that the PKC1 pathway was hardly activated in a tps1 mutant that is unable to accumulate any trehalose. Furthermore, it was observed that an increase of the extracellular osmolarity during a continuous heat stress prevented the activation of the pathway. The results of these analyses support our hypothesis that under heat stress conditions the activation of the PKC1 pathway is triggered by an increase in intracellular osmolarity, due to the accumulation of trehalose, rather than by the increase in temperature as such.     

The Nicotinamide Biosynthetic Pathway Is a By-Product of the RNA World

Many of the biosynthetic pathways, especially those leading to the coenzymes, must have originated very early, perhaps before enzymes were available to catalyze their synthesis. While a number of enzymatic reactions in metabolism are known to proceed nonenzymatically, there are no examples of entire metabolic sequences that can be achieved in this manner. The most primitive pathway for nicotinic acid biosynthesis is the reaction of aspartic acid with dihydroxyacetone phosphate. We report here that nicotinic acid (NAc) and its metabolic precursor, quinolinic acid (QA), are produced in yields as high as 7% in a six-step nonenzymatic sequence from aspartic acid and dihydroxyacetone phosphate (DHAP). The biosynthesis of ribose phosphate could have produced DHAP and other three carbon compounds. Aspartic acid could have been available from prebiotic synthesis or from the ribozyme synthesis of pyrimidines. These results suggest that NAD could have originated in the RNA world and that the nonenzymatic biosynthesis of the cofactor nicotinamide could have been an inevitable consequence of life based on carbohydrates and amino acids. The enzymes of the modern pathway were later added in any order. Received: 22 May 2000 / Accepted: 7 August 2000