Genetic evidence that polysumoylation bypasses the need for a SUMO-targeted Ub ligase

Saccharomyces cerevisiae cells lacking the Slx5-Slx8 SUMO-targeted Ub ligase display increased levels of sumoylated and polysumoylated proteins, and they are inviable in the absence of the Sgs1 DNA helicase. One explanation for this inviability is that one or more sumoylated proteins accumulate to toxic levels in sgs1Δ slx5Δ cells. To address this possibility, we isolated a second-site suppressor of sgs1Δ slx5Δ synthetic lethality and identified it as an allele of the ULP2 SUMO isopeptidase. The suppressor, ulp2-D623H, behaved like the ulp2Δ allele in its sensitivity to heat, DNA replication stress, and DNA damage. Surprisingly, deletion of ULP2, which is known to promote the accumulation of poly-SUMO chains, suppressed sgs1Δ slx5Δ synthetic lethality and the slx5Δ sporulation defect. Further, ulp2Δ's growth sensitivities were found to be suppressed in ulp2Δ slx5Δ double mutants. This mutual suppression indicates that SLX5-SLX8 and ULP2 interact antagonistically. However, the suppressed strain sgs1Δ slx5Δ ulp2-D623H displayed even higher levels of sumoylated proteins than the corresponding double mutants. Thus, sgs1Δ slx5Δ synthetic lethality cannot be due simply to high levels of bulk sumoylated proteins. We speculate that the loss of ULP2 suppresses the toxicity of the sumoylated proteins that accumulate in slx5Δ-slx8Δ cells by permitting the extension of poly-SUMO chains on specific target proteins. This additional modification might attenuate the activity of the target proteins or channel them into alternative pathways for proteolytic degradation. In support of this latter possibility we find that the WSS1 isopeptidase is required for suppression by ulp2Δ.     

Assembly and expression of an intrinsic factor IX activator complex on the surface of cultured human endothelial cells.

Endothelial cells expose specific receptors for blood clotting factors and, upon perturbation, can initiate and propagate the reactions of the extrinsic pathway of blood coagulation leading to fibrin formation on the cell surface. The existence of an intrinsic mechanism of Factor IX activation on cultured human umbilical vein cells (HUVECs) was investigated by studies of the interaction between HUVECs and two proteins of the contact activation system, the cofactor high molecular weight kininogen (H-kininogen) and the zymogen Factor XI. In the presence of zinc ions (10-300 microM), 125I-labeled H-kininogen bound to HUVECs in a time-dependent, reversible, and saturable manner, with calcium ions exerting an inhibitory effect on the zinc-dependent binding. Analysis of the binding data by the LIGAND computer program indicated that HUVECs, in the presence of 2 mM CaCl2 and 100 microM ZnCl2 at 37 degrees C, bound 1.14 x 10(7) H-kininogen molecules per cell with an apparent dissociation constant of 55 nM. HUVEC-bound H-kininogen functions as the cell surface receptor for both 125I-labeled Factor XI and 125I-labeled Factor XIa, since HUVECs cultured in contact factor-depleted serum do not detectably bind either the zymogen or the enzyme in the absence of H-kininogen and zinc ions. In the presence of saturating concentrations of H-kininogen, 2 mM CaCl2 and 100 microM ZnCl2, the binding of 125I-labeled Factor XI and Factor XIa to HUVECs was time-dependent, reversible, and saturable, with apparent dissociation constants of 4.5 and 1.5 nM, respectively. HUVEC-bound complexes of H-kininogen and Factor XI generated Factor XIa activity only after the addition of purified Factor XIIa, and cell-bound Factor XIa in turn activated Factor IX, as documented by a 3H-labeled activation peptide release assay for 3H-Factor IX activation. The results indicate that cultured HUVECs provide a surface for the assembly and expression of an intrinsic Factor IX activator complex that may participate in the initiation of blood coagulation at sites of vascular injury.     

Avermectin B1a: an irreversible activator of the gamma-aminobutyric acid-benzodiazepine-chloride-ionophore receptor complex

Avermectin B_1a, an antihelminthic macrocyclic lactone, has been previously shown to reduce muscle membrane resistance by stimulating γ-aminobutyric acid-mediated chloride conductance. Since the benzodiazepine receptor is coupled to a receptor for γ-aminobutyric acid and related chloride ionophore, the effects of Avermectin B_1a on [³H]diazepam binding to the benzodiazepine receptor were studied. In well-washed membrane fragments from rat cerebral cortex, Avermectin B_1a markedly increased the binding of [³H]diazepam to benzodiazepine receptors. This effect was qualitatively similar to that observed with either γ-aminobutyric acid or chloride ion and was partially reversed by the γ-aminobutyric acid receptor antagonist, bicuculline. In contrast to the effects of γ-aminobutyric acid and chloride, the enhanced binding of [³H]benzodiazepine elicited by Avermectin B_1a was $\text{not}$ reversed by extensive washing of the membrane preparation. Avermectin B_1a appears to irreversibly modify benzodiazepine receptors at a γ-aminobutyric acid-chloride recognition site and may be valuable in biochemical studies of the regulation of benzodiazepine receptor function.     

Independent recruitment of mediator and SAGA by the activator Met4

Mediator is a key RNA polymerase II (Pol II) cofactor in the regulation of eukaryotic gene expression. It is believed to function as a coactivator linking gene-specific activators to the basal Pol II initiation machinery. In support of this model, we provide evidence that Mediator serves in vivo as a coactivator for the yeast activator Met4, which controls the gene network responsible for the biosynthesis of sulfur-containing amino acids and S-adenosylmethionine. In addition, we show that SAGA (Spt-Ada-Gcn5-acetyltransferase) is also recruited to Met4 target promoters, where it participates in the recruitment of Pol II by a mechanism involving histone acetylation. Interestingly, we find that SAGA is not required for Mediator recruitment by Met4 and vice versa. Our results provide a novel example of functional interplay between Mediator and coactivators involved in histone modification.     

RNA polymerase and an activator form discrete subcomplexes in a transcription initiation complex

Using high-resolution atomic force microscopy (AFM) we show that in a ternary complex of an activator protein, FIS, and RNA polymerase containing the sigma(70) specificity factor at the Escherichia coli tyrT promoter the polymerase and the activator form discrete, but connected, subcomplexes in close proximity. This is the first time that a ternary complex between an activator, a sigma(70) polymerase holoenzyme and promoter DNA has been visualised. Individually FIS and RNA polymerase wrap approximately 80 and 150 bp of promoter DNA, respectively. We suggest that the architecture of the ternary complex provides a general paradigm for the facilitation of direct, but weak, interactions between polymerase and an activator.     

Transgenic overexpression of transforming growth factor alpha bypasses the need for c-Ha-ras mutations in mouse skin tumorigenesis.

The induction of skin papillomas in mice can be divided into two different stages. Chemical initiation frequently elicits mutations in the Ha-ras gene, leading to the constitutive activation of ras. The second step, promotion, involves repetitive topical application of phorbol esters or wounding, leading to epidermal hyperproliferation and papilloma formation. We have found that overexpression of transforming growth factor alpha (TGF-alpha) in the basal epidermal layer of transgenic mice yielded papillomas directly upon wounding or 12-O-tetradecanoylphorbol-13-acetate treatment without the need for an initiator. Moreover, papillomas from TGF-alpha mice did not exhibit mutations in the Ha-ras gene. Interestingly, TGF-alpha acted synergistically with 12-O-tetradecanoylphorbol-13-acetate to enhance epidermal hyperproliferation. Our results demonstrate a central role for TGF-alpha overexpression in tumorigenesis and provide an important animal model for the study of skin tumorigenesis.     

Assembly of the nucleolus: in need of revision

Our current view of the nucleolus has been shaped by the concept that the organization of this prominent compartment within the nucleus is primarily dictated by its function, the making of ribosome subunits. Whether ribosome biogenesis is framed by a dedicated nucleolar scaffold has remained unclear. In this issue of The EMBO Journal, Caudron‐Herger and colleagues present evidence for a nucleolar skeleton composed of non‐coding RNA enriched in Alu repeat elements.     

Presence of an activator of pyruvate dehydrogenase in human circulation: elevation following a glucose load and possible relation to an insulin mediator

Our earlier observation that the chemical mediator of insulin action stimulates lipid synthesis in primary cultures of rat hepatocytes prompted us to examine its presence in human serum and its regulation by changes in insulin levels. Serum samples were obtained from normal subjects following an oral 100 gm glucose tolerance test (GTT; n = 10). An acid soluble, heat stable and charcoal non-absorbable substance was extracted from different sera and tested for their ability to stimulate liver mitochondrial pyruvate dehydrogenase (PDH). This substance obtained from GTT samples at 1/1000 final dilution caused significantly higher stimulation of PDH when compared to that obtained from fasting samples. These results demonstrate the presence of an activator of PDH (molecular weight approximately 1000-2000) in human circulation. Since the activator of PDH is modulated by physiological perturbation such as oral glucose ingestion, known to cause changes in circulating insulin levels, it may possibly be related to insulin mediator.     

GAGA factor and the TFIID complex collaborate in generating an open chromatin structure at the Drosophila melanogaster hsp26 promoter

The upstream regulatory region of the Drosophila melanogaster hsp26 gene includes two DNase I-hypersensitive sites (DH sites) that encompass the critical heat shock elements. This chromatin structure is required for heat shock-inducible expression and depends on two (CT)n*(GA)n elements bound by GAGA factor. To determine whether GAGA factor alone is sufficient to drive formation of the DH sites, we have created flies with an hsp26/lacZ transgene wherein the entire DNA segment known to interact with the TFIID complex has been replaced by a random sequence. The replacement results in a loss of heat shock-inducible hsp26 expression and drastically diminishes nuclease accessibility in the chromatin of the regulatory region. Chromatin immunoprecipitation experiments show that the decrease in TFIID binding does not reduce GAGA factor binding. In contrast, the loss of GAGA factor binding resulting from (CT)n mutations decreases TFIID binding. These data suggest that both GAGA factor and TFIID are necessary for formation of the appropriate chromatin structure at the hsp26 promoter and predict a regulatory mechanism in which GAGA factor binding precedes and contributes to the recruitment of TFIID.     

Developmental role and regulation of cortex, a meiosis-specific anaphase-promoting complex/cyclosome activator

During oogenesis in metazoans, the meiotic divisions must be coordinated with development of the oocyte to ensure successful fertilization and subsequent embryogenesis. The ways in which the mitotic machinery is specialized for meiosis are not fully understood. cortex, which encodes a putative female meiosis-specific anaphase-promoting complex/cyclosome (APC/C) activator, is required for proper meiosis in Drosophila. We demonstrate that CORT physically associates with core subunits of the APC/C in ovaries. APC/C^CORT targets Cyclin A for degradation prior to the metaphase I arrest, while Cyclins B and B3 are not targeted until after egg activation. We investigate the regulation of CORT and find that CORT protein is specifically expressed during the meiotic divisions in the oocyte. Polyadenylation of cort mRNA is correlated with appearance of CORT protein at oocyte maturation, while deadenylation of cort mRNA occurs in the early embryo. CORT protein is targeted for degradation by the APC/C following egg activation, and this degradation is dependent on an intact D-box in the C terminus of CORT. Our studies reveal the mechanism for developmental regulation of an APC/C activator and suggest it is one strategy for control of the female meiotic cell cycle in a multicellular organism.     

Assembly stoichiometry of the GluK2/GluK5 kainate receptor complex

Ionotropic glutamate receptors assemble as homo- or heterotetramers. One well-studied heteromeric complex is formed by the kainate receptor subunits GluK2 and GluK5. Retention motifs prevent trafficking of GluK5 homomers to the plasma membrane, but coassembly with GluK2 yields functional heteromeric receptors. Additional control over GluK2/GluK5 assembly seems to be exerted by the aminoterminal domains, which preferentially assemble into heterodimers as isolated domains. However,the stoichiometry of the full-length GluK2/GluK5 receptor complex has yet to be determined, as is the case for all non-NMDA glutamate receptors. Here, we address this question, using a single-molecule imaging technique that enables direct counting of the number of each GluK subunit type in homomeric and heteromeric receptors in the plasma membranes of live cells. We show that GluK2 and GluK5 assemble with 2:2 stoichiometry. This is an important step toward understanding the assembly mechanism, architecture, and functional consequences of heteromer formation in ionotropic glutamate receptors.