Clavanin permeabilizes target membranes via two distinctly different pH-dependent mechanisms

The pH dependence of the antimicrobial and membrane activity of clavanin A, a peptide antibiotic that is rich in histidines and glycines, was analyzed in growth and membrane leakage experiments. Clavanin A more effectively inhibited the growth of the test organism Lactobacillus sake when the pH of the medium was lowered. Whereas the wild-type peptide efficiently released fluorophores from unilamellar vesicles at neutral pH according to a nonspecific permeabilization mechanism, it did not permeabilize model bilayers at low pH. It was therefore suggested that this peptide uses a distinct mode of action under acidic conditions different than that used around neutral pH. However, at low pH, the membrane is still the target for clavanin A, as the peptide collapsed both vital transmembrane proton gradients and ion gradients under these conditions. Clavanin A did not act as a ionophore across phospholipid bilayers, indicating that membrane constituents other than membrane phospholipids are involved in the dissipation of transmembrane ion gradients. Membrane proteins that generate transmembrane ion gradients are suggested to be the targets for clavanin A at low pH. In addition to the histidines, the three glycine residues of clavanin A are shown to play an important role in the specific mode of interaction with these membrane targets. These residues may induce a flexible hydrophobic conformation that allows the peptide to exert different membrane activities. This study demonstrates that clavanin A is a special membrane-active peptide that has access to two markedly distinct pH-dependent modes of actions.     

Xist Exon 7 Contributes to the Stable Localization of Xist RNA on the Inactive X-Chromosome

To equalize X-linked gene dosage between the sexes in mammalian females, Xist RNA inactivates one of the two X-chromosomes. Here, we report the crucial function of Xist exon 7 in X-inactivation. Xist exon 7 is the second-largest exon with a well-conserved repeat E in eutherian mammals, but its role is often overlooked in X-inactivation. Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells. Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells. Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.     

Organelle-specific control of intracellular transport: distinctly targeted isoforms of the regulator Klar

Microtubule-based transport in cells is powered by a small set of distinct motors, yet timing and destination of transport can be controlled in a cargo-specific manner. The mechanistic basis for this specificity is not understood. To address this question, we analyzed the Drosophila Klarsicht (Klar) protein that regulates distinct microtubule-based transport processes. We find that localization of Klar to its cargoes is crucial for Klar function. Using mutations, we identify functionally important regions of Klar that confer distinct cargo specificity. In ovaries, Klar is present on the nuclear envelope, a localization that requires the C-terminal KASH domain. In early embryos, Klar is attached to lipid droplets, a localization mediated by a novel C-terminal domain encoded by an alternatively spliced exon. In cultured cells, these two domains are sufficient for targeting to the correct intracellular location. Our analysis disentangles Klar's modular organization: we propose that a core region integral to motor regulation is attached to variable domains so that the cell can target regulators with overlapping, yet distinct functions to specific cargoes. Such isoform variation may be a general strategy for adapting a common regulatory mechanism to specifically control motion and positioning of multiple organelles.     

YY1 tethers Xist RNA to the inactive X nucleation center

The long noncoding Xist RNA inactivates one X?chromosome in the female mammal. Current models posit that Xist induces silencing as it spreads along X and recruits Polycomb complexes. However, the mechanisms for Xist loading and spreading are currently unknown. Here, we define the nucleation center for Xist RNA and show that YY1 docks Xist particles onto the X chromosome. YY1 is a "bivalent" protein, capable of binding both RNA and DNA through different sequence motifs. Xist's exclusive attachment to the inactive X is determined by an epigenetically regulated trio of YY1 sites as well as allelic origin. Specific YY1-to-RNA and YY1-to-DNA contacts are required to load Xist particles onto X. YY1 interacts with Xist RNA through Repeat C. We propose that YY1 acts as adaptor between regulatory RNA and chromatin targets.     

Seasonal variation of leaf glutamine synthetase isoforms in temperate deciduous trees strongly suggests different functions for the enzymes

Changes in the activities of leaf glutamine synthetase (GS) isoforms were followed in four temperate deciduous trees from full leaf expansion to senescence (May to November). In the early part of the season, total GS activity was high in all species, with values ranging from 90 to 200 μmol h⁻¹ g⁻¹ fw. During this early period this activity comprised only the activity of the chloroplastic (GS₂) isoform in all species. These high GS₂ activities are consistent with the role of GS₂ in the re-assimilation of photorespired ammonia. The early high values also coincided with high nitrate reductase activity in one of the species, the highly nitrophilous species Sambucus nigra, with values of up to 16μmol h⁻¹ g⁻¹ fw. This indicates that GS₂ is also important in the assimilation of ammonia produced from nitrate reduction. From mid- to late-season, the cytosolic isoform (GS₁) was detected in all four species and became increasingly more active in comparison to GS₂. By the time of senescence it was the dominant enzyme of the two forms in both S. nigra and Carpinus betulus. The results provide strong support for recent findings that GS₁ is an important enzyme for the mobilization of nitrogen for translocation or storage.     

Identification of two distinct isoforms of stathmin and characterization of their respective phosphorylated forms

Stathmin is a ubiquitous soluble protein (Mr approximately 19,000, pI approximately 6.2-5.5) whose phosphorylation is associated with the intracellular mechanisms involved in the regulations of cell differentiation and functions by extracellular effectors. Its purification from rat brain and the preparation of specific antibodies allowed us to identify a set of immunologically related unphosphorylated (N1, N2) and phosphorylated (P1, P2a, P2b, P3) proteins of decreasing isoelectric points. All these proteins yielded identical silver-stained or 32P-radioactive peptide maps with the protease V8 from Staphylococcus aureus, indicating that they are also structurally related. In vitro phosphorylation with the exogenous catalytic subunit of the cAMP-dependent protein kinase, as well as dephosphorylation with alkaline phosphatase, indicated that P1, P2, and P3 derived from N1 and N2 by progressive phosphorylation. Phosphorylation of individual proteins extracted from semi-preparative two-dimensional polyacrylamide gels demonstrated the existence of two distinct isoforms of stathmin, alpha and beta: N1 and N2 are their respective unphosphorylated forms (alpha O and beta O), whereas proteins P1-P3 could be resolved as at least three increasingly phosphorylated forms of both alpha and beta stathmin (alpha 1, alpha 2, alpha(3) and beta 1, beta 2, beta(3]. In intact pituitary GH4C1 cells, hormones like thyrotropin-releasing hormone and vasoactive intestinal peptide induced a similar conversion from N1 and N2 to P1, P2, and P3. The phosphorylation of both alpha and beta isoforms of stathmin is therefore a physiologically significant response to specific extracellular regulatory agents. In conclusion, stathmin represents a family of at least two distinct protein isoforms, whose respective phosphorylation and expression might play a role in its likely function as an intracellular relay of various converging extracellular signals.     

The existence of two different forms of apo-electron-transferring flavoprotein

The association process of FAD and apo-electron-transferring flavoprotein (apoETF) from hog kidney was investigated. The reaction schemes which involve the association-dissociation of the protein species could be excluded by the light scattering data, which indicated that the molecular weights of apoETF and holoETF are identical. The binding reaction between FAD and a large excess of apoETF was monophasic and obeyed pseudo-first order kinetics. On the other hand, the reaction between apoETF and a large excess of FAD was biphasic: the fast phase obeyed a pseudo-first order reaction, and the rate of the slow phase was almost independent of FAD concentration. These results suggest the existence of two different forms of apoETF, as represented in the following reaction scheme: [formula: see text] where "F" is FAD, "H" is holoETF, and "A" and "A" are the different forms of apoETF. The kinetic parameters were determined as k-1 = 3.9 x 10(4) M-1.s-1, k-1 approximately 10(-5) s-1, k+2 = 1.0 x 10(-3) s-1, and k-2 = 3.1 x 10(-3) s-1, in 50 mM potassium phosphate buffer, pH 7.6, containing 0.3 mM EDTA, and 5% v/v glycerol, at 7 degrees C. The elution patterns of apoETF on molecular sieve chromatography were very different from that of holoETF although the true molecular weights were identical. This result suggests that the structure of apoETF differs greatly from that of holoETF.     

Expression of Xist RNA is sufficient to initiate macrochromatin body formation

MacroH2A1 is a histone variant that is found as a component of the inactive X chromosome where it is detected as a dense accumulation called a macrochromatin body (MCB). Macrochromatin bodies co-localize with Xist RNA, which is an untranslated RNA that is expressed exclusively from the inactive X chromosome of placental mammals. However, no studies to date have investigated whether Xist RNA expression is necessary or sufficient to cause the formation of MCBs. Here we show that expression of Xist RNA is sufficient to cause the formation of MCBs even when Xist is expressed from an inducible transgene at ectopic autosomal sites. Macrochromatin bodies form at sites of transgenic Xist expression in differentiating mouse ES cell lines and transgenic fibroblasts, but MCBs cannot form in undifferentiated ES cells even after prolonged Xist expression. The kinetics of MCB formation revealed that Xist expression precedes MCB formation and that differentiating ES cells undergo a rapid and synchronous transition that renders them competent to form MCBs. Once MCBs have formed, continued expression of Xist is required for their maintenance. These results show that Xist RNA and macroH2A1 function in a common pathway. Expression of Xist in a permissive nuclear environment is sufficient to initiate a chromatin-remodeling event culminating in the incorporation of macroH2A1. The results also strongly suggest the existence of additional regulatory factors for X inactivation that are regulated developmentally. In addition, we present evidence that macroH2A1 density is not simply a measure of the general degree of DNA compaction.     

Coordinate gene regulation by two different catenins

In this issue of Developmental Cell, McCrea and colleagues report that p120-catenin regulates the same Wnt target genes as beta-catenin in the Xenopus embryo (). These findings raise the exciting possibility that these two related proteins function in parallel to mediate cadherin-associated regulation of gene expression.