An InCytes from MBC Selection: Importin β Regulates the Seeding of Chromatin with Initiation Sites for Nuclear Pore Assembly

The nuclear envelope of higher eukaryotic cells reforms at the exit from mitosis, in concert with the assembly of nuclear pore complexes (NPCs). The first step in postmitotic NPC assembly involves the “seeding” of chromatin with ELYS and the Nup107-160 complex. Subsequent steps in the assembly process are poorly understood and different mechanistic models have been proposed to explain the formation of the full supramolecular structure. Here, we show that the initial step of chromatin seeding is negatively regulated by importin β. Direct imaging of the chromatin attachment sites reveals single sites situated predominantly on the highest substructures of chromatin surface and lacking any sign of annular structures or oligomerized pre-NPCs. Surprisingly, the inhibition by importin β is only partially reversed by RanGTP. Importin β forms a high-molecular-weight complex with both ELYS and the Nup107-160 complex in cytosol. We suggest that initiation sites for NPC assembly contain single copies of chromatin-bound ELYS/Nup107-160 and that the lateral oligomerization of these subunits depends on the recruitment of membrane components. We predict that additional regulators, besides importin β and Ran, may be involved in coordinating the initial seeding of chromatin with subsequent steps in the NPC assembly pathway.     

Chemo-enzymatic synthesis of the exocyclic olefin isomer of thymidine monophosphate

Exocyclic olefin variants of thymidylate (dTMP) recently have been proposed as reaction intermediates for the thymidyl biosynthesis enzymes found in many pathogenic organisms, yet synthetic reports on these materials are lacking. Here we report two strategies to prepare the exocyclic olefin isomer of dTMP, which is a putative reaction intermediate in pathogenic thymidylate biosynthesis and a novel nucleotide analog. Our most effective strategy involves preserving the existing glyosidic bond of thymidine and manipulating the base to generate the exocyclic methylene moiety. We also report a successful enzymatic deoxyribosylation of a non-aromatic nucleobase isomer of thymine, which provides an additional strategy to access nucleotide analogs with disrupted ring conjugation or with reduced heterocyclic bases. The strategies reported here are straightforward and extendable towards the synthesis of various pyrimidine nucleotide analogs, which could lead to compounds of value in studies of enzyme reaction mechanisms or serve as templates for rational drug design.     

Construction of a genome-anchored,high-density genetic map for melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.) and identification of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">melonis</Emphasis> race 1 resistance QTL

Key message

Four QTLs and an epistatic interaction were associated with disease severity in response to inoculation with Fusarium oxysporum f. sp. melonis race 1 in a recombinant inbred line population of melon.

Abstract

The USDA Cucumis melo inbred line, MR-1, harbors a wealth of alleles associated with resistance to several major diseases of melon, including powdery mildew, downy mildew, Alternaria leaf blight, and Fusarium wilt. MR-1 was crossed to an Israeli cultivar, Ananas Yok’neam, which is susceptible to all of these diseases, to generate a recombinant inbred line (RIL) population of 172 lines. In this study, the RIL population was genotyped to construct an ultra-dense genetic linkage map with 5663 binned SNPs anchored to the C. melo genome and exhibits the overall high quality of the assembly. The utility of the densely genotyped population was demonstrated through QTL mapping of a well-studied trait, resistance to Fusarium wilt caused by Fusarium oxysporum f. sp. melonis (Fom) race 1. A major QTL co-located with the previously validated resistance gene Fom-2. In addition, three minor QTLs and an epistatic interaction contributing to Fom race 1 resistance were identified. The MR-1 × AY RIL population provides a valuable resource for future QTL mapping studies and marker-assisted selection of disease resistance in melon.

     

How does the exosite of rhomboid protease affect substrate processing and inhibition?

Rhomboid proteases constitute a family of intramembrane serine proteases ubiquitous in all forms of life. They differ in many aspects from their soluble counterparts. We applied molecular dynamics (MD) computational approach to address several challenging issues regarding their catalytic mechanism: How does the exosite of GlpG rhomboid protease control the kinetics efficiency of substrate hydrolysis? What is the mechanism of inhibition by the non‐competitive peptidyl aldehyde inhibitors bound to the GlpG rhomboid active site (AS)? What is the underlying mechanism that explains the hypothesis that GlpG rhomboid protease is not adopted for the hydrolysis of short peptides that do not contain a transmembrane domain (TMD)? Two fundamental features of rhomboid catalysis, the enzyme recognition and discrimination of substrates by TMD interactions in the exosite, and the concerted mechanism of non‐covalent pre‐catalytic complex to covalent tetrahedral complex (TC) conversion, provide answers to these mechanistic questions.     

The effect of blue light on stomatal oscillations and leaf turgor pressure in banana leaves

Stomatal oscillations are cyclic opening and closing of stomata, presumed to initiate from hydraulic mismatch between leaf water supply and transpiration rate. To test this assumption, mismatches between water supply and transpiration were induced using manipulations of vapour pressure deficit (VPD) and light spectrum in banana (Musa acuminata). Simultaneous measurements of gas exchange with changes in leaf turgor pressure were used to describe the hydraulic mismatches. An increase of VPD above a certain threshold caused stomatal oscillations with variable amplitudes. Oscillations in leaf turgor pressure were synchronized with stomatal oscillations and balanced only when transpiration equaled water supply. Surprisingly, changing the light spectrum from red and blue to red alone at constant VPD also induced stomatal oscillations – while the addition of blue (10%) to red light only ended oscillations. Blue light is known to induce stomatal opening and thus should increase the hydraulic mismatch, reduce the VPD threshold for oscillations and increase the oscillation amplitude. Unexpectedly, blue light reduced oscillation amplitude, increased VPD threshold and reduced turgor pressure loss. These results suggest that additionally, to the known effect of blue light on the hydroactive opening response of stomata, it can also effect stomatal movement by increased xylem–epidermis water supply.     

Capture of AT-rich chromatin by ELYS recruits POM121 and NDC1 to initiate nuclear pore assembly

Assembly of the nuclear pore, gateway to the genome, from its component subunits is a complex process. In higher eukaryotes, nuclear pore assembly begins with the binding of ELYS/MEL-28 to chromatin and recruitment of the large critical Nup107-160 pore subunit. The choreography of steps that follow is largely speculative. Here, we set out to molecularly define early steps in nuclear pore assembly, beginning with chromatin binding. Point mutation analysis indicates that pore assembly is exquisitely sensitive to the change of only two amino acids in the AT-hook motif of ELYS. The dependence on AT-rich chromatin for ELYS binding is borne out by the use of two DNA-binding antibiotics. AT-binding Distamycin A largely blocks nuclear pore assembly, whereas GC-binding Chromomycin A(3) does not. Next, we find that recruitment of vesicles containing the key integral membrane pore proteins POM121 and NDC1 to the forming nucleus is dependent on chromatin-bound ELYS/Nup107-160 complex, whereas recruitment of gp210 vesicles is not. Indeed, we reveal an interaction between the cytoplasmic domain of POM121 and the Nup107-160 complex. Our data thus suggest an order for nuclear pore assembly of 1) AT-rich chromatin sites, 2) ELYS, 3) the Nup107-160 complex, and 4) POM121- and NDC1-containing membrane vesicles and/or sheets, followed by (5) assembly of the bulk of the remaining soluble pore subunits.     

Peptidyl epoxides extended in the P' direction as cysteine protease inhibitors: effect on affinity and mechanism of inhibition

Endo peptidyl epoxides, in which the central epoxidic moiety replaces the scissile amide bond of a P(3)-P(3)' peptide, were designed as cysteine proteases inhibitors. The additional P'-S' interactions, relative to those of an exo peptidyl epoxide of the same P(3)-P(1) sequence, significantly improved affinity to the enzymes papain and cathepsin B, but also changed the mode of inhibition from active-site directed inactivation to reversible competitive inhibition. Computational models rationalize the binding affinity and the inhibition mechanism.     

The gibberellin-induced, cysteine-rich protein GIP2 from Petunia hybrida exhibits in planta antioxidant activity

Numerous GAST-like genes have been identified in various plant species. All code for small proteins with a conserved C-terminal region in which 12 cysteines are located in exactly the same positions. We have previously identified five gibberellin (GA)-induced GAST1-like genes in petunia, GIP1-5. GIP2 is expressed in elongating zones, and its suppression in transgenic petunia plants inhibits stem elongation, suggesting a role for the protein in GA-induced cell growth. However, nothing is known about the biochemical activity of GIP2 or any other GAST-like protein. As all contain putative catalytic disulfide bonds (putative redox-active cysteines), we speculated that they might be involved in redox regulation. Expression analysis of GIP2, GIP4 and GIP5 revealed that they are induced by H(2)O(2). To study whether GIP2 modulates H(2)O(2) levels, we generated transgenic petunia plants expressing GIP2 under the regulation of the ubiquitous CaMV 35S promoter. The transgene reduced H(2)O(2) levels in leaves following wounding. It also reduced the levels of H(2)O(2) in guard cells following osmotic stress and ABA treatments, leading to the suppression of stomatal closure. In addition, the transgene promoted stem and corolla elongation. As reactive oxygen species (ROS) are involved in cell elongation, we suggest that GIP2 affects growth by regulating the levels of ROS. As all known GAST-like proteins contain putative redox-active cysteines, they may all act as antioxidants.