Fission yeast Dma1 requires RING domain dimerization for its ubiquitin ligase activity and mitotic checkpoint function

In fission yeast (Schizosaccharomyces pombe), the E3 ubiquitin ligase Dma1 delays cytokinesis if chromosomes are not properly attached to the mitotic spindle.Dma1contains a C-terminal RING domain, and we have found that the Dma1 RING domain forms a stable homodimer. Although the RING domain is required for dimerization, residues in the C-terminal tail are also required to help form or stabilize the dimeric structure because mutation of specific residues in this region disrupts Dma1 dimerization. Further analyses showed that Dma1 dimerization is required for proper localization at spindle pole bodies and the cell division site, E3 ligase activity, and mitotic checkpoint function. Thus, Dma1 forms an obligate dimer via its RING domain, which is essential for efficient transfer of ubiquitin to its substrate(s). This study further supports the mechanistic paradigm that many RING E3 ligases function as RING dimers. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc

Oligomerization but Not Membrane Bending Underlies the Function of Certain F-BAR Proteins in Cell Motility and Cytokinesis

SummaryF-BAR proteins function in diverse cellular processes by linking membranes to the actin cytoskeleton. Through oligomerization, multiple F-BAR domains can bend membranes into tubules, though the physiological importance of F-BAR-to-F-BAR assemblies is not yet known. Here, we investigate the F-BAR domain of the essential cytokinetic scaffold, Schizosaccharomyces pombe Cdc15, during cytokinesis. Challenging a widely held view that membrane deformation is a fundamental property of F-BARs, we report that the Cdc15 F-BAR binds, but does not deform, membranes in vivo or in vitro, and six human F-BAR domains—including those from Fer and RhoGAP4—share this property. Nevertheless, tip-to-tip interactions between F-BAR dimers are critical for Cdc15 oligomerization and high-avidity membrane binding, stabilization of contractile ring components at the medial cortex, and the fidelity of cytokinesis. F-BAR oligomerization is also critical for Fer and RhoGAP4 physiological function, demonstrating its broad importance to F-BAR proteins that function without membrane bending

Spindle assembly in the absence of a RanGTP gradient requires localized CPC activity

Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Current Biology 19 (2009): 1210-1215, doi:10.1016/j.cub.2009.05.061.During animal cell division, a gradient of GTP-bound Ran is generated around mitotic chromatin. It is generally accepted that this RanGTP gradient is essential for organizing the spindle since it locally activates critical spindle assembly factors. Here, we show in Xenopus egg extract, where the gradient is best characterized, that spindles can assemble in the absence of a RanGTP gradient. Gradient-free spindle assembly occurred around sperm nuclei but not around chromatin-coated beads and required the chromosomal passenger complex (CPC). Artificial enrichment of CPC activity within hybrid bead arrays containing both immobilized chromatin and the CPC supported local microtubule assembly even in the absence of a RanGTP gradient. We conclude that RanGTP and the CPC constitute the two major molecular signals that spatially promote microtubule polymerization around chromatin. Furthermore, we hypothesize that the two signals mainly originate from discreet physical sites on the chromosomes to localize microtubule assembly around chromatin: a RanGTP signal from any chromatin, and a CPC-dependent signal predominantly generated from centromeric chromatin.This work was supported by the American Cancer Society (grant PF0711401 to T.J. Maresca), the National Cancer Institute (grant CA078048-09 to T.J. Mitchison) and the National Institutes of Health (grant F32GM080049 to J.C. Gatlin and grant GM24364 to E.D. Salmon)

Analysis of Buoyancy-Driven Ventilation of Hydrogen from Buildings: Preprint

When hydrogen gas is used or stored within a building, as with a hydrogen-powered vehicle parked in a residential garage, any leakage of unignited H2 will mix with indoor air and may form a flammable mixture. One approach to safety engineering relies on buoyancy-driven, passive ventilation of H2 from the building through vents to the outside

The Tubulation Activity of a Fission Yeast F-BAR Protein Is Dispensable for Its Function in Cytokinesis

F-BAR proteins link cellular membranes to the actin cytoskeleton in many biological processes. Here we investigated the function of the Schizosaccharomyces pombe Imp2 F-BAR domain in cytokinesis and find that it is critical for Imp2\u27s role in contractile ring constriction and disassembly. To understand mechanistically how the F-BAR domain functions, we determined its structure, elucidated how it interacts with membranes, and identified an interaction between dimers that allows helical oligomerization and membrane tubulation. Using mutations that block either membrane binding or tubulation, we find that membrane binding is required for Imp2\u27s cytokinetic function but that oligomerization and tubulation, activities often deemed central to F-BAR protein function, are dispensable. Accordingly, F-BARs that do not have the capacity to tubulate membranes functionally substitute for the Imp2 F-BAR, establishing that its major role is as a cell-cycle-regulated bridge between the membrane and Imp2 protein partners, rather than as a driver of membrane curvature

Analysis of Buoyancy-Driven Ventilation of Hydrogen from Buildings

The scope of work for this project includes safe building design, vehicle leak in residential garage, continual slow leak, passive, buoyancy-driven ventilation (versus mechanical), and steady-state concentration of hydrogen versus vent size

The Prp19 U-box crystal structure suggests a common dimeric architecture for a class of oligomeric E3 ubiquitin ligases

Prp19 is an essential splicing factor and a member of the U-box family of E3 ubiquitin ligases. Prp19 forms a tetramer via a central coiled-coil domain. Here, we show the U-box domain of Prp19 exists as a dimer within the context of the Prp19 tetramer. A high-resolution structure of the homodimeric state of the Prp19 U-box was determined by X-ray crystallography. Mutation of the U-box dimer interface abrogates U-box dimer formation and is lethal in vivo. The structure of the U-box dimer enables construction of a complete model of Prp19 providing insights into how the tetrameric protein functions as an E3 ligase. Finally, comparison of the Prp19 U-box homodimer with the heterodimeric complex of BRCA1/BARD1 RING-finger domains uncovers a common architecture for a family of oligomeric U-box and RING-finger E3 ubiquitin ligases, which has mechanistic implications for E3 ligase-mediated polyubiquitination and E4 polyubiquitin ligases. © 2006 American Chemical Society

Systematic Two-Hybrid and Comparative Proteomic Analyses Reveal Novel Yeast Pre-mRNA Splicing Factors Connected to Prp19

Prp19 is the founding member of the NineTeen Complex, or NTC, which is a spliceosomal subcomplex essential for spliceosome activation. To define Prp19 connectivity and dynamic protein interactions within the spliceosome, we systematically queried the Saccharomyces cerevisiae proteome for Prp19 WD40 domain interaction partners by two-hybrid analysis. We report that in addition to S. cerevisiae Cwc2, the splicing factor Prp17 binds directly to the Prp19 WD40 domain in a 1∶1 ratio. Prp17 binds simultaneously with Cwc2 indicating that it is part of the core NTC complex. We also find that the previously uncharacterized protein Urn1 (Dre4 in Schizosaccharomyces pombe) directly interacts with Prp19, and that Dre4 is conditionally required for pre-mRNA splicing in S. pombe. S. pombe Dre4 and S. cerevisiae Urn1 co-purify U2, U5, and U6 snRNAs and multiple splicing factors, and dre4Δ and urn1Δ strains display numerous negative genetic interactions with known splicing mutants. The S. pombe Prp19-containing Dre4 complex co-purifies three previously uncharacterized proteins that participate in pre-mRNA splicing, likely before spliceosome activation. Our multi-faceted approach has revealed new low abundance splicing factors connected to NTC function, provides evidence for distinct Prp19 containing complexes, and underscores the role of the Prp19 WD40 domain as a splicing scaffold

A Natural Framework for Solar and 17 keV Neutrinos

Motivated by recent experimental claims for the existence of a 17 keV neutrino and by the solar neutrino problem, we construct a class of models which contain in their low-energy spectrum a single light sterile neutrino and one or more Nambu-Goldstone bosons. In these models the required pattern of breaking of lepton-number symmetry takes place near the electroweak scale and all mass heirarchies are technically natural. The models are compatible with all cosmological and astrophysical constraints, and can solve the solar neutrino problem via either the MSW effect or vacuum oscillations. The deficit in atmospheric muon neutrinos seen in the Kamiokande and IMB detectors can also be explained in these models.Comment: 23 page