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

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

Cdk1 phosphorylation of the kinetochore protein Nsk1 prevents error-prone chromosome segregation

Phosphorylation of the kinetochore component Nsk1 by Cdk1 antagonizes its localization to and function at the kinetochore and spindle during early mitosis

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

Cryo-EM Structures of Centromeric Tri-nucleosomes Containing a Central CENP-A Nucleosome

The histone H3 variant CENP-A is a crucial epigenetic marker for centromere specification. CENP-A forms a characteristic nucleosome and dictates the higher-order configuration of centromeric chromatin. However, little is known about how the CENP-A nucleosome affects the architecture of centromeric chromatin. In this study, we reconstituted tri-nucleosomes mimicking a centromeric nucleosome arrangement containing the CENP-A nucleosome, and determined their 3D structures by cryoelectron microscopy. The H3-CENP-A-H3 tri-nucleosomes adopt an untwisted architecture, with an outward-facing linker DNA path between nucleosomes. This is distinct from the H3-H3-H3 tri-nucleosome architecture, with an inward-facing DNA path. Intriguingly, the untwisted architecture may allow the CENP-A nucleosome to be exposed to the solvent in the condensed chromatin model. These results provide a structural basis for understanding the 3D configuration of CENP-A-containing chromatin, and may explain how centromeric proteins can specifically target the CENP-A nucleosomes buried in robust amounts of H3 nucleosomes in centromeres

Biased Brownian motion as a mechanism to facilitate nanometer-scale exploration of the microtubule plus end by a kinesin-8

Kinesin-8s are plus-end–directed motors that negatively regulate microtubule (MT) length. Well-characterized members of this subfamily (Kip3, Kif18A) exhibit two important properties: (i) They are “ultraprocessive,” a feature enabled by a second MT-binding site that tethers the motors to a MT track, and (ii) they dissociate infrequently from the plus end. Together, these characteristics combined with their plus-end motility cause Kip3 and Kif18A to enrich preferentially at the plus ends of long MTs, promoting MT catastrophes or pausing. Kif18B, an understudied human kinesin-8, also limits MT growth during mitosis. In contrast to Kif18A and Kip3, localization of Kif18B to plus ends relies on binding to the plus-end tracking protein EB1, making the relationship between its potential plus-end–directed motility and plus-end accumulation unclear. Using single-molecule assays, we show that Kif18B is only modestly processive and that the motor switches frequently between directed and diffusive modes of motility. Diffusion is promoted by the tail domain, which also contains a second MT-binding site that decreases the off rate of the motor from the MT lattice. In cells, Kif18B concentrates at the extreme tip of a subset of MTs, superseding EB1. Our data demonstrate that kinesin-8 motors use diverse design principles to target MT plus ends, which likely target them to the plus ends of distinct MT subpopulations in the mitotic spindle.Samsung (Firm) (Scholarship