MINDY-1 is a member of an evolutionarily conserved and structurally distinct new family of Deubiquitinating enzymes

Deubiquitinating enzymes (DUBs) remove ubiquitin (Ub) from Ub-conjugated substrates to regulate the functional outcome of ubiquitylation. Here we report the discovery of a new family of DUBs, which we have named MINDY (motif interacting with Ub-containing novel DUB family). Found in all eukaryotes, MINDY-family DUBs are highly selective at cleaving K48-linked polyUb, a signal that targets proteins for degradation. We identify the catalytic activity to be encoded within a previously unannotated domain, the crystal structure of which reveals a distinct protein fold with no homology to any of the known DUBs. The crystal structure of MINDY-1 (also known as FAM63A) in complex with propargylated Ub reveals conformational changes that realign the active site for catalysis. MINDY-1 prefers cleaving long polyUb chains and works by trimming chains from the distal end. Collectively, our results reveal a new family of DUBs that may have specialized roles in regulating proteostasis

The Mitotic Exit Network and Cdc14 phosphatase initiate cytokinesis by counteracting CDK phosphorylations and blocking polarised growth

Polarisation of the actin cytoskeleton must cease during cytokinesis, to support efficient assembly and contraction of the actomyosin ring at the site of cell division, but the underlying mechanisms are still understood poorly in most species. In budding yeast, the Mitotic Exit Network (MEN) releases Cdc14 phosphatase from the nucleolus during anaphase, leading to the inactivation of mitotic forms of cyclin-dependent kinase (CDK) and the onset of septation, before G1-CDK can be reactivated and drive re-polarisation of the actin cytoskeleton to a new bud. Here, we show that premature inactivation of mitotic CDK, before release of Cdc14, allows G1-CDK to divert the actin cytoskeleton away from the actomyosin ring to a new site of polarised growth, thereby delaying progression through cytokinesis. Our data indicate that cells normally avoid this problem via the MEN-dependent release of Cdc14, which counteracts all classes of CDK-mediated phosphorylations during cytokinesis and blocks polarised growth. The dephosphorylation of CDK targets is therefore central to the mechanism by which the MEN and Cdc14 initiate cytokinesis and block polarised growth during late mitosis. © 2012 European Molecular Biology Organization | All Rights Reserved.We are grateful to Cancer Research UK for funding this work. ASD joined the University of Cantabria as a recipient of a Ramón y Cajal contract (call 2010) and now receives funding from the Cantabria International Campus and via grant BFU2011-23193 from the Spanish 'Ministerio de Economía y Competitividad' (co-funded by the European Regional Development Fund).Peer Reviewe

Hof1 and Rvs167 Have Redundant Roles in Actomyosin Ring Function during Cytokinesis in Budding Yeast

This is an open-access article distributed under the terms of the Creative Commons Attribution License.The Hof1 protein (Homologue of Fifteen) regulates formation of the primary septum during cytokinesis in the budding yeast Saccharomyces cerevisiae, whereas the orthologous Cdc15 protein in fission yeast regulates the actomyosin ring by using its F-BAR domain to recruit actin nucleators to the cleavage site. Here we show that budding yeast Hof1 also contributes to actin ring assembly in parallel with the Rvs167 protein. Simultaneous deletion of the HOF1 and RVS167 genes is lethal, and cells fail to assemble the actomyosin ring as they progress through mitosis. Although Hof1 and Rvs167 are not orthologues, they both share an analogous structure, with an F-BAR or BAR domain at the amino terminus, capable of inducing membrane curvature, and SH3 domains at the carboxyl terminus that bind to specific proline-rich targets. The SH3 domain of Rvs167 becomes essential for assembly of the actomyosin ring in cells lacking Hof1, suggesting that it helps to recruit a regulator of the actin cytoskeleton. This new function of Rvs167 appears to be independent of its known role as a regulator of the Arp2/3 actin nucleator, as actin ring assembly is not abolished by the simultaneous inactivation of Hof1 and Arp2/3. Instead we find that recruitment to the bud-neck of the Iqg1 actin regulator is defective in cells lacking Hof1 and Rvs167, though future studies will be needed to determine if this reflects a direct interaction between these factors. The redundant role of Hof1 in actin ring assembly suggests that the mechanism of actin ring assembly has been conserved to a greater extent across evolution than anticipated previously. © 2013 Nkosi et al.The authors are grateful to Cancer Research United Kingdom who funded this work. ASD joined the University of Cantabria as a recipient of a Ramón y Cajal contract (call 2010) and now receives funding from the Cantabria International Campus and via grant BFU2011-23193 from the Spanish "Ministerio de Economía y Competitividad" (co-funded by the European Regional Development Fund).Peer Reviewe

Recruitment of Inn1 to the bud-neck is defective in the complete absence of Hof1 and Rvs167.

<p>(<b>A</b>) The indicated strains were synchronised in G1 phase at 24°C, before expression of <i>GAL-UBR1</i> (Ubr1 is the E3 ligase for N-end rule pathway that mediates ubiquitylation of the heat inducible degron) and degradation of Hof1-td and Cyk3-td at 37°C. Cells were then released from G1 arrest and samples taken at the indicated times to determine the proportion of bi-nucleate cells (i) and the percentage of cells with rings or spots of Inn1 at the bud-neck (ii), as cells completed the cell cycle. (<b>B</b>) Images from the experiment described in (A). The Inn1-GFP rings in <i>hof1-td cyk3-td</i> were frequently less bright than those observed in control cells. The scale bars correspond to 2 µm.</p

Physical interactions between Rvs167 and Inn1.

<p>(<b>A</b>) Truncated alleles of Rvs167 and Inn1 were used to show that the region of Rvs167 after the BAR domain (Rvs167 241-482) can interact in a 2-hybrid assay with the Proline-rich region of Inn1 after its C2 domain (Inn1 135-409). (<b>B</b>) Scheme explaining how the indicated protein fragments were expressed in cultures of <i>E. coli</i> cells, and then mixed to allow the purification of protein complexes. After induction with IPTG, pairs of cultures were mixed as indicated in (D) below, and used to purify protein complexes between the induced proteins, via Strep-Tactin Superflow and Ni-NTA agarose resins (see Methods). (<b>C</b>) Immunoblots showing induction of the various protein fragments listed in (B). The tagged proteins were detected with anti-Streptag or anti-His antibodies. In each case, a non-specific band corresponding to an unknown <i>E. coli</i> protein is included to provide a loading control. (<b>D</b>) Inn1 135-409 can interact directly to form a stable complex with the SH3 domains of Hof1 and Cyk3, as well as with Rvs167 241-482. Pairs of <i>E. coli</i> cell cultures expressing the indicated protein fragments were mixed and used to purify putative protein complexes as shown in (B). The final purified fractions were analysed by SDS-PAGE and the gels were stained with colloidal Coomassie blue.</p

Recruitment of Iqg1 to the bud-neck is defective in cells lacking Hof1 and Rvs167.

<p>(<b>A</b>) Cells were processed as described above for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057846#pone-0057846-g004" target="_blank">Figure 4</a>, so that the recruitment of GFP-Iqg1 to the bud-neck (i) could be monitored as cells passed through mitosis (ii). (<b>B</b>) Images from the experiment described in (A). The scale bars correspond to 2 µm.</p