17 research outputs found
A toolbox of stable integration vectors in the fission yeast Schizosaccharomyces pombe
Schizosaccharomyces pombe is a widely used model organism to study many aspects of eukaryotic cell physiology. Its popularity as an experimental system partially stems from the ease of genetic manipulations, where the innate homology-targeted repair is exploited to precisely edit the genome. While vectors to incorporate exogenous sequences into the chromosomes are available, most are poorly characterized. Here we show that commonly used fission yeast vectors, which upon integration produce repetitive genomic regions, yield unstable genomic loci. We overcome this problem by designing a new series of Stable Integration Vectors (SIV) that target four different prototrophy genes. SIV produce non-repetitive, stable genomic loci and integrate predominantly as single copy. Additionally, we develop a set of complementary auxotrophic alleles that preclude false-positive integration events. We expand the vector series to include antibiotic resistance markers, promoters, fluorescent tags and terminators, and build a highly modular toolbox to introduce heterologous sequences. Finally, as proof of concept, we generate a large set of ready-to-use, fluorescent probes to mark organelles and cellular processes with a wide range of applications in fission yeast research
Tethering of SCF<sup>Dia2</sup> to the replisome promotes efficient ubiquitylation and disassembly of the CMG helicase
SummaryDisassembly of the Cdc45-MCM-GINS (CMG) DNA helicase, which unwinds the parental DNA duplex at eukaryotic replication forks, is the key regulated step during replication termination but is poorly understood [1, 2]. In budding yeast, the F-box protein Dia2 drives ubiquitylation of the CMG helicase at the end of replication, leading to a disassembly pathway that requires the Cdc48 segregase [3]. The substrate-binding domain of Dia2 comprises leucine-rich repeats, but Dia2 also has a TPR domain at its amino terminus that interacts with the Ctf4 and Mrc1 subunits of the replisome progression complex [4, 5], which assembles around the CMG helicase at replication forks [6]. Previous studies suggested two disparate roles for the TPR domain of Dia2, either mediating replisome-specific degradation of Mrc1 and Ctf4 [4] or else tethering SCFDia2 (SCF [Skp1/cullin/F-box protein]) to the replisome to increase its local concentration at replication forks [5]. Here, we show that SCFDia2 does not mediate replisome-specific degradation of Mrc1 and Ctf4, either during normal S phase or in response to replication stress. Instead, the tethering of SCFDia2 to the replisome progression complex increases the efficiency of ubiquitylation of the Mcm7 subunit of CMG, both in vitro and in vivo. Correspondingly, loss of tethering reduces the efficiency of CMG disassembly in vivo and is synthetic lethal in combination with a disassembly-defective allele of CDC48. Residual ubiquitylation of Mcm7 in dia2-ΔTPR cells is still CMG specific, highlighting the complex regulation of the final stages of chromosome replication, about which much still remains to be learned
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
Hof1 and Rvs167 Have Redundant Roles in Actomyosin Ring Function during Cytokinesis in Budding Yeast
Hof1 and Rvs167 Have Redundant Roles in Actomyosin Ring Function during Cytokinesis in Budding Yeast
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.Funding: The authors are grateful to Cancer Research United Kingdom who funded this work. ASO joined the University of Cantabria as a recipient of a Ramón y Cajal contract (call 2010) and now receives funding from the Cantabria lnternational Campus and via grant BFU2011-23193 from the Spanish "Ministerio de Economía y Competitividad" (co-funded by the European Regional Development Fund). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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
Inn1 can still be recruited to the bud-neck in the absence of the SH3 domains of Rvs167 and Hof1.
<p>(<b>A</b>) The indicated strains were released from G1-arrest at 24°C and allowed to progress through the cell cycle. The proportion of binucleate cells was monitored in parallel with recruitment of Inn1 to the bud-neck. (<b>B</b>) Examples of cells with Inn1-GFP rings at the bud-neck are shown for the 90’ time-point in (A). The scale-bars indicate 2 µm.</p
Actin ring assembly still occurs following inactivation of Arp2/3.
<p>(<b>A</b>) The indicated strains were arrested in G2-M phase with nocodazole at 24°C, before expression of <i>GAL-UBR1</i> and incubation at 37°C for 60’ to inactivate Hof1-td and Arp2-2. Cells were then released at 37°C into fresh medium lacking nocodazole, and processed as described above for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057846#pone-0057846-g005" target="_blank">Figure 5</a>. (<b>B</b>) Images of cells with actin rings (marked by arrows), from the 30-minute timepoint in the experiment in (A). The scale bars correspond to 2 µm.</p