Telomere anchoring at the nuclear periphery requires the budding yeast Sad1-UNC-84 domain protein Mps3

Positioning of telomeres at the nuclear periphery can have dramatic effects on gene expression by establishment of heritable, transcriptionally repressive subdomains. However, little is known about the integral membrane proteins that mediate telomere tethering at the nuclear envelope. Here, we find a previously unrecognized function for the Saccharomyces cerevisiae Sad1-UNC-84 domain protein Mps3 in regulating telomere positioning in mitotic cells. Our data demonstrate that the nucleoplasmic N-terminal acidic domain of Mps3 is not essential for viability. However, this acidic domain is necessary and sufficient for telomere tethering during S phase and the silencing of reporter constructs integrated at telomeres. We show that this is caused by the role of the Mps3 acidic domain in binding and localization of the silent information regulator protein Sir4 to the nuclear periphery. Thus, Mps3 functions as an integral membrane anchor for telomeres and is a novel nuclear receptor for the Sir4 pathway of telomere tethering and gene inactivation

The Polo-related kinase Cdc5 activates and is destroyed by the mitotic cyclin destruction machinery in S. cerevisiae

AbstractBackground: Following chromosome segregation in anaphase, ubiquitin-dependent degradation of mitotic cyclins contributes to the exit from mitosis. A key step in this process is catalyzed by a ubiquitin–protein ligase known as the anaphase-promoting complex (APC), the regulation of which is poorly understood. The Polo-related protein kinase Cdc5 in Saccharomyces cerevisiae might encode a regulator of the APC, because cdc5 mutant cells arrest with a late mitotic phenotype similar to that observed in cells with defective cyclin destruction.Results: We investigated the role of Cdc5 in the regulation of mitotic cyclin degradation. In cdc5-1 mutant cells, we observed a defect in the destruction of cyclins and a reduction in the cyclin–ubiquitin ligase activity of the APC. Overexpression of CDC5 resulted in increased APC activity and mitotic cyclin destruction in asynchronous cells or in cells arrested in metaphase. CDC5 mutation or overexpression did not affect the degradation of the APC substrate Pds 1, which is normally degraded at the metaphase-to-anaphase transition. Cyclin-specific APC activity in cells overexpressing CDC5 was reduced in the absence of the APC regulatory proteins Hct 1 and Cdc20. In G1, Cdc5 itself was degraded by an APC-dependent and Hct1-dependent mechanism.Conclusions: We conclude that Cdc5 is a positive regulator of cyclin-specific APC activity in late mitosis. Degradation of Cdc5 in G1 might provide a feedback mechanism by which the APC destroys its activator at the onset of the next cell cycle

The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope

The spindle pole body (SPB) is the sole site of microtubule nucleation in Saccharomyces cerevisiae; yet, details of its assembly are poorly understood. Integral membrane proteins including Mps2 anchor the soluble core SPB in the nuclear envelope. Adjacent to the core SPB is a membrane-associated SPB substructure known as the half-bridge, where SPB duplication and microtubule nucleation during G1 occurs. We found that the half-bridge component Mps3 is the budding yeast member of the SUN protein family (Sad1-UNC-84 homology) and provide evidence that it interacts with the Mps2 C terminus to tether the half-bridge to the core SPB. Mutants in the Mps3 SUN domain or Mps2 C terminus have SPB duplication and karyogamy defects that are consistent with the aberrant half-bridge structures we observe cytologically. The interaction between the Mps3 SUN domain and Mps2 C terminus is the first biochemical link known to connect the half-bridge with the core SPB. Association with Mps3 also defines a novel function for Mps2 during SPB duplication

Cdc28/Cdk1 Regulates Spindle Pole Body Duplication through Phosphorylation of Spc42 and Mps1

AbstractDuplication of the Saccharomyces cerevisiae spindle pole body (SPB) once per cell cycle is essential for bipolar spindle formation and accurate chromosome segregation during mitosis. We have investigated the role that the major yeast cyclin-dependent kinase Cdc28/Cdk1 plays in assembly of a core SPB component, Spc42, to better understand how SPB duplication is coordinated with cell cycle progression. Cdc28 is required for SPB duplication and Spc42 assembly, and we found that Cdc28 directly phosphorylates Spc42 to promote its assembly into the SPB. The Mps1 kinase, previously shown to regulate Spc42 phosphorylation and assembly, is also a Cdc28 substrate, and Cdc28 phosphorylation of Mps1 is needed to maintain wild-type levels of Mps1 in cells. Analysis of nonphosphorylatable mutants in SPC42 and MPS1 indicates that direct Spc42 phosphorylation and indirect regulation of Spc42 through Mps1 are two overlapping pathways by which Cdc28 regulates Spc42 assembly and SPB duplication during the cell cycle

Histone H2BK123 monoubiquitination is the critical determinant for H3K4 and H3K79 trimethylation by COMPASS and Dot1

Histone H2B monoubiquitination by Rad6/Bre1 is required for the trimethylation of both histone H3K4 and H3K79 by COMPASS and Dot1 methyltransferases, respectively. The dependency of methylation at H3K4 and H3K79 on the monoubiquitination of H2BK123 was recently challenged, and extragenic mutations in the strain background used for previous studies or epitope-tagged proteins were suggested to be the sources of this discrepancy. In this study, we show that H3K4 and H3K79 methylation is solely dependent on H2B monoubiquitination regardless of any additional alteration to the H2B sequence or genome. Furthermore, we report that Y131, one of the yeast histone H2A/H2B shuffle strains widely used for the last decade in the field of chromatin and transcription biology, carries a wild-type copy of each of the HTA2 and HTB2 genes under the GAL1/10 promoter on chromosome II. Therefore, we generated the entire histone H2A and H2B alanine-scanning mutant strains in another background, which does not express wild-type histones

The SUN Protein Mps3 Is Required for Spindle Pole Body Insertion into the Nuclear Membrane and Nuclear Envelope Homeostasis

The budding yeast spindle pole body (SPB) is anchored in the nuclear envelope so that it can simultaneously nucleate both nuclear and cytoplasmic microtubules. During SPB duplication, the newly formed SPB is inserted into the nuclear membrane. The mechanism of SPB insertion is poorly understood but likely involves the action of integral membrane proteins to mediate changes in the nuclear envelope itself, such as fusion of the inner and outer nuclear membranes. Analysis of the functional domains of the budding yeast SUN protein and SPB component Mps3 revealed that most regions are not essential for growth or SPB duplication under wild-type conditions. However, a novel dominant allele in the P-loop region, MPS3-G186K, displays defects in multiple steps in SPB duplication, including SPB insertion, indicating a previously unknown role for Mps3 in this step of SPB assembly. Characterization of the MPS3-G186K mutant by electron microscopy revealed severe over-proliferation of the inner nuclear membrane, which could be rescued by altering the characteristics of the nuclear envelope using both chemical and genetic methods. Lipid profiling revealed that cells lacking MPS3 contain abnormal amounts of certain types of polar and neutral lipids, and deletion or mutation of MPS3 can suppress growth defects associated with inhibition of sterol biosynthesis, suggesting that Mps3 directly affects lipid homeostasis. Therefore, we propose that Mps3 facilitates insertion of SPBs in the nuclear membrane by modulating nuclear envelope composition

The Inhibition of Polo Kinase by Matrimony Maintains G2 Arrest in the Meiotic Cell Cycle

Many meiotic systems in female animals include a lengthy arrest in G2 that separates the end of pachytene from nuclear envelope breakdown (NEB). However, the mechanisms by which a meiotic cell can arrest for long periods of time (decades in human females) have remained a mystery. The Drosophila Matrimony (Mtrm) protein is expressed from the end of pachytene until the completion of meiosis I. Loss-of-function mtrm mutants result in precocious NEB. Coimmunoprecipitation experiments reveal that Mtrm physically interacts with Polo kinase (Polo) in vivo, and multidimensional protein identification technology mass spectrometry analysis reveals that Mtrm binds to Polo with an approximate stoichiometry of 1:1. Mutation of a Polo-Box Domain (PBD) binding site in Mtrm ablates the function of Mtrm and the physical interaction of Mtrm with Polo. The meiotic defects observed in mtrm/+ heterozygotes are fully suppressed by reducing the dose of polo+, demonstrating that Mtrm acts as an inhibitor of Polo. Mtrm acts as a negative regulator of Polo during the later stages of G2 arrest. Indeed, both the repression of Polo expression until stage 11 and the inactivation of newly synthesized Polo by Mtrm until stage 13 play critical roles in maintaining and properly terminating G2 arrest. Our data suggest a model in which the eventual activation of Cdc25 by an excess of Polo at stage 13 triggers NEB and entry into prometaphase

B-Cyclin/CDKs Regulate Mitotic Spindle Assembly by Phosphorylating Kinesins-5 in Budding Yeast

Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCFCdc4 ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that, in budding yeast, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APCCdh1). We have determined, however, that the Kip1 and Cin8 proteins are present at wild-type levels in the absence of Clb/Cdc28 kinase activity. Here, we show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28 and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's in vivo localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, we have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCFCdc4 target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, we propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors

To Make a Long Spindle Short: Nuclear Envelope Breakdown during Meiosis

In fission yeast, the nuclear envelope (NE) remains intact during mitosis and meiosis I but is compromised during meiosis II. In this issue of Cell Reports, Flor-Parra et al. (2018) demonstrate that this NE alteration regulates meiosis II spindle disassembly and the ploidy of meiotic products. : In fission yeast, the nuclear envelope (NE) remains intact during mitosis and meiosis I but is compromised during meiosis II. In this issue of Cell Reports, Flor-Parra et al. (2018) demonstrate that this NE alteration regulates meiosis II spindle disassembly and the ploidy of meiotic products