Inositol Pyrophosphate-Controlled Kinetochore Architecture and Mitotic Entry in S. pombe

Inositol pyrophosphates (IPPs) comprise a specific class of signaling molecules that regulate central biological processes in eukaryotes. The conserved Vip1/PPIP5K family controls intracellular IP8 levels, the highest phosphorylated form of IPPs present in yeasts, as it has both inositol kinase and pyrophosphatase activities. Previous studies have shown that the fission yeast S. pombe Vip1/PPIP5K family member Asp1 impacts chromosome transmission fidelity via the modulation of spindle function. We now demonstrate that an IP8 analogue is targeted by endogenous Asp1 and that cellular IP8 is subject to cell cycle control. Mitotic entry requires Asp1 kinase function and IP8 levels are increased at the G2/M transition. In addition, the kinetochore, the conductor of chromosome segregation that is assembled on chromosomes is modulated by IP8. Members of the yeast CCAN kinetochore-subcomplex such as Mal2/CENP-O localize to the kinetochore depending on the intracellular IP8-level: higher than wild-type IP8 levels reduce Mal2 kinetochore targeting, while a reduction in IP8 has the opposite effect. As our perturbations of the inositol polyphosphate and IPP pathways demonstrate that kinetochore architecture depends solely on IP8 and not on other IPPs, we conclude that chromosome transmission fidelity is controlled by IP8 via an interplay between entry into mitosis, kinetochore architecture, and spindle dynamics.Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)Medical Research Council (MRC)Peer Reviewe

The Asp1 pyrophosphatase from S. pombe hosts a [2Fe-2S]2+ cluster in vivo

AbstractThe Schizosaccharomyces pombe Asp1 protein is a bifunctional kinase/pyrophosphatase that belongs to the highly conserved eukaryotic diphosphoinositol pentakisphosphate kinase PPIP5K/Vip1 family. The N-terminal Asp1 kinase domain generates specific high-energy inositol pyrophosphate (IPP) molecules, which are hydrolyzed by the C-terminal Asp1 pyrophosphatase domain (Asp1365−920). Thus, Asp1 activities regulate the intracellular level of a specific class of IPP molecules, which control a wide number of biological processes ranging from cell morphogenesis to chromosome transmission. Recently, it was shown that chemical reconstitution of Asp1371−920 leads to the formation of a [2Fe-2S] cluster; however, the biological relevance of the cofactor remained under debate. In this study, we provide evidence for the presence of the Fe–S cluster in Asp1365−920 inside the cell. However, we show that the Fe–S cluster does not influence Asp1 pyrophosphatase activity in vitro or in vivo. Characterization of the as-isolated protein by electronic absorption spectroscopy, mass spectrometry, and X-ray absorption spectroscopy is consistent with the presence of a [2Fe-2S]2+ cluster in the enzyme. Furthermore, we have identified the cysteine ligands of the cluster. Overall, our work reveals that Asp1 contains an Fe–S cluster in vivo that is not involved in its pyrophosphatase activity.</jats:p

The PPIP5K Family Member Asp1 Controls Inorganic Polyphosphate Metabolism in S. pombe

Inorganic polyphosphate (polyP) which is ubiquitously present in both prokaryotic and eukaryotic cells, consists of up to hundreds of orthophosphate residues linked by phosphoanhydride bonds. The biological role of this polymer is manifold and diverse and in fungi ranges from cell cycle control, phosphate homeostasis and virulence to post-translational protein modification. Control of polyP metabolism has been studied extensively in the budding yeast Saccharomyces cerevisiae. In this yeast, a specific class of inositol pyrophosphates (IPPs), named IP7, made by the IP6K family member Kcs1 regulate polyP synthesis by associating with the SPX domains of the vacuolar transporter chaperone (VTC) complex. To assess if this type of regulation was evolutionarily conserved, we determined the elements regulating polyP generation in the distantly related fission yeast Schizosaccharomyces pombe. Here, the VTC machinery is also essential for polyP generation. However, and in contrast to S. cerevisiae, a different IPP class generated by the bifunctional PPIP5K family member Asp1 control polyP metabolism. The analysis of Asp1 variant S. pombe strains revealed that cellular polyP levels directly correlate with Asp1-made IP8 levels, demonstrating a dose-dependent regulation. Thus, while the mechanism of polyP synthesis in yeasts is conserved, the IPP player regulating polyP metabolism is diverse

A Functional Yeast-Based Screen Identifies the Host Microtubule Cytoskeleton as a Target of Numerous <i>Chlamydia pneumoniae</i> Proteins

Bacterial pathogens have evolved intricate ways to manipulate the host to support infection. Here, we systematically assessed the importance of the microtubule cytoskeleton for infection by Chlamydiae, which are obligate intracellular bacteria that are of great importance for human health. The elimination of microtubules in human HEp-2 cells prior to C. pneumoniae infection profoundly attenuated the infection efficiency, demonstrating the need for microtubules for the early infection processes. To identify microtubule-modulating C. pneumoniae proteins, a screen in the model yeast Schizosaccharomyces pombe was performed. Unexpectedly, among 116 selected chlamydial proteins, more than 10%, namely, 13 proteins, massively altered the yeast interphase microtubule cytoskeleton. With two exceptions, these proteins were predicted to be inclusion membrane proteins. As proof of principle, we selected the conserved CPn0443 protein, which caused massive microtubule instability in yeast, for further analysis. CPn0443 bound and bundled microtubules in vitro and co-localized partially with microtubules in vivo in yeast and human cells. Furthermore, CPn0443-transfected U2OS cells had a significantly reduced infection rate by C. pneumoniae EBs. Thus, our yeast screen identified numerous proteins encoded using the highly reduced C. pneumoniae genome that modulated microtubule dynamics. Hijacking of the host microtubule cytoskeleton must be a vital part of chlamydial infection

CPn0572, the C. pneumoniae ortholog of TarP, reorganizes the actin cytoskeleton via a newly identified F-actin binding domain and recruitment of vinculin.

Chlamydia pneumoniae is one of the two major species of the Chlamydiaceae family that have a profound effect on human health. C. pneumoniae is linked to a number of severe acute and chronic diseases of the upper and lower respiratory tract including pneumonia, asthma, bronchitis and infection by the pathogen might play a role in lung cancer. Following adhesion, Chlamydiae secrete effector proteins into the host cytoplasm that modulate the actin cytoskeleton facilitating internalization and infection. Members of the conserved TarP protein family comprise such effector proteins that polymerize actin, and in the case of the C. trachomatis TarP protein, has been shown to play a critical role in pathogenesis. In a previous study, we demonstrated that, upon bacterial invasion, the C. pneumoniae TarP family member CPn0572 is secreted into the host cytoplasm and recruits and associates with actin via an actin-binding domain conserved in TarP proteins. We have now extended our analysis of CPn0572 and found that the CPn0572 actin binding and modulating capability is more complex. With the help of the fission yeast system, a second actin modulating domain was identified independent of the actin binding domain. Microscopic analysis of HEp-2 cells expressing different CPn0572 deletion variants mapped this domain to the C-terminal part of the protein as CPn0572536-755 binds F-actin in vitro and colocalizes with aberrantly thickened actin cables in vivo. Finally, microscopic and bioinformatic analysis revealed the existence of a vinculin binding sequence in CPn0572. Our findings contribute to the understanding of the function of the TarP family and underscore the existence of several actin binding domains and a vinculin binding site for host actin modulation

S. pombe CLASP needs dynein, not EB1 or CLIP170, to induce microtubule instability and slows polymerization rates at cell tips in a dynein-dependent manner

The Schizosaccharomyces pombe CLIP170-associated protein (CLASP) Peg1 was identified in a screen for mutants with spindle formation defects and a screen for molecules that antagonized EB1 function. The conditional peg1.1 mutant enabled us to identify key features of Peg1 function. First, Peg1 was required to form a spindle and astral microtubules, yet destabilized interphase microtubules. Second, Peg1 was required to slow the polymerization rate of interphase microtubules that establish end-on contact with the cortex at cell tips. Third, Peg1 antagonized the action of S. pombe CLIP170 (Tip1) and EB1 (Mal3). Fourth, although Peg1 resembled higher eukaryotic CLASPs by physically associating with both Mal3 and Tip1, neither Tip1 nor Mal3 was required for Peg1 to destabilize interphase microtubules or for it to associate with microtubules. Conversely, neither Mal3 nor Tip1 required Peg1 to associate with microtubules or cell tips. Consistently, while mal3.Δ and tip1.Δ disrupted linear growth, corrupting peg1 (+) did not. Fifth, peg1.1 phenotypes resembled those arising from deletion of the single heavy or both light chains of fission yeast dynein. Furthermore, all interphase phenotypes arising from peg1 (+) manipulation relied on dynein function. Thus, the impact of S. pombe CLASP on interphase microtubule behavior is more closely aligned to dynein than EB1 or CLIP170

The Fission Yeast Kinetochore Component Spc7 Associates with the EB1 Family Member Mal3 and Is Required for Kinetochore–Spindle Association

A critical aspect of mitosis is the interaction of the kinetochore with spindle microtubules. Fission yeast Mal3 is a member of the EB1 family of microtubule plus-end binding proteins, which have been implicated in this process. However, the Mal3 interaction partner at the kinetochore had not been identified. Here, we show that the mal3 mutant phenotype can be suppressed by the presence of extra Spc7, an essential kinetochore protein associated with the central centromere region. Mal3 and Spc7 interact physically as both proteins can be coimmunoprecipitated. Overexpression of a Spc7 variant severely compromises kinetochore–microtubule interaction, indicating that the Spc7 protein plays a role in this process. Spc7 function seems to be conserved because, Spc105, a Saccharomyces cerevisiae homolog of Spc7, identified by mass spectrometry as a component of the conserved Ndc80 complex, can rescue mal3 mutant strains