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

The Vip1 Inositol Polyphosphate Kinase Family Regulates Polarized Growth and Modulates the Microtubule Cytoskeleton in Fungi

<div><p>Microtubules (MTs) are pivotal for numerous eukaryotic processes ranging from cellular morphogenesis, chromosome segregation to intracellular transport. Execution of these tasks requires intricate regulation of MT dynamics. Here, we identify a new regulator of the <i>Schizosaccharomyces pombe</i> MT cytoskeleton: Asp1, a member of the highly conserved Vip1 inositol polyphosphate kinase family. Inositol pyrophosphates generated by Asp1 modulate MT dynamic parameters independent of the central +TIP EB1 and in a dose-dependent and cellular-context-dependent manner. Importantly, our analysis of the <i>in vitro</i> kinase activities of various <i>S. pombe</i> Asp1 variants demonstrated that the C-terminal phosphatase-like domain of the dual domain Vip1 protein negatively affects the inositol pyrophosphate output of the N-terminal kinase domain. These data suggest that the former domain has phosphatase activity. Remarkably, Vip1 regulation of the MT cytoskeleton is a conserved feature, as Vip1-like proteins of the filamentous ascomycete <i>Aspergillus nidulans</i> and the distantly related pathogenic basidiomycete <i>Ustilago maydis</i> also affect the MT cytoskeleton in these organisms. Consistent with the role of interphase MTs in growth zone selection/maintenance, all 3 fungal systems show aspects of aberrant cell morphogenesis. Thus, for the first time we have identified a conserved biological process for inositol pyrophosphates.</p></div

Asp1 MT regulation functions independently of Mal3.

<p>(A) Serial dilution patch test (10⁴–10¹ cells) of the indicated strains grown for 5 days at 25°C on YE5S without (−) or with (+) TBZ. (B) Serial dilution patch test (10⁴–10¹ cells) of the indicated strains grown for 5 days at 25°C on YE5S without (−) or with (+) MBC. (C) Serial dilution patch test of the <i>mal3</i>Δ transformants grown under plasmid selective conditions at 25°C for 5 or 9 days without (−) or with (+) TBZ, respectively. (D) Serial dilution patch tests (10⁵–10¹ cells) of the indicated strains grown at 25°C on MM without (−) or with (+) TBZ. (E) Photomicrographs of living wild-type, <i>mal3</i>Δ and <i>mal3</i>Δ <i>asp1^H397A</i> cells grown at 30°C expressing <i>nmt81::gfp-atb2</i>⁺. Bar, 5 µm. (F) MT relative fluorescent intensity (<i>mal3</i>Δ strain, 1+/−0.28, n = 26; <i>mal3</i>Δ <i>asp1^H397A</i> strain, 1.25+/−0.46, n = 16; arbitrary units; *, <i>P</i><0.05 as determined using Welch-Test). (G) Movement of outmost outbound Mal3-GFP comets (see diagram). Speed of comets (nm/sec): wild-type, 56±30, n = 93; <i>asp1^H397A</i>, 56±31.6, n = 69; <i>asp1^D333A</i>, 83.7±43,8, n = 75. * p<0.0005 for <i>asp1^D333A</i> versus wild-type (Welch-test).</p

Function of the Asp1 phosphatase-like domain.

<p>(A) Diagrammatic representation of the Asp1 protein with the conserved phosphatase signature motif (motif: RH(GNA)XR-HD in Asp1 RHADR-HI)(top) and the Asp1 variants used. Top: (B) Generation of IP₇ by GST-Asp1 with varying amounts (2,4,8 µg) of Asp1^365-920. Enzymatic reaction was carried out as for 1C. −, component not present; +, component present. (C) Serial dilution patch tests (10⁴–10¹ cells) of the <i>asp1</i>Δ (deletion) strain expressing the indicated <i>asp1</i> variants via the <i>nmt1⁺</i> promoter. This promoter is repressed in the presence of thiamine and de-repressed in its absence. Cells were grown for 6 days at 25°C on plasmid selective minimal medium without (−) or with (+) TBZ. (D) Serial dilution patch tests (10⁵–10¹ cells) of the <i>asp1</i>Δ strain expressing either <i>asp1^1-364</i>, <i>asp1^365-920</i> or <i>asp1^1-364</i> and <i>asp1^365-920</i>. Cells were grown for 7 days at 25°C on plasmid selective minimal medium without (−) or with (+) TBZ.</p

Loss of UmAsp1 causes defects in filamentous growth.

<p>(A) Edges of colonies of the indicated AB33 derived strains grown on charcoal plates. Aerial hyphae are emanating from the colony. (B) Photomicrographs (DIC) of the indicated strains grown for 8 hrs under filament inducing conditions. Wild-type and UmAsp1-GFP filaments form characteristic empty sections at the basal pole. White arrow: growth zone; white star: yeast cell (bar, 10 µm). (C) Bar diagram showing percentage of filaments exhibiting normal or disturbed growth. Bars show the mean of three independent experiments with n>100 cells (error bar, SEM; ** p = 0.0108). (D) Examples of UmAsp1-GFP yeast cells and filaments (8 hours post induction) (bar, 10 µm). (E) Bar chart showing mean average fluorescence intensity of UmAsp1-GFP in yeast and hyphae (yeast, n = 10 cells and hyphae, n = 7 cells; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004586#pgen.1004586.s011" target="_blank">Figure S11</a> for example photomicrographs). Error bars indicate standard deviation (*** p<0.001 unpaired t-test). (F) Western blot analysis of protein extracts of strain AB33 Umasp1-GFP after induction (0–8 hrs) of filamentous growth. Tub1 served as a loading control (hpi, hours post induction).</p

Asp1 kinase function affects MT organization.

<p>(A) Live cell images of the indicated strains expressing <i>nmt81::gfp-atb2⁺</i>. The same imaging and image-processing conditions were used for all strains. Bar, 5 µm. (B) Percentage of MTs depolymerising at a cell end or at the lateral cortex/in the cytoplasm. Wild-type: n = 102, <i>asp1^H397A</i>: n = 218, <i>asp1^D333A</i>: n = 166, <i>asp1</i>Δ: n = 131. ** P<0.005 for <i>asp1^D333A</i> or <i>asp1</i>Δ compared to wild-type as determined using χ²-test. (C) MT pausing time (sec) at cell ends in the indicated strains. Overall MT pausing time of these strains is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004586#pgen-1004586-t001" target="_blank">table 1</a>. We arbitrarily defined the 4 categories to show the variability within this system. Wild-type: n = 100, <i>asp1^H397A</i>: n = 67, <i>asp1^D333A</i>: n = 75. (D) MT pausing time (sec) at cell ends in the <i>asp1^H397A</i> strain transformed with a vector control or expressing pasp1^1-364. Overall MT pausing time of these strains is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004586#pgen-1004586-t001" target="_blank">table 1</a>. Cells were grown in plasmid-selective minimal medium. <i>asp1^H397A</i>+vector: n = 105, <i>asp1^H397A</i>+p<i>asp1^1-364</i>: n = 110. pasp1^1-364 denotes plasmid-borne expression of Asp1^1-364 via the <i>nmt1⁺</i> promoter under promoter de-repressing conditions. (E) Live cell images of the <i>nmt81</i>::<i>gfp</i>-<i>atb2</i>⁺ expressing <i>asp1^H397A</i> strain transformed with the vector control or expressing pasp1^1-364. Images shown are 10 sec intervals. Asterisks (*) denote MTs touching the cell end. Bar, 5 µm.</p

Strains used in this study.

<p>Strains used in this study.</p

Model for the regulation of Asp1 kinase function by the C-terminal phosphatase domain.

<p>(A) <i>In vitro</i> IP₇ output of wild-type Asp1 (top), Asp1^H397A (middle) and Asp1 plus Asp1^365-920 (phosphatase domain only). ++++ - +, high to low IP₇ output. (B) Two possible modes of action are shown. (I) The phosphatase domain could modulate the function of the kinase domain directly leading to reduced inositol pyrophosphate generation. (II) The phosphatase domain has enzymatic activity using the inositol pyrophosphate generated by the kinase domain as a substrate. IPP = inositol pyrophosphate. (C) MT stability correlates directly with intracellular inositol pyrophosphate levels.</p