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Genetics, Vol. 175, 1213-1227, March 2007, Copyright © 2007
doi:10.1534/genetics.106.069252
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Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637
1 Corresponding author: Department of Molecular Genetics and Cell Biology, University of Chicago, 920 E. 58th St., Chicago, IL 60637.
E-mail: re-esposito{at}uchicago.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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occur independently, demonstrating that Spo1 acts at distinct steps. Loss of Spo1 is suppressed by high-copy glycosylphosphatidylinositol (GPI) proteins, dependent on sequence, timing, and strength of induction in meiosis. Since phosphatidylinositol (PI) serves as both an anchor component and a lipase substrate, we hypothesized that GPI-protein expression might substitute for Spo1 by decreasing levels of its potential substrates, PI and phosphatidylinositol phosphates (PIPs). Partial spo1 complementation by PLB3 (encoding a unique PLB capable of cleaving PI) and relatively strong Spo1 binding to PI(4)P derivatives (via a novel N-terminal lysine-rich fragment essential for Spo1 function) are consistent with this view. Epistasis of SPO1 mutations to those in SPO14 (encoding a PLD involved in signaling) and physical interaction of Spo1 with Spo23, a protein regulating PI synthesis required for wild-type sporulation, further support this notion. Taken together these findings implicate PI and/or PIPs in Spo1 function and suggest the existence of a novel Spo1-dependent meiosis-specific signaling pathway required for progression of MI, MII, and spore formation via regulation of the SPB.
We previously provided evidence that SPO1, a gene implicated in meiotic SPB morphogenesis, potentially functions in coordinating the divisions with gamete development (TEVZADZE et al. 2000). SPO1 is required at three stages of meiosis where proper SPB morphogenesis is essential: SPB duplication at MI and MII and at spore formation. It is predicted to encode a phospholipase B (PLB) homolog (TEVZADZE et al. 1996) induced early in meiosis and expressed through ascus formation, consistent with its requirement for proper SPB function at successive steps of development. A conserved serine within the lipase active site [essential for PLB biochemical activity (SHARP et al. 1994)] is required for its meiotic function (TEVZADZE et al. 2000). Unlike the other known budding yeast PLB enzymes, Plb1, Plb2, Plb3 (LEE et al. 1994; FYRST et al. 1999; MERKEL et al. 1999), and Nte1 (ZACCHEO et al. 2004), Spo1 is the only one that is meiosis specific.
In initial genetic studies we found that the spo1 sporulation defect is partially suppressed by high-copy CWP1 (TEVZADZE et al. 2000). This gene encodes a cell wall protein with a C-terminal segment characteristic of glycosylphosphatidylinositol (GPI)-anchored proteins (SHIMOI et al. 1995). GPIs are inositol-containing glycolipids, which covalently attach these proteins to the cell wall or external surface of the plasma membrane in mammals, yeast, and protozoa (IKEZAWA 2002; MAYOR and RIEZMAN 2004). Some phospholipases, including Plb1 and Plb2 (LEE et al. 1994; FYRST et al. 1999; MERKEL et al. 1999), but not Spo1, are anchored this way. The structure of the GPI anchor is well conserved in all eukaryotes. Here we report spo1 suppression by two other GPI proteins, Spo19 and Cwp2, dependent on level and timing of expression, and provide new evidence supporting the idea that the putative Spo1 lipase likely acts on phosphatidylinositol (PI) (or its phosphorylated derivatives) in a novel meiosis-specific signaling pathway coordinating successive stages of meiosis.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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and KC8 were employed for propagation and maintenance of plasmid DNA and BL21(DE3) for bacterial expression of yeast proteins. The S. cerevisiae strains used in this study are listed in Table 1.
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, forming >60% spores in the W303 background (TEVZADZE et al. 2000). pGT118 and pGT119 contain the SPO1 ORF tagged with six copies of the myc epitope at the C terminus immediately before the STOP codon cloned into pRS426 (CHRISTIANSON et al. 1992) and pRS306, a URA3-marked integrative plasmid (SIKORSKI and HIETER 1989), respectively. pGT119 was linearized by StuI to direct integration into the ura3-1 locus. The tagged construct complements spo1 at levels comparable to the untagged locus (>50% asci). pS25, carrying a 1.0-kb SalI internal fragment of SPO14 cloned into pRS306, was used to create a SPO14 gene disruption/deletion marked with URA3 (HONIGBERG et al. 1992). pGT34, used to construct a TRP1-marked deletion of SPO1, and pGT43, used to make a URA3-marked deletion of CWP1, were described previously (TEVZADZE et al. 2000).
ER targeting and Spo1 localization plasmids:
YIplac128-T and YIp-lac204/TKC-GFP-HDEL were generously provided by B. S. Glick (University of Chicago). YIplac128-T, an integrative plasmid carrying LEU2, contains a strong constitutive promoter from TPI1, which encodes triose-phosphate isomerase. pJP19 and pJP22 contain an untagged SPO1 ORF from pGT106 and the SPO1-6MYC ORF from pGT118, respectively, cloned into the SacI–BamHI sites of YIplac128-T under the control of the TPI1 promoter (the SacI sites were engineered immediately upstream of the SPO1 ATG). These plasmids were linearized by ClaI for integration into leu2-3,114. The YIp-lac204/TKC-GFP-HDEL plasmid, marked by TRP1, contains a GFP insert. The GFP coding sequence was modified by adding the N-terminal 135-bp sequence from the KAR2 ORF (for targeting to the ER lumen) and the C-terminal sequence encoding HDEL, a tetrapeptide that ensures retention of GFP in the ER lumen (ROSSANESE et al. 2001). This plasmid was linearized with EcoRV for integration into TRP1.
Mutated SPO1 promoter plasmids:
The "urs-SPO1" allele contains multiple mutations disrupting two potential overlapping URS1 core sequences (GGCGGC), one of which starts at +268 and the other at +271 (see Figure 2A, RESULTS). The mutations were introduced at positions +268, +269, +271, +272, +274, +275, and +276 (G–A transitions) and +270 and +273 (C–T transitions). This allele was cloned into the high-copy plasmid pRS426 and into the integrating vector pRS306 to create pGT62 and pGT64, respectively. The "uasH-SPO1" allele also contains multiple mutations in the UASH sequence (227GCGTGTTGAAAG238) at positions +227 (G–C), +229 (G–T), +236 (A–T), +237 (A–C), and +238 (G–T). This allele was also cloned into pRS426 and pRS306 to construct plasmids pGT63 and pGT65, respectively. Mutations in both URS1 and UASH cause a loss of spo1 complementation.
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Sporulation landmarks:
Sporulation of yeast cells was carried out as reported in KLAPHOLZ et al. (1985). Briefly, cells inoculated into YPA liquid medium at 
5 x 104 cells/ml were grown to a density of 1 x 107 cells/ml, washed twice with distilled water, and resuspended in 600 ml of SPII medium (supplemented with 75 µg/ml amino acids required for growth) in 4-liter flasks at 4–5 x 107 cells/ml. Sporulation cultures were incubated with vigorous aeration (250 rpm) in a New Brunswick rotary shaker at 30°. Under these conditions, the wild-type diploid strain produces >75% asci within 60 hr after transfer to sporulation medium. Recombination assays, visualization of yeast nuclei by 4',6-diamidino-2-phenylindole (DAPI) staining, fluorescence assays for dityrosine accumulation in yeast spore walls (BRIZA et al. 1986), immunofluorescence staining of formaldehyde-fixed yeast cells, and preparation of thin sections for electron microscopy were performed as described elsewhere, with modifications reported earlier (TEVZADZE et al. 2000).
DNA, RNA, and protein analysis:
DNA assays:
Isolation of genomic DNA for Southern blot analysis was performed as described (HOFFMAN and WINSTON 1987). Transformation of yeast utilized the one-step (CHEN et al. 1992; JOHNSTON 1994) or high-efficiency lithium acetate protocols (GIETZ and WOODS 1994; GIETZ and SCHIESTL 1995). RbCl-mediated E. coli transformation of DH5 was performed as in ANO and SHODA (1992) with the following modifications in media and buffer composition. Briefly, cells were grown in 100 ml 
-broth (2% Trypton, 0.5% yeast extract, 0.4% MgSO4, and 10 mM KCl) to OD550 = 0.48, incubated on ice for 10 min, pelleted, and resuspended in 30 ml cold TJB1 (100 mM RbCl, 50 mM MnCl2, 30 mM KAc, 10 mM CaCl2, and 15% glycerol pH 5.8). The suspension was incubated on ice for 5 min, pelleted, and resuspended in 4 ml cold TJB2 (10 mM MOPS, 10 mM RbCl, 75 mM CaCl2, 15% glycerol pH 7.0). DNA was added to 100 µl cells, and the mixture was kept on ice for 30 min and transferred to 42° for 45 sec. Finally, 400 µl SOC was added and incubated with aeration at 37° for 1 hr.
RNA analysis:
Approximately 4 x 108 yeast cells were collected by centrifugation and quick frozen by submerging in liquid nitrogen. Total RNA was prepared by the glass bead/phenol protocol (AUSUBEL et al. 1991). Transcript levels were quantified using 15–20 µg RNA per sample by S1 nuclease protection assays and normalized against DED1 mRNA, as described (TEVZADZE et al. 2000). Gels were exposed to a PhosphorImager screen and quantitated using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). All RNA probes were synthesized in SP6, T3, or T7 in vitro transcription reactions (AUSUBEL et al. 1991). Probes were made as follows: DED1, 0.2-kb fragment generated from AflII digestion of pGT31 (TEVZADZE et al. 2000) or a 0.45-kb fragment generated from XhoI digestion of the same plasmid; SPO1, a 0.8-kb PvuII fragment from pGT29 (TEVZADZE et al. 2000), which contains 0.6 kb of the SPO1 transcript; and SPO14, a 0.4-kb BglII–SalI fragment from pS25 (HONIGBERG et al. 1992).
Protein assays:
Western analyses and immunoprecipitation techniques were performed by standard procedures. Endoglycosidase H (EndoH) was obtained from ProZyme and enzymatic digestion performed as suggested by the manufacturer. After enzymatic treatment, Spo1–6myc samples were run on a 7% PAGE gel (optimal for detecting the difference between the untreated and the EndoH-treated samples).
Expression of Spo1 fragments fused to protein G was performed in BL21(DE3). Cells were grown from OD600 = 0.1–0.5 in LB plus 100 mg/ml ampicillin (for selection of the fusion plasmid) and 40 mg/ml kanamycin (for selection of the plasmid expressing T7 polymerase required for expression of the fusion). Expression was induced by addition of IPTG (Sigma, St. Louis) to a final concentration of 1 mM. After 4 hr, cells were harvested, lysed, and fusion proteins purified as described (DAMES et al. 2005). Purified protein concentrations were determined by comparison to 1- to 10-µg standards on PAGE gels, and samples were diluted to 0.5 µg/ml for lipid-binding assays.
Lipid-binding assays:
Lipid-binding protein overlay assays were performed using phosphatidylinositol phosphate (PIP) strips or PIP arrays provided by Echelon, according to a protocol provided by the manufacturer (www.echelon-inc.com). PIP strips are nitrocellulose filters containing 100-pmol spots of 15 various lipids: PI and seven of its phosphorylated derivatives, phosphatidic acid (PA), lysophosphatidic acid, lysophosphocholine, phosphatidylethanolamine, phosphatidylcholine, sphingosine-1-phosphate, and phosphatidylserine. Manufacturer information indicates that binding is reproducible for all but PA. PIP arrays that allow a quantitative estimate of lipid-binding strength contain a range of seven different concentrations (100, 50, 25, 12.5, 6.25, 3.13, and 1.56 pmol) for each of the following 8 lipids (Figure 7, A and B): PtdIns, PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2, and PtdIns(3,4,5)P3. Binding to strips or arrays was assayed by ECL (Amersham, Arlington Heights, IL). Signal intensities for individual lipid spots were calculated using the NIH Image 1.62 software and normalized to the blank (PIP strips) or "no binding" (PI, arrays) spots.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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mutants arrest at three stages of meiotic development. The major arrest occurs at MI SPB duplication, followed by two minor arrest points at MII and spore formation (TEVZADZE et al. 2000). These findings are consistent with the finding that spo1-1 ts, (which sporulates normally at 23°, a permissive temperature, and exhibits a null phenotype at 34°, a restrictive one) has a temperature-sensitive period extending throughout most of sporulation (MOENS 1974). They do not, however, distinguish whether the minor arrest points are a downstream consequence of the initial defect (e.g., abnormal SPBs formed at MI) or reflect a functional requirement for SPO1 at multiple stages. To determine which of these occurs, the ts allele was used in a temperature-shift experiment to monitor whether the later arrest points can occur independently of the first one. If SPO1 is required only at MI, then bypassing this arrest at a permissive temperature should allow the mutant to avoid subsequent arrest when switched to a restrictive temperature (i.e., after execution of the MI function). Conversely, if Spo1 is required at several stages during sporulation, then increased numbers of arrested cells should accumulate at the second and third arrest points after a shift to restrictive conditions. Figure 1A demonstrates that the latter is the case. Shifting cells to 34° after 8 hr of incubation at 23° (which allows completion of MI) results in a significant increase in binucleate cells that fail to progress to MII (compare >40% binucleate cells for the temperature-shifted strains to 20% for those sporulated at 34°). Accumulation of an increased number of tetranucleate cells likely represents an asynchronous fraction that bypassed the first two arrest points at MI and MII at 23° by the time of the shift, but not the third one at spore formation (compare >20% MII cells after temperature shift to <10% in strains maintained at 34°; Figure 1, A and B). Similarly to strains incubated constantly at 34°, the cultures shifted to 34° at 8 hr yielded no asci even after another 90 hr of incubation in sporulation medium (the control constantly at 23° produces 75% asci by 96 hr).
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The Spo1 transcript and protein accumulate in a similar pattern throughout meiosis:
The regulatory region of SPO1 has an unusual and complex organization. In contrast to most yeast genes, the cis-acting regulatory sites (two URS1 elements and a UASH site) all reside within the SPO1 ORF. At least two other yeast genes have URS1 transcriptional regulatory sites within their coding regions [SPO11, a meiosis-specific gene (ATCHESON 1991), and LPD1, required for amino acid catabolism (ZAMAN et al. 1992)]. In addition, genomewide studies of meiosis-specific transcripts that undergo splicing (DAVIS et al. 2000) indicate that SPO1 has a larger ORF than initially reported (TEVZADZE et al. 1996), due to a previously undetected intron. Both the UASH site and the two URS1 sites, which overlap in their core sequence (GGCGG), are located in the second exon (Figure 2A).
SPO1 transcription depends upon these cis-acting elements (Figure 2B) and other factors that regulate early meiotic transcription (Figure 2, C and D). One of them, Ume6, a zinc finger protein that binds directly to the URS1 core (STRICH et al. 1994; ANDERSON et al. 1995), interacts with a component of histone deacetylase, Sin3/Ume4 (KADOSH and STRUHL 1997), to repress early genes during vegetative growth (STRICH et al. 1994; BOWDISH et al. 1995; STEBER and ESPOSITO 1995). Ume6 also interacts with Ime1 (RUBIN-BEJERANO et al. 1996), a key inducer of meiosis (KASSIR et al. 1988), to both relieve Sin3-mediated mitotic repression and induce meiotic transcription (WASHBURN and ESPOSITO 2001).
The data in Figure 2, B–D, show that SPO1, with its unusual internal promoter, is nonetheless regulated similarly to other early meiosis-specific genes (ATCHESON et al. 1987; BUCKINGHAM et al. 1990; VERSHON et al. 1992; PITTMAN et al. 1993; STRICH et al. 1994). For example, mutations in either the URS1 elements or in UME6 cause derepressed expression in vegetative cells as well as reduced meiotic induction (Figure 2, B and D) while mutations in UASH [recognized by the Abf1 transcription factor (PRINZ et al. 1995; GAILUS-DURNER et al. 1996)], dramatically affect meiotic induction and have only a slight, if any, defect in repression (Figure 2B). Finally, absence of the Sin3/Ume4 corepressor causes SPO1 derepression and a delay in meiotic induction (Figure 2C). Although Ime1 and Ume6 are both needed for SPO1 transcription, their own expression is unaffected by Spo1 (TEVZADZE et al. 2000).
Analysis of Spo1 protein expression, using a fully functional Spo1-6myc allele, indicates that the protein begins to accumulate at 3 hr and starts to decline at 15 hr as asci form (Figure 2, E and F). The pattern closely follows transcript accumulation, which spans the time when Spo1 is required at MI, MII, and spore formation on the basis of mutant phenotype analysis. In addition to the PLB active site, which is required for its meiotic function (TEVZADZE et al. 2000), Spo1 also contains another conserved motif (89SGGGYR94) defined as the "oxyanion hole," critical for lipase activity (D. SIX, personal communication; Figure 3). Intriguingly, the Spo1 oxyanion hole is encoded by the same sequence (265TCTGGCGGCGGGTACAGA282) that contains the two overlapping URS1 regulatory sites. Altering the URS1 sites and oxyanion hole by replacing 90Gly Gly Gly92 with Asp Asp Lys causes both derepressed expression and loss of spo1 complementation (monitored by light microscopy and dityrosine spore fluorescence). Since constitutive expression per se has no effect on Spo1 function in meiosis (SPO1 expressed from the TPI1 promoter shows full spo1 complementation; next section), we conclude that the null phenotype of the urs1 and uash spo1 mutants likely results from amino acid replacements in the oxyanion hole rather than changes in transcription. The need for both the active site as well as the oxyanion hole for Spo1 meiotic function provides additional support for the view that Spo1 acts in vivo as a meiosis-specific PLB. Furthermore, the maintenance of transcription regulatory sites in the coding region provides an unusual example of direct coupling between distinct systems controlling transcriptional regulation and protein function.
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High-copy GPI proteins partially suppress spo1:
Potential downstream targets, interactors and/or regulators of the likely Spo1 lipase were initially sought by screening for high-copy suppression of spo1. The first suppressor recovered, CWP1 (TEVZADZE et al. 2000), encodes a GPI-anchored cell wall protein (SHIMOI et al. 1995; TEVZADZE et al. 2000). More extensive studies below show that CWP1 acts at all three arrest points. Suppression more than doubles the level of MI and MII cells over the spo1 control and permits relatively efficient spore formation (20% asci for high-copy CWP1 compared to 60% for high-copy SPO1 and <0.1% for the empty vector in both null and ts mutants at 34°). Significantly, CWP1, which we previously showed is dispensable for sporulation (TEVZADZE et al. 2000), plays no apparent role in redundant Spo1-independent pathways during sporulation, since the formation of bi- and tetranucleate cells is identical in spo1 and spo1 cwp1 strains (data not shown).
Suppression screens using the spo1-1 ts (Ile103Phe) allele in this study identified a novel meiosis-specific gene (YPL130w) along with SPO1 and CWP1. This gene, encoding another GPI protein (CARO et al. 1997) required for sporulation, is designated here SPO19. Expression of the last 40 amino acid residues of Spo19 fused to a vegetative promoter suggests that the protein localizes to the cell wall, similar to Cwp1 (HAMADA et al. 1999). High-copy SPO19 has no effect on spo1, but exhibits a low reproducible suppression of the ts allele (compare 3–5% asci for high-copy SPO19 to <0.1% for the ts mutant alone at the restrictive temperature, 34°). The sporulation behavior and meiosis-specific mid/late transcription of this gene suggest that it suppresses the ts phenotype only at minor arrest points (MII and/or spore formation). Interestingly, a complete deletion of SPO19 itself has a ts sporulation defect. Null mutants sporulate normally at 23° but fail to form asci at 30° and 34° in liquid media (Figure 5A), with the ts profile shifting slightly on solid media where asci form at both 23° and 30° but not 34°. The sporulation behavior of spo19 implies the existence of another Spo19-independent but redundant ts Spo function that acts at 23° but not at 30° and/or 34°.
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Promoter strength and time of expression are critical for high-copy GPI-protein suppression:
The recovery of two high-copy suppressors encoding GPI proteins, one of which is dispensable for meiosis (Cwp1), raises several questions: Is the GPI attachment sequence itself sufficient for suppression? Why do other GPI proteins (e.g., Cwp2) not appear to suppress spo1? Why are the stages of suppression by Cwp1 and Spo19 different? First, the role of the GPI sequence in suppression was examined using constructs containing either (1) a truncated CWP1 allele without the GPI-attachment sequence or (2) a high-copy plasmid expressing only the GPI-attachment sequence from a CWP1 or GAL1 promoter (containing the N-terminal ER-targeting signal sequence from CWP1 inserted between the promoter and the anchor sequence for proper localization). Both constructs fail to suppress spo1. On the basis of these findings we conclude that both the GPI-attachment sequence and protein-specific sequence information are critical for suppression.
Next, promoter-swapping experiments were done to test whether the level and time of GPI-protein expression are important for suppression. For example, while both CWP1 and SPO19 are upregulated in midmeiosis, the continuous presence of the CWP1 transcript in vegetative cells and early sporulation may account for why this gene suppresses all three arrest points, in contrast to SPO19, which is specifically transcribed in meiosis over a shorter interval (Figure 6A; see also PRIMIG et al. 2000). The lack of suppression by constitutively transcribed CWP2 might similarly be related to the absence of any meiotic regulation. Strikingly, promoter-swapping analysis shows that when the CWP2 ORF is driven by the CWP1 promoter it now suppresses spo1 at significantly higher levels (6% asci for CWP1pr-CWP2 to <0.1% for CWP2; Figure 6B). Notably, a small but reproducible increase in ascus formation is also seen in isogenic spo1-1 ts strains bearing high-copy SPO19 driven from the native or CWP1 promoter (5% vs. 7%, respectively). Even higher suppression levels (from 17% asci to 26%, respectively) are seen in a related spo1-1 ts strain [used in the cloning of SPO1 (TEVZADZE et al. 2000)]. More efficient suppression (and complementation by SPO1) was also detected for other constructs in this strain (25% asci for CWP1, 1% for CWP2, 18% for CWP1pr-CWP2, and 48% for high-copy SPO1). Taken together, these results demonstrate that the efficiency of suppression by specific GPI proteins indeed depends not only on the presence of the GPI anchor and specific protein sequence, but also on expression levels, time of meiotic induction, and background modifiers.
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These ideas were first genetically tested employing another locus, PLB3, known to alter PI levels in vivo. Among the three previously characterized PLB enzymes (expressed constitutively during growth and sporulation), Plb3 is the only one utilizing PI as a substrate (MERKEL et al. 1999). The results show that PLB3 expressed from the SPO1 promoter (including internal URS1 and UAS sites, see Figure 2A) partially complements spo1, resulting in a >30-fold increase in spore production (3% asci compared to <0.1% for empty vector or vector containing only the SPO1 promoter and regulatory sequences). The failure to complement at higher levels may result from localization differences and/or the specific activity of the protein. For example, Plb3, a GPI protein targeted to the ER, subsequently anchors to the plasma membrane (CARO et al. 1997; MERKEL et al. 1999), while Spo1, not a GPI protein, localizes to the ER lumen and then to the periplasmic space (this study).
The absence of PLB3 alone leads to a mild but significant meiotic defect detected by reduced sporulation, reduced spo1 complementation by pSPO1, and a small but reproducible decrease in spo1 suppression (Figure 6C). These results are compatible with the idea that lack of Plb3 activity increases PI (or PIP) above a critical level so that neither Spo1 activity nor enhanced GPI-anchor synthesis (e.g., by high-copy CWP1) lowers it enough to permit wild-type sporulation. Strikingly, Spo1 and Plb3 are the only PLBs required for sporulation, with Spo1 being essential and Plb3 playing a minor role. These findings further strengthen the view that Spo1 acts as a meiosis-specific Plb3 and that PI- (or PIP)-specific phospholipases are important for meiotic progression.
At present we favor the model described above as the simplest explanation of the data. However, it should be emphasized that the role of Spo1 may be more complex (see DISCUSSION). In addition to functioning as a lipase (using PI and/or PIPs or other phospholipids as substrates), its activity on different substrates could be stimulated by PIPs and/or it could also act as a PIP-transfer protein in a manner similar to Spo14, a PLD involved in signaling known to have lipase activity (enhanced by PIP binding) as well as PIP transfer function (SCIORRA et al. 1999; RUDGE et al. 2001). These alternatives could also potentially reduce PIP levels with similar consequences for suppression and meiotic progression as suggested above. Furthermore, Spo1, like Plb3, may also cleave PS (MERKEL et al. 1999), as well as participate in a PI signaling network. To gain further insight into Spo1 function the following studies were thus undertaken.
Spo1 binds phosphatidylinositol (4)P mono- and polyphosphates via a lysine-rich motif at the N terminus:
The idea that Spo1 utilizes PI (or PIP) as its substrate (or that its activity is enhanced by PIPs) received support from an independent proteome array analysis demonstrating that Spo1 is among 30 proteins that bind PI(4)P and PI(4,5)P2 (ZHU et al. 2001). Below we pursue the lipid-binding behavior of Spo1 in more detail and in addition identify the domain essential for PIP binding. These studies utilized a soluble protein G–Spo1 fusion (lacking the 24 N-most terminal amino acids of Spo1) expressed in bacteria and were initially performed with lipid strips containing 15 different lipids spotted at 100-pmol concentrations and later with arrays with a wide range of PIP concentrations ("Echelon"). The strips (Figure 7A, left) show that Spo1 binds to PIPs (not unphosphorylated PI) and to PA (not Lyso-PA) and exhibits only very weak binding to PS. Thus, we conclude that both phosphorylation of the inositol ring and the presence of the sn-2 acyl chain are important for binding. The arrays (Figure 7B), which allow better discrimination of binding specificities, further demonstrate that Spo1 preferentially binds PIPs phosphorylated in the fourth position, including PI(3,4)P2 and PI(3,4,5)P3, which were previously detected only as weak binders in the study by ZHU et al. (2001).
Surprisingly, assays with purified protein fragments derived from various deletion derivatives indicated that an internal fragment (residues 78–144), containing the oxyanion hole (89SGGGYR94) and the lipase active site (117YIAGLSGG124), does not bind lipids at all. In contrast, a nearby upstream fragment lacking these two motifs (residues 24–67) retains lipid-binding properties (Figure 7A, center). This novel region of 44 amino acids at the N terminus of Spo contains a lysine cluster motif (47KKKKIKTKNK56) critical for both Spo1 lipid-binding properties and its meiotic function. A full-size protein lacking the cluster neither binds lipids nor complements spo1 (Figure 7, A and C), while a 5-kDa fragment containing the 47KKKKIK52 sequence (followed by 53RRIN*57, generated by PCR errors) binds at wild-type levels (not shown). Significantly, K47 and K48 are conserved in Spo1, Plb1, and Plb2, while only K48 is present in Plb3 as well (Figure 3). Recently, a mammalian protein kinase CK2, which contains a similar lysine stretch (KKKKIKR), was also found to bind PIPs likely by positively charged lysine residues electrostatically interacting with the negatively charged head group of phosphorylated inositol (KOROLCHUK et al. 2005).
SPO14, critical for SPB modification during meiosis, acts in the same pathway as SPO1:
Prior meiotic analysis of SPO14 demonstrates that it is required for MII spindle development and spore formation (HONIGBERG et al. 1992). It encodes a phospholipase PLD that cleaves PC, stimulated by binding of PI(4,5)P2 to the protein (ROSE et al. 1995a,b; SCIORRA et al. 1999). Electron microscopy analysis in our laboratory (G. G. TEVZADZE, unpublished results) and recently reported elsewhere (NAKANISHI et al. 2006), shows that it is specifically needed for MII SPB modification and separation, as well as for initiation of the prospore membrane synthesis at SPBs. Interestingly, Spo14 is also required for Sec14-independent secretory pathways during vegetative growth (SREENIVAS et al. 1998) as ts mutants of SEC14, an essential gene encoding a PI transfer protein (XIE et al. 1998), yield significantly fewer revertants in a spo14 background. Several spo14 alleles were subsequently isolated that support Sec14-independent secretion, but not sporulation, identifying two distinct functions of the Spo14 PLD during vegetative growth and sporulation (RUDGE et al. 2001). Given Spo14's lipase activity, PIP-binding ability, and requirement for progression through two of the same stages of sporulation as Spo1 (MII and spore formation), we inquired whether it might interact directly or indirectly with Spo1.
First, to test whether Spo1 might also be involved in secretory pathways we assayed sec14 revertants in strains expressing Spo1 during mitosis (under the constitutive promoter TPI1, see MATERIALS AND METHODS) and found no effect (1.2 ± 0.6/106 colonies for sec14-1 TPI-SPO1 compared to 1.0 ± 0.6/106 for sec14-1 or sec14-1 spo1). On the basis of this criterion Spo1 does not appear to play a similar role in secretion when expressed in vegetative cells. In contrast, genetic epistasis analysis suggests that SPO1 and SPO14 act in the same pathway/network during meiosis. The double mutant has a phenotype identical to spo1 with most cells being blocked at the mononucleate stage (Figure 8, C and D). Comparison of transcription profiles indicates that while SPO1 is expressed well before SPO14, epistasis does not stem from altered regulation of SPO14 as its expression is unchanged in spo1 and thus independent of SPO1 (Figure 8, A and B).
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60 kDa protein (Ybr-250w), was identified as a Spo1 interactor in high-throughput two-hybrid analysis (UETZ et al. 2000). Like SPO19 and SPO14, it is induced in midmeiosis (PRIMIG et al. 2000). We confirmed that it physically interacts with Spo1 in meiosis by co-immunoprecipitation of FLAG-tagged Ybr250w with Spo1-6myc (Figure 9). Further analysis of a null revealed that it has a moderate sporulation defect (59% vs. 74% asci at 30° and 57% vs. 75% asci at 34° for isogenic ybr250w and wild-type strains sporulating in liquid media for >90 hr). Given its sporulation phenotype we have designated this gene SPO23. Interestingly, SPO23/YBR250w was recently identified as one of the loci affecting expression of PIS1 (GARDOCKI et al. 2005), a gene encoding PI synthase essential for PI biosynthesis (NIKAWA et al. 1987), implying that it is involved in regulation of lipid signaling. The precise role of Spo23 in meiosis [e.g., whether it acts as a cofactor for Spo1 lipase activity or facilitates interaction with potential substrate PI(P) molecules] remains to be determined.
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DISCUSSION |
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A model for the role of Spo1 in meiosis:
The above observations were integrated into a model for the role of the Spo1 lipase during meiosis (Figure 10). The model proposes that Spo1 functions as a meiosis-specific PLB, cleaving PI(4)Ps in a novel signal pathway regulating the timing and coordination of the MI and MII nuclear divisions and spore formation. Accordingly, decreased concentrations of PI(4)Ps below a certain threshold and increased levels of Lyso-products and fatty acids (known products of PLB and PLA2 activities) would serve as negative and positive signals, respectively, for meiotic progression. These signals may in turn directly or indirectly regulate the activities of the SPB in nucleating spindle and prospore membrane formation. The genetic and biochemical basis for this model and its implications are discussed further below.
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Evidence that Spo1 acts as a meiotic PI-specific PLB:
Three lines of evidence support the view that Spo1 lipase activity is required in meiosis. First, Spo1 displays significant similarity to known PLB enzymes and contains two conserved motifs required for lipase activity essential for Spo1 function. Second, Plb3, a previously characterized PI-specific PLB, partially replaces Spo1 when expressed at high copy. None of the other PLBs (which act on phosphatidylethanolamine, PE, and phosphatidylcholine, PC), have the capacity to substitute for Spo1. Third, Spo1 strongly and specifically binds PI(4)P mono- and polyphosphates, well-known signaling molecules. While binding of a lipid species does not define it as a substrate (e.g., it may be a cofactor), the failure of Spo1 to bind to other conventional PLB substrates (unphosphorylated PI, PC, PE, etc.) makes it more likely that PI(4)Ps and especially PI(4,5)P2 are cleaved by Spo1. Final proof awaits direct in vitro assays of Spo1 activity, which have been hampered thus far by insufficient recovery of soluble protein. While two other strong binders, PI(3,4)P2 and PI(3,4,5)P3, have not been detected in budding yeast during vegetative growth (DE CAMILLI et al. 1996; VANHAESEBROECK et al. 1997), it remains to be determined whether they accumulate during sporulation. Furthermore, a recent report suggests that PI(3,4,5)P3 may exist in Schizosaccharomyces pombe (COOKE 2004; DIVECHA and HALSTEAD 2004; MITRA et al. 2004).
Finally, a 5-kDa N-terminal fragment was identified as necessary and sufficient for Spo1 binding to PIPs. It does not have any similarity to known lipid-binding domains, e.g., PH, PX, FYVE (LEMMON 2003), or to the PI(4,5)P2-binding region of Spo14 (SCIORRA et al. 1999). It thus represents a novel PI(4)P-specific binding domain essential for Spo1 function. Within this region, a lysine cluster 47KKKKIK52 is sufficient for binding.
The evidence that PI levels coordinate and control meiotic progression:
The notion that threshold levels of PI (and its derivatives) regulate meiosis and spore development provides a plausible explanation for high-copy suppression of spo1 mutants by GPI proteins. These utilize PI in anchor synthesis, potentially reducing levels of PI (and PI(4)Ps) to partially substitute for the loss of Spo1 activity. One protein, Cwp1, apparently has no other role in meiosis and is important only when Spo1 is absent. In contrast, the other, Spo19, appears to be integrated into the normal regulatory network controlling MII and spore formation. As high-copy expression of Spo19 specifically suppresses a ts allele, it may directly interact with Spo1 to facilitate its function at these times. Since not all GPI-anchor proteins suppress spo1 and the sequence of the protein as well as timing and level of expression is critical, their localization to the cell wall in conjunction with the presence of Spo1 in the periplasmic space may be significant. Accordingly, temporal as well as spatial reduction in concentration of PI and/or PIPs may generate crucial signals.
The model described above predicts that the absence or the enhanced activity of PI(4)P-specific lipid kinases or phosphatases could also potentially substitute for Spo1 activity. On the other hand, mutant phenotypes for these functions likely will have pleiotropic effects leading not only to meiotic defects but also to impaired growth. This may be why high-copy and mutant spo1 suppressor screens did not recover such alleles. Future studies to examine the effect of mutations in specific enzymes [e.g., the Mss4 kinase required for PI(4,5)P2 synthesis and the Inp51 phosphatase, which reduces PI(4,5)P2) levels] on meiotic stages where Spo1 is required may nevertheless provide useful insights.
The evidence that SPO1 acts in a PIP-signaling pathway regulating successive stages of meiosis:
SPO1 acts at several transition points where SPBs undergo morphogenic changes during meiosis. These include SPB duplication and subsequent spindle development at MI and MII and initiation of prospore membrane synthesis at modified outer plaques. Studies of genetic interaction between SPO1 and SPO14, a gene encoding phospholipase D (ROSE et al. 1995a,b), suggest that these genes act in the same pathway with SPO1 first required at MI (TEVZADZE et al. 2000) and SPO14 needed later for MII, SPB modification, and spore formation (HONIGBERG et al. 1992; RUDGE and ENGEBRECHT 1999; RUDGE et al. 2004; RIEDEL et al. 2005; NAKANISHI et al. 2006). Recent identification and analysis of additional components of a SPO1-dependent pathway, e.g., Spo23, a physical interactor with Spo1 (this study), and Spo73, another PIP-binding protein acting downstream of the Spo1 lipase (TEVZADZE et al. 2003; our unpublished results), lend further credence to the notion of a signaling network controlling meiotic progression.
Additional evidence in favor of this idea is provided by identification of several other signaling genes required for meiotic progression and spore formation. These include MAP and CDK kinases such as: (1) Smk1, a MAPK kinase required for spore morphogenesis (KRISAK et al. 1994; WAGNER et al. 1999); (2) Sps1, a serine–threonine kinase required for proper localization of enzymes involved in the prospore membrane synthesis (FRIESEN et al. 1994; IWAMOTO et al. 2005); (3) Cak1, a CDK kinase required for meiotic DNA synthesis and expression of meiosis-specific loci, e.g., IME1 (KALDIS et al. 1998; SCHABER et al. 2002; MCDONALD et al. 2005); and (4) Mps1, a dual-specificity kinase required for mitotic and meiotic SPB duplication as well as later stages of meiosis (STRAIGHT et al. 2000). How all these functions interface with one another in this relatively simple model system is not yet fully understood. Analysis of their genetic and biochemical interaction with genes defined in the Spo1 pathway described in this study should further uncover conserved signaling mechanisms coordinating the nuclear divisions with gamete maturation in yeast and higher eukaryotes.
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ACKNOWLEDGEMENTS |
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LITERATURE CITED |
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