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Vol. 20, Issue 1, 134-145, January 1, 2009
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*Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794-5215; and Laboratory of Biological Chemistry, Graduate School of Agricultural and Life Science, The University of Tokyo, Tokyo 113-8657, Japan
Submitted June 18, 2008;
Revised October 6, 2008;
Accepted October 9, 2008
Monitoring Editor: Mark J. Solomon
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mutants are defective in prospore membrane closure. Ssp1, a protein found at the leading edge of the prospore membrane, is stabilized in ama1 mutants. Inactivation of a conditional form of Ssp1 can partially rescue the sporulation defect of the ama1 mutant, indicating that an essential function of Ama1 is to lead to the removal of Ssp1. Depletion of Cdc15 causes a defect in meiotic exit. We find that prospore membrane closure is also defective in Cdc15 and that this defect can be overcome by expression of a form of Ama1 in which multiple consensus cyclin-dependent kinase phosphorylation sites have been mutated. These results demonstrate that APCAma1 functions to coordinate the exit from meiosis II with cytokinesis. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Neiman, 2005). The process of spore formation is driven by a cell division in which the daughter cells are formed in the cytoplasm of the mother cell. As cells enter meiosis II, the cytoplasmic faces of the spindle pole bodies are modified so that they become centers of membrane nucleation (Moens, 1971; Moens and Rapport, 1971). Four membranes, termed prospore membranes, are formed, one at each spindle pole (Moens, 1971; Neiman, 1998). As haploid chromosome sets separate within the nucleus in meiosis II, each of the prospore membranes grows to engulf the region of the nucleus adjacent to it. Closure of a prospore membrane around a nascent haploid nucleus completes cell division and is equivalent to cytokinesis in mitotic growth. Once the prospore membrane has closed, the prospore then matures by the deposition of spore wall material into the luminal space between the two membranes derived from the prospore membrane (Lynn and Magee, 1970).
As the prospore membrane expands, its growth is guided in part by proteins found at the lip of the growing membrane, termed the leading edge protein coat (Moreno-Borchart et al., 2001). Three components of this coat are known: Don1, Ady3, and Ssp1 (Knop and Strasser, 2000; Moreno-Borchart et al., 2001; Nickas and Neiman, 2002). The function of Don1 is unknown, although it may be the most peripheral member of the complex, because it requires both Ady3 and Ssp1 for localization to the leading edge (Moreno-Borchart et al., 2001; Nickas and Neiman, 2002). Ady3 may function primarily in promoting mitochondrial segregation into the spore (Suda et al., 2007). The critical constituent of the leading edge complex is Ssp1. This protein is required for localization of both Ady3 and Don1 to the leading edge (Moreno-Borchart et al., 2001). Moreover, in the absence of SSP1 prospore membrane growth is abnormal and spore formation is blocked (Nag et al., 1997; Moreno-Borchart et al., 2001).
Ectopic overexpression of SSP1 in vegetative cells has been shown to block cell growth by interfering with the fusion of secretory vesicles to the plasma membrane (Maier et al., 2007). During sporulation Ssp1 is degraded around the time of prospore membrane closure and mutations that stabilize the protein inhibit sporulation (Maier et al., 2007). These results have led to the proposal that removal of Ssp1 from the leading edge regulates the timing of cytokinesis during sporulation (Maier et al., 2007).
The anaphase promoting complex (APC) is a multisubunit E3 ubiquitin ligase essential for progression through mitosis (Morgan, 1999). The activity of this complex is regulated by accessory subunits of the Cdc20/Fizzy family that direct it to specific substrates (Morgan, 1999). In vegetatively growing S. cerevisiae, Cdc20 and Cdh1 regulate APC activity (Visintin et al., 1997). Although both Cdc20 and Cdh1 direct the degradation of multiple, overlapping targets, each controls the degradation of a specific target essential for cell division: mitotic cyclins for Cdh1 and securin for Cdc20 (Thornton and Toczyski, 2003). During meiosis, Cdc20 is again important for controlling APC activity (Katis et al., 2004; Oelschlaegel et al., 2005). No meiotic role for Cdh1 has been described; however, a third family member, AMA1, is expressed specifically in meiotic cells (Chu et al., 1998; Cooper et al., 2000).
Deletion of AMA1 does not block meiosis, but rather the formation of spores; prospore membranes are formed, but the subsequent formation of spore walls is blocked (Coluccio et al., 2004). This result suggests that the critical target(s) of APCAma1 is a protein(s) whose degradation is required to allow spore wall assembly. The identity of these putative targets is unknown.
Ama1 is subject to regulation at several levels. Both transcriptional control and meiosis-specific splicing ensure that the protein is expressed only during sporulation (Chu et al., 1998; Cooper et al., 2000). Although Ama1 can associate with the APC and direct the degradation of securin both in vivo and in vitro, in a wild-type meiosis APCCdc20 is primarily responsible for securin degradation (Oelschlaegel et al., 2005). The activity of APCAma1 is held in check by the Mnd2 subunit of the APC and by the activity of the Clb-Cdc28 kinase (Oelschlaegel et al., 2005; Penkner et al., 2005). Failure to restrict APCAma1 leads to premature loss of cohesin and chromosome missegregation (Oelschlaegel et al., 2005; Penkner et al., 2005). Although APCAma1 may contribute to the turnover of Pds1 and cyclins during meiosis, the actions of Mnd2 and Clb-Cdc28 ensure that APCAma1 remains inactive until late meiosis II when Mnd2 dissociates from the APC and Clb-kinase activity decreases, consistent with the primary function of APCAma1 in postmeiotic cells (Dahmann and Futcher, 1995; Rabitsch et al., 2001; Coluccio et al., 2004; Oelschlaegel et al., 2005; Carlile and Amon, 2008).
Meiosis also differs from mitosis in the circuitry that regulates exit from the division. In mitotic cells, exit requires the activity of the Cdc14 protein (Wood and Hartwell, 1982; Taylor et al., 1997). Two separate pathways, termed FEAR and MEN, collaborate to regulate Cdc14 (Dumitrescu and Saunders, 2002). CDC14 is also necessary in meiosis, but regulation of Cdc14 in meiosis is largely or wholly mediated by the FEAR network (Marston et al., 2003; Kamieniecki et al., 2005). The MEN component CDC15 is required for sporulation, but this seems to be independent of CDC14 (Pablo-Hernando et al., 2007). In meiotic cells depleted of Cdc15, chromosome segregation proceeds normally as judged by 4,6-diamidino-2-phenylindole (DAPI) staining, but prospore membrane growth is abnormal. Also, spindle disassembly at the end of meiosis II is abnormal, and microtubules accumulate rather than disappear (Pablo-Hernando et al., 2007). This last result suggests that a late step in exit from meiosis is defective in this mutant.
The terminal phenotype of ama1 mutants led to the suggestion that AMA1 may be required to trigger spore wall assembly after the completion of meiosis (Coluccio et al., 2004). We report here that ama1 mutants are defective at a slightly earlier stage of spore formation, the closure of the prospore membrane. This defect in cytokinesis may account for the spore wall assembly defect in the mutant. The ama1 mutant stabilizes the leading edge protein complex so that the Ssp1 protein persists in postmeiotic cells and the ring structure of the complex remains intact. The stabilization of Ssp1 is likely directly responsible for the ama1 cytokinesis defect, because inactivation of a conditional allele of SSP1 can partially rescue the ama1 sporulation defect. A cdc15 mutant defective in exit from meiosis II also displays defects in prospore membrane closure. This closure defect can be suppressed by expression of a mutant form of Ama1 in which all the Cdc28 consensus phosphorylation sites have been mutated. These observations suggest that the primary function of APCAma1 is to coordinate exit from meiosis II with cytokinesis during spore formation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The strains used in this study are listed in Table 1. All strains are in the SK-1 background except for AN390 and the JSP strains, which are hybrids between SK-1 and the S288c background. ADY12 and ADY13 were constructed by polymerase chain reaction (PCR)-mediated knockout of AMA1 in AN117-4B and AN117-16D, respectively, by using pFa6A CgTRP1 as a template for PCR and oligonucleotides (oligos) F1AMA1 and R1AMA1 in AN117-4B and AN117-16D. ADY64 and ADY65 were constructed in the same manner except pFa6A MX6HIS3 (Longtine et al., 1998) was the PCR template. To create TC37 and TC38, oligos HT362 and HT87 were used to amplify the hemagglutinin (HA)-tagging cassette in pFA6a-3xHA-HisMX6 (Longtine et al., 1998), and the product was used to transform strain ANT117-4B and AN117-16D, respectively. All PCR-mediated integrations were confirmed by genomic PCR with appropriate primers. ADY183 and ADY184 were obtained as segregants from a cross of TC38 and ADY64. ADY183-AMA1 and ADY184-AMA1 are ADY183 and ADY184 transformed with EcoRV-digested pRS306AMA1. To construct strains ADY216, ADY217, ADY218, and ADY220 the degron cassette in plasmid pKL187PSSP1 was amplified using primers SSP1DegronF and SSP1DegronR and transformed into ADY12, ADY13, AN117-4B, and AN117-16D, respectively. The plasmid pKL142 (Sanchez-Diaz et al., 2004), carrying GAL promoter driven UBR1, was linearized by digestion with PmeI and integrated into strains ADY216, ADY217, ADY218, ADY220, AN117-4B, and AN117-16D to generate ADY221, ADY222, ADY223, ADY224, ADY229, and ADY231, respectively. To create ADY225, ADY226, ADY227, ADY228, ADY230, and ADY232, the plasmid p926 was linearized with NdeI and used to transform ADY221, ADY222, ADY223, ADY224, ADY229, and ADY231, respectively. ADY239 and ADY240 were created by transforming ADY12 and ADY13, with p926 followed by pKL142.
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), and mating of ama1::HIS3 TEF2::GFP::his5+ segregants from this cross produced JSP22. To generate the CLB2pr-CDC15 strains, the GFP collection strain carrying TEF2::GFP was crossed with AN117-4B, and a segregant from this cross, JSP62, was crossed to a CLB2pr-CDC15 haploid (Pablo-Hernando et al., 2007). JSP64 and JSP65 are segregants from this latter cross. Transformation of JSP64 and JSP65 with PstI linearized YIplac128-AMA1pr-AMA1 or YIplac128-AMA1pr-AMA1-m8 (Oelschlaegel et al., 2005) was used to generate the haploid parents for JSP99 and JSP104.
Plasmids
Plasmids used in this study are listed in Table 2. To construct pRS426-R20, the coding region of monomeric red fluorescent protein (mRFP) in a template pTi mRFP (Gao et al., 2005) was amplified using primers HNO941 and HNO942, the product was digested with XbaI and EcoRI and used to replace the GFP sequence in similarly digested pGFP-N-FUS-SPO20151-273 (Nakanishi et al., 2004). An EcoRI-XhoI fragment carrying the coding region of mRFP-SPO20151-273 was then cloned from this construct into pRS426-TEF (Mumberg et al., 1995). pKL187PSSP1 is a modified pKL187 (Sanchez-Diaz et al., 2004) containing the SSP1 promoter in place of the CUP1 promoter. A 700-base pair fragment carrying the SSP1 promoter was amplified form pRS314-SSP1-HA by using primers SSP1FPromoterMfe1 and SSP1RpromoterEcoR1. The PCR product was digested with Mfe1 and EcoR1 and ligated to the vector backbone of similarly digested pKL187. p926 (gift from A. Amon, Massachusetts Institute of Technology, Cambridge, MA) is pRS306-Pgpd1-GAL4.ER (Benjamin et al., 2003), an integrative plasmid containing a GAL4-endoplasmic reticulum (ER) fusion under the control of the GPD1 promoter. pRS306-AMA1pr-AMA1 was constructed as follows. To remove the sporulation-specific intron (base pairs 1184–1276), a 5' fragment of AMA1 open reading frame (ORF) was amplified with primers FAMA1EcoRI (+631) and RNdeI and blunt-end ligated into pBluescript at the EcoRV restriction enzyme site to create pBluescriptAma1A. This was followed by amplification of a 3' AMA1 ORF fragment with primers FAMA1Nde1 and RAMA1Pst1stop. This fragment was digested with NdeI and PstI and ligated to similarly digested pBluescriptAma1A. The removal of the intron sequence yielded a conservative amino acid change at position 236 from a cysteine to a serine. This intronless sequence lacks the 5' end of the coding sequence. To place the full-length AMA1 sequence under its own promoter, we used overlap polymerase chain reaction (PCR). We amplified a 5' fragment of AMA1 by using primers ADO5 and ADO10. This product was mixed with a vector carrying the intronless AMA1 3' sequence and amplified with primers ADO3 and ADO5, which yielded a 2-kb DNA fragment. Separately, oligos PAMA1F and PAMA1R were used to amplify 500 base pairs of the upstream sequence of the AMA1 gene, and this promoter region was cloned into the XhoI and Kpn1 sites of pRS306 to create pRS306-AMA1pr. The full-length intronless AMA1 ORF was then inserted into a pRS306-AMA1pr, at the XhoI and SpeI sites creating plasmid pRS306-AMA1pr-AMA1. The AMA1 in this plasmid was shown to be functional based on its ability to complement the ama1/ama1 phenotype (data not shown). Plasmids pRS306-AMA1pr-AMA1-IA and pRS306-AMA1pr-AMA1-IR were constructed by reamplifying the intronless AMA1 ORF by using the oligo pairs Ama1pFXhoI and Ama1StopIA, or Ama1pFXhoI and Ama1StopIR, respectively. The Ama1StopIA and Ama1StopIR oligos incorporate the mutations at the extreme C terminus of the coding region. The PCR fragments carrying the mutant alleles were then cloned into pRS306-AMA1-pr as XhoI-SpeI fragments. pRS426-AMA1pr-AMA1-IR was created by moving a KpnI and SpeI fragment carrying the gene from pRS306-AMA1pr-AMA1-IR into pRS426 (Christianson et al., 1992).
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). Briefly, strains were grown at 30°C overnight in YPD or in selective medium if they contained plasmids. The cultures were then diluted to a cell density of 0.2 at OD660 in YP media containing 2% potassium acetate and incubated at 30°C overnight. Cells were then washed once in distilled water and then resuspended in sporulation medium (2% potassium acetate) at a cell density of 1.2 at OD660, and these cultures were incubated at 30°C. For experiments using the degronSSP1, 25 nM β-estradiol (Sigma-Aldrich, St. Louis, MO) was added to the sporulating cells at the time of transfer to sporulation medium. Cells were cultured at 23°C for 2 h and then moved to restrictive temperature.
Ether Tests
Cells were sporulated at permissive (25°C) and restrictive (35°C) temperatures for the degron cassette in the presence or absence of 25 nM β-estradiol (Sigma-Aldrich). Serial dilutions of sporulated cells from each culture condition were spotted onto two duplicate YPD plates, and one plate was exposed to ether vapor for 5 min by inversion over an ether-soaked paper filter. Plates were photographed after incubation at 30°C for 1 d.
Immunoblotting and Immunofluorescence
For the Western analysis of Ssp1, cell extracts were prepared as described previously (Moreno-Borchart et al., 2001). Briefly, cells were lysed, and proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose. Ssp1 was detected using anti-HA antibody 12CA5 (Roche Diagnostics, Indianapolis, IN) at 1 µg/ml. Monclonal anti-porin (Invitrogen, Carlsbad, CA) and polyclonal goat anti-Clb5 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were also used at 1 µg/ml. For detection, the secondary antibody IR Dye 680 goat anti-mouse (LI-COR Biosciences, Lincoln, NE) and donkey anti-goat antibodies conjugated to IR Dye 800 were used at 1:10,000 dilutions of 0.5 mg/ml stocks. Membranes were scanned using Odyssey Infrared Imaging system (LI-COR Biosciences), and the signal intensities for all three proteins measured (Ssp1 and porin were measured on the same membrane, Clb5 from a separate membrane with equal volumes of each sample loaded). To allow comparison of levels between wild-type and ama1 cells, the porin signals in each lane were normalized to the value in the wild-type time zero lane and then the Ssp1 and Clb5 intensities at each time point were normalized to the porin signal at that same time point. Indirect immunofluorescence of the β-glucan layer was performed as described previously (Tachikawa et al., 2001).
Time-Lapse Fluorescence Microscopy
Time-lapse imaging was done as follows. Sporulation media containing 1.5% agarose-S was dropped on the glass surface of a glass-bottomed dish. Solidified media were removed from dish, and cells were spotted on a flat surface of media, put again on a dish to sandwich cells between glass and media. Images were captured on an Axiovert 100 microscope (Carl Zeiss, Thornwood, NY) at 2-min intervals for wild type and at 4-min intervals for the ama1 mutant by using of IPLab 3.6.5a software (Scanalytics, Rockville, MD). At each time point, 12 Z-sections were collected at 0.5-µm intervals. The temperature was kept at 28°C. Deconvolution was performed using an EPR system (Scanalytics) and three-dimensional stacks using IPLab 3.6.5a.
Fluorescence Loss in Photobleaching (FLIP) Assays
For FLIP microscopy, strains AN390 and JSP22 were first transformed with pRS424-R20 and pRS426-R20, respectively. A thin layer of 1.5% agarose containing 1% potassium acetate and 2 mM NaHCO3 was prepared. A 1.5-cm square of this agarose was cut out, and sporulated cells were spotted onto this square. The agarose square was then placed cell-side down onto a glass-bottomed Petri dish (MatTek, Ashland, MA), and cells were observed on an LSM 510 inverted microscope (Carl Zeiss).
For photobleaching, a 488-nm argon laser was used at 100% power. A cytoplasmic area was photobleached for 8 s with 75 pulses per bleaching. To analyze fluorescence intensity, LSM 510 META software version 3.2 (Carl Zeiss) was used.
For the FLIP assay, cells displaying mRFP-Spo2051-91 fluorescence (a prospore membrane marker) were selected. An area of the mother cell cytoplasm outside of the prospore membrane was photobleached four times over a period of 14 min, and the GFP fluorescence of Tef2-GFP was monitored every 15 s. Using the software, the fluorescence intensities were then measured in several areas in the cell: 1) the site of bleaching in the cytoplasm, 2) a cytoplasmic area separate from the bleached area and outside of the prospore membrane, 3) an area inside the prospore membrane, and 4) an area of cytoplasm in an unbleached, neighboring cell.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mutants). These studies demonstrated that as prospore membranes expanded they progressed through a series of discrete morphological stages (Figure 1 and Supplemental Movie 1). They began as small horseshoe-shaped structures that expanded into small round structures and initially maintained that round shape as they expanded. As meiosis II progressed, the membranes elongated into a tubular shape. After extending as tubes, the membranes widened in the middle to form an oval before a rapid transition back to a round shape. This final change in shape may correspond to the closure of the prospore membrane.
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), consistent with the idea that the rounding up of the membrane correlates with cytokinesis.
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mutants were examined in the same way, different behaviors of the prospore membrane and Don1-GFP were seen. Membrane expansion was initially normal, but the duration of the tubular phase was greatly extended (Figure 2B and Supplemental Movie 3). Moreover, even though in many cells the membranes eventually rounded up, the Don1-GFP staining never dispersed as in wild type. Rather, discrete foci of Don1-GFP fluorescence, and occasionally intact rings, persisted throughout the time course. At later times, abnormal prospore membrane structures began to accumulate in the mutant. Don1 localization is dependent on Ssp1 (Moreno-Borchart et al., 2001). Therefore, Don1-GFP serves as a marker for Ssp1 localization in these cells. If disassembly of the leading edge complex and rounding up of the prospore membrane are linked to closure, these observations suggest that cytokinesis may be defective in the ama1 mutant.
FLIP Assay for Closure Reveals a Defect in ama1 Mutants
To directly assess whether AMA1 has a role in cytokinesis, we developed a FLIP assay for membrane closure based on the ability of a GFP-tagged protein to diffuse between the presumptive ascal and spore cytoplasms. A strain expressing both a GFP-tagged TEF2 gene, encoding translation elongation factor 2, as well as mRFP-Spo2051-91 was sporulated. During meiosis II a small region of the cytoplasm outside of the prospore membranes was repeatedly photobleached with pulses from a laser and the fluorescence intensity of the Tef2-GFP signal at spots both inside and outside of the prospore membranes was monitored over time (Figure 3A). Cells were examined at different stages of prospore membrane growth, as defined by the shape and size of the membrane.
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mutant. As in wild-type cells, Tef2-GFP diffused freely throughout the cytoplasm in ama1 cells with small or tubular prospore membranes (Figure 3B). However, in ama1 cells only 
30% of the round or oval prospore membranes were closed off to the ascal cytoplasm (Table 3 and Figure 3B). At least half of the membranes examined clearly remained open. The remaining 20% gave ambiguous results, in which loss of fluorescence due to photobleaching was intermediate between the obviously open or closed membranes. This may represent cells where closure is incomplete, leaving a small diffusion-limiting opening, or simply be a result of the technical difficulty of photobleaching the ascal cytoplasm alone in cells with abnormal prospore membranes. That some of the prospore membranes do close suggests either that there may be an AMA1-independent means of removing the leading edge complex, for example by some functional overlap with CDC20, or possibly an alternative pathway to membrane closure. Nonetheless, the abundance of open prospore membranes demonstrates that ama1 mutants are defective in the cytokinesis at the end of meiosis II.
Ssp1 Protein Levels Persist in the ama1 Mutant
Our microscopy results indicate that ama1 mutants are defective both in prospore membrane closure and in disassembly of the leading edge complex. The leading edge component Ssp1 has been shown to antagonize membrane fusion when expressed in vegetative cells leading to the suggestion that closure of the prospore membrane requires removal of Ssp1 from the leading edge (Maier et al., 2007). In this light, one simple explanation for our results would be that APCAma1 promotes degradation of Ssp1 and this degradation allows prospore membrane closure.
To determine whether AMA1 influences the stability of the Ssp1 protein, the levels of a HA-tagged Ssp1 protein were examined across sporulation time courses in wild-type and ama1 strains (Figure 4). To correlate protein levels with the events of meiosis, cells were also fixed at each time point, stained with DAPI, and the nuclear morphology was examined in the fluorescence microscope. In wild-type cells, Ssp1 protein began to accumulate after 3 h in sporulation medium, coincident with the onset of the meiotic divisions. Consistent with earlier reports (Maier et al., 2007), Ssp1 levels peaked after 6 h and then fell sharply as the population reached the end of meiosis. By contrast, in the ama1 cells, Ssp1 levels increased as in wild type, but the protein accumulated to a much greater extent than in wild-type cells. At later time points, after the bulk of cells in the population had completed meiosis II, Ssp1 levels declined in the ama1 mutant strain, but even after 12 h the level of Ssp1 was comparable with the peak level in wild-type cells. Thus, although some degradation of Ssp1 is seen, loss of AMA1 leads to an accumulation and a persistence of the Ssp1 protein at the end of meiosis (Figure 4B).
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mutants have been reported to have defects in meiotic progression in some backgrounds (Cooper et al., 2000), it was possible that this accumulation and persistence of Ssp1 was an indirect consequence of a meiotic defect in the ama1 strain. To examine this possibility, we also measured the abundance of Clb5, a protein degraded at the completion of meiosis II (Carlile and Amon, 2008), in the two strains (Figure 4C). The levels of Clb5 in both strains were comparable throughout the time course, indicating that the accumulation of Ssp1 is not a consequence of a more general defect in protein turnover at the end of meiosis II in the ama1 mutant.
The ama1 Sporulation Defect Can Be Partially Rescued by a Conditional SSP1
If AMA1-mediated turnover of Ssp1 is required for prospore membrane closure and this cytokinesis defect is responsible for the subsequent spore wall formation phenotypes of ama1, then mutation of SSP1 might be expected to rescue the ama1 sporulation defect. Because SSP1 is essential for proper prospore membrane assembly, a conditional allele of SSP1 is required so that Ssp1 protein can be inactivated after prospore membranes have formed and expanded. A point mutation creating a conditional allele of SSP1 has been reported previously (Esposito et al., 1970; Maier et al., 2007), but in our strain background the phenotype of this mutant was very mild, and no restoration of sporulation was observed when it was combined with ama1 (Diamond, unpublished observations). We therefore sought to engineer a conditional allele of SSP1 by using a degron cassette (Sanchez-Diaz et al., 2004). Fusion of one copy of ubiquitin followed by a temperature-sensitive form of dihydrofolate reductase (DHFR) to the amino terminus of a heterologous protein can be used to generate proteins whose stability is temperature dependent in vivo (Sanchez-Diaz et al., 2004). Cleavage of the ubiquitin moiety reveals an amino-terminal arginine residue on the DHFRts and, upon shift to elevated temperature, the DHFRts fusion protein is degraded by the N-end rule pathway. Efficient turnover in this system also requires overexpression of the ubiquitin ligase Ubr1 (Sanchez-Diaz et al., 2004). To induce Ubr1 in our strains, both a GAL promoter driven UBR1 and a plasmid expressing a fusion of the Gal4 transcription factor to the hormone-binding domain of the human estrogen receptor were integrated into the genome. This Gal4 fusion can induce transcription only in the presence of steroid ligands such as β-estradiol (Picard, 1999). A strain expressing the fusion form of SSP1 (hereafter degssp1) as its only source of Ssp1 and carrying the GAL-UBR1 and Gal4-ER plasmids displays conditional sporulation; sporulation is blocked only in the presence of both estradiol, to induce UBR1, and elevated temperature to destabilize Ssp1 (Figure 5A).
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strain failed to produce spores at either temperature, and the degssp1 strain displayed temperature-sensitive sporulation. By contrast, the ama1 degssp1 strain displayed very weak sporulation at low temperature that was markedly improved by raising the temperature to 35°C. Tests of different temperature regimes and incubation times before temperature upshift indicated that the protocol used in these experiments provided the best level of sporulation in the double mutant (Diamond, unpublished observations). Although this level of sporulation was only 10% of the wild type, no spores are ever seen in the ama1 mutant alone. Thus, this represents a significant suppression of ama1 by degssp1. Reproducibly, at 35°C, slightly higher sporulation was seen in the ama1 degssp1 strain than in the strain carrying degssp1 alone (Figure 5C). Thus, reciprocally, deletion of AMA1 improves sporulation of degssp1 cells. The accumulation of Ssp1-HA seen in ama1 mutants (Figure 4) suggests that this improvement of degssp1 sporulation may be due to stabilization of degSsp1 that has escaped from N-end rule mediated degradation.
When ama1 cells are sporulated they fail to synthesize spore wall components. As spore wall assembly is required for the generation of visible spores, the incomplete rescue of ama1 by degssp1 could represent strong suppression of the cytokinesis defect masked by a subsequent spore wall synthesis phenotype. To address this possibility, spore wall synthesis in the ama1 degssp1 mutant was examined using anti-β-1,3-glucan antibodies (Figure 5E). Examination of anti-β-1,3-glucan staining in the mutant demonstrated that the number of β-1,3-glucan containing prospores varied between asci. The distribution of β-glucan staining was comparable with the distribution of visible spores as seen in differential interference contrast microscopy (DIC) (Diamond, unpublished observations). Because β-glucan deposition is required for acquisition of refractility and occurs early in spore wall formation (Tachikawa et al., 2001; Coluccio et al., 2004), this result suggests that those prospores that bypass the cytokinesis block in the ama1 degssp1 strain go on to complete spore wall synthesis.
A Motif Important for Interaction with the APC Is Essential for Ama1 Function
Cdc20/Fizzy family members carry two motifs, a C-box and a carboxy-terminal isoleucine and arginine (IR) that are important for interaction with the APC (Schwab et al., 2001; Vodermaier et al., 2003). The IR motif has been shown to bind to the APC subunit Cdc27, and mutation of the IR motif in Cdh1 or Cdc20 blocks their interaction with the APC in vitro and inactivates Cdh1 in vivo (Vodermaier et al., 2003; Kraft et al., 2005; Oelschlaegel et al., 2005). Similarly, mutation of the carboxy-terminal arginine residue of Ama1 to an alanine blocks its ability to interact with the APC in vitro (Oelschlaegel et al., 2005). We examined the phenotype of this arginine to alanine mutation (AMA1R593A) in vivo. Despite the strong effect of this mutation in vitro, cells expressing AMA1R593A from the chromosome as the only form of AMA1 showed only a mild sporulation defect (Figure 6). By contrast, deletion of both the carboxy-terminal isoleucine and arginine residues (IR) strongly reduced sporulation. When this AMA1IR allele was overexpressed from a high copy plasmid, the mutant was able to partially rescue the sporulation defect of ama1. These observations demonstrate that the carboxy-terminal IR motif is important, although not essential, for Ama1 function in vivo. This suggests that interaction with APC is necessary for Ama1 to promote sporulation but that, in contrast to the in vitro studies, in vivo the IR motif enhances but is not absolutely required for this interaction.
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; Pablo-Hernando et al., 2007). Although homozygous CLB2pr-CDC15 cells progress through meiosis with normal kinetics, the cells fail to form spores and display other phenotypes, including defective spindle disassembly, that suggest a failure to properly exit from meiosis II (Pablo-Hernando et al., 2007).
APCAma1 activity is inhibited during meiosis by both the Mnd2 subunit of APC and by Clb-Cdc28 kinase and both of these antagonistic activities are down-regulated as cells complete meiosis (Dahmann and Futcher, 1995; Oelschlaegel et al., 2005; Carlile and Amon, 2008). If, as a consequence of the failure to exit meiosis properly, a CLB2pr-CDC15 mutant does not release the inhibition of APCAma1, we would expect to find a prospore membrane closure defect in these cells. To test this possibility, we used the FLIP assay to examine prospore membrane closure in a CLB2pr-CDC15 strain. As reported previously (Pablo-Hernando et al., 2007), the CLB2pr-CDC15 cells displayed a prospore membrane growth defect, with many cells displaying only one or two membranes that are full size by the end of meiosis II. Because small membranes are usually open in wild-type cells, we limited our analysis of closure to the largest membrane in each cell. In the CLB2pr-CDC15 strain, 53% of these membranes were closed and 24% were open, with the remaining membranes in the indeterminate class (Table 4). Thus, depletion of Cdc15 results in a prospore membrane closure defect.
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; Penkner et al., 2005). By contrast, mutation of eight consensus Cdc28 phosphorylation sites in the Ama1 protein to alanines (Ama1m8) does not cause any obvious phenotype (Oelschlaegel et al., 2005). We therefore integrated either AMA1 or AMA1-m8 into the CLB2pr-CDC15 strain and examined prospore membrane closure by using the FLIP assay. Integration of an extra two copies of AMA1 into these cells did not significantly alter the fraction of prospore membranes that were closed. However, introduction of the AMA1-m8 allele, increased the fraction of membranes that close in the CLB2pr-CDC15 cells to 80%. Although modest, a chi-square test indicates that the fraction of closed prospore membranes in the presence of AMA1-m8 is significantly different from either of the other two strains (p < 0.001). These results indicate that the closure defect in the CLB2pr-CDC15 cells is caused by a failure to activate APCAma1 possibly due to direct phosphorylation of Ama1 by the Cdc28 kinase.
In addition to the prospore membrane closure defect, CLB2pr-CDC15 cells display defects in prospore membrane growth, meiotic spindle disassembly, and fail to form spores (Pablo-Hernando et al., 2007). Expression of AMA1-m8 did not rescue any of these other phenotypes (Suda and Park, unpublished observations). Thus, the lack of APCAma1 activity is responsible only for the closure defect in these cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Using a FLIP assay, we provide direct evidence that the morphological changes in the prospore membrane that correlate with removal of the leading edge complex are coincident with closure of the prospore membrane. In an ama1 mutant, Ssp1 degradation and leading edge complex disassembly is delayed and cytokinesis is impaired. This provides direct support for the idea that removal of Ssp1 from the leading edge is required for membrane closure (Maier et al., 2007).
A conserved IR dipeptide at the extreme amino terminus is a hallmark of Cdc20/Fizzy proteins (Vodermaier et al., 2003). However, the effects of mutations in this motif vary in vivo. Deletion of this motif in the yeast Cdh1 creates a null allele (Kraft et al., 2005), whereas cells carrying a deletion of the IR residues of Cdc20 are viable, with only modest effects on function of the protein (Thornton et al., 2006). Ama1 falls between these extremes. Deletion of the IR tail does greatly reduce sporulation, but overexpression of this allele can restore activity. The differences between these different APC targeting subunits in sensitivity to carboxy-terminal mutations suggest that in addition to the conserved C-box and IR motifs they may each make unique contacts with the APC that effect the relative importance of the conserved motifs.
Activity of APCAma1 is regulated by both the Mnd2 subunit of the APC and Clb-Cdc28 kinase activity (Oelschlaegel et al., 2005; Penkner et al., 2005). In the presence of Mnd2, Ama1 cannot promote ubiquitylation of substrates either in vivo or in vitro (Oelschlaegel et al., 2005). The Mnd2 protein, however, dissociates from the APC during anaphase II, suggesting that the activity of APCAma1 might be up-regulated at that time (Oelschlaegel et al., 2005). Similarly, Clb-Cdc28 kinase activity down-regulates APCAma1 and Clb-Cdc28 kinase activity drops at the end of meiosis II (Dahmann and Futcher, 1995; Oelschlaegel et al., 2005; Carlile and Amon, 2008). Our finding that mutation of the consensus Cdc28 phosphorylation sites of Ama1 allows prospore membrane closure in the CLB2pr-CDC15 mutant suggests that Cdc28 directly phosphorylates Ama1. Relief of these two different inhibitions might trigger APCAma1 activity at the end of meiosis. This is analogous to the manner in which APCCdh1 activity in mitotic cells is restricted until anaphase by the combination of Cdc28 phosphorylation of Cdh1 and the binding of the APCCdh1 inhibitor Acm1 (Zachariae et al., 1998; Martinez et al., 2006; Ostapenko et al., 2008).
In the case of Cdh1, phosphorylation seems to be the predominant brake on APCCdh1 activity, as mutation of the phosphorylation sites creates a constitutively active, lethal allele (Zachariae et al., 1998). By contrast, though we provide evidence that mutation of the sites in Ama1 results in an allele that is dominantly active late in meiosis, in an otherwise wild type cell, the AMA1-m8 mutant does not produce a phenotype (Oelschlaegel et al., 2005). Mutation of MND2, however, is sufficient to activate APCAma1 (Oelschlaegel et al., 2005; Penkner et al., 2005). One interpretation of these observations is that MND2 provides the primary restraint on APCAma1 activity early in meiosis and that inhibition by Clb-kinase late in meiosis, as Mnd2 activity is lost, allows the activation of APCAma1 to be timed more precisely to exit from meiosis II. In light of the report that different Clb-Cdc28 complexes are active at different times of meiosis (Carlile and Amon, 2008), it will be of interest to determine if APCAma1 is subject to down-regulation by Clb-kinases generally or only those that are active during meiosis II.
Our finding that depletion of Cdc15 results in a cytokinesis defect that can be relieved by a nonphosphorylatable allele of AMA1 is consistent with the idea that completion of meiosis leads to activation of APCAma1. These observations, and previous work indicating that removal of Ssp1 from the prospore membrane is important for prospore membrane closure (Maier et al., 2007) suggest a model in which Ama1 functions to coordinate exit from meiosis II with cytokinesis. The disappearance of Mnd2 and Clb-Cdc28 kinase activity at the end of meiosis activates APCAma1 leading to the destruction of Ssp1 and disassembly of the leading edge complex. The removal of Ssp1 then allows the prospore membrane to close (Figure 7).
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). Furthermore, though the Ssp1 sequence contains matches to both the consensus KEN and D boxes found in many APC substrates, both these sites lie outside the C-terminal domain that has been shown to be required for Ssp1 degradation (Maier et al., 2007); and mutation of the KEN box does not produce an obvious phenotype (Diamond, unpublished). Therefore, although the simple model is appealing, it is possible that, analogous to the way in which APCCdc20 regulates cohesin stability through the action of separase (Ciosk et al., 1998; Uhlmann et al., 1999), APCAma1 acts through some additional protein to regulate Ssp1 turnover.
Another issue still to be addressed is the connection between the failure of cytokinesis in ama1 and the spore wall phenotype of the mutant. Our FLIP studies reveal that a significant fraction of the prospore membranes in ama1 cells eventually close, possibly due to slower, AMA1-independent turnover of Ssp1. Nonetheless, these prospores do not develop spore walls. In wild-type cells, the onset of spore wall development only occurs after cytokinesis. It may be that the normal closure process generates a signal that initiates wall assembly and this signal is not generated by the abnormal closure of membranes in the ama1 cells. Alternatively, APCAma1 may have additional roles in spore wall development after cytokinesis. The APC subunit Swm1 was originally identified as a mutant with a spore wall defect (Ufano et al., 1999), and an APC subunit has also been identified as a spore wall mutant in Schizosaccharomyces pombe (Kakihara et al., 2003), suggesting that the APC is important for proper wall assembly. Moreover, AMA1 has been found to be required for the activation of the mitogen-activated protein kinase Smk1, which regulates spore wall assembly (Krisak et al., 1994; Huang et al., 2005; McDonald et al., 2005). This last observation strongly suggests the existence of additional APCAma1 targets besides Ssp1, and may explain why inactivation of degSsp1 only provides a partial suppression of the ama1 sporulation defect.
Finally, the mechanism by which prospore membrane closure is achieved also requires further exploration. In vegetative yeast cells, cytokinesis is driven by a combination actomyosin ring mediated ingression of the plasma membrane as well as deposition of septal wall material. Neither of these mechanisms is likely to operate in prospore membrane closure as actin is not found at the leading edge, and there is no significant deposition of wall material until well after prospore membrane closure (Coluccio et al., 2004; Taxis et al., 2006). However, the final separation into distinct cells in a mitotic division requires rearrangement of membrane bilayers and topologically, it represents the same situation as closure of the prospore membrane. Several different membrane fusion associated functions including the exocyst, soluble N-ethylmaleimide-sensitive factor attachment protein receptors, and the ESCRT complex have been implicated in this final stage of cell separation during division in multicellular eukaryotes (Lauber et al., 1997; Jantsch-Plunger and Glotzer, 1999; Gromley et al., 2005; Carlton and Martin-Serrano, 2007). Exactly how division is achieved remains obscure.
Similarly, the mechanism of prospore membrane closure remains to be determined. Ssp1 seems to be antagonistic to membrane fusion (Maier et al., 2007), and our data are consistent with the proposal that removal of Ssp1 promotes closure of the membrane, but this leaves open the question of how closure is achieved. Given the parallels to the final stages of cytokinesis in mitotic cells, the identification of proteins that directly mediate closure of the membrane could provide insight into the general mechanism of cytokinesis.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Aaron M. Neiman (aaron.neiman{at}sunysb.edu)
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