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Eukaryotic Cell, June 2003, p. 431-445, Vol. 2, No. 3
1535-9778/03/$08.00+0     DOI: 10.1128/EC.2.3.431-445.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Ady4p and Spo74p Are Components of the Meiotic Spindle Pole Body That Promote Growth of the Prospore Membrane in Saccharomyces cerevisiae

Mark E. Nickas,1 Cindi Schwartz,2 and Aaron M. Neiman1*

Department of Biochemistry and Cell Biology and Institute for Cell and Developmental Biology, State University of New York at Stony Brook, Stony Brook, New York 11794-5215,1 Boulder Laboratory for 3-D Electron Microscopy of Cells, Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 803092

Received 30 January 2003/ Accepted 28 March 2003


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Spore formation in Saccharomyces cerevisiae occurs via the de novo synthesis of the prospore membrane during the second meiotic division. Prospore membrane formation is triggered by assembly of a membrane-organizing center, the meiotic outer plaque (MOP), on the cytoplasmic face of the spindle pole body (SPB) during meiosis. We report here the identification of two new components of the MOP, Ady4p and Spo74p. Ady4p and Spo74p interact with known proteins of the MOP and are localized to the outer plaque of the SPB during meiosis II. MOP assembly and prospore membrane formation are abolished in spo74{Delta}/spo74{Delta} cells and occur aberrantly in ady4{Delta}/ady4{Delta} cells. Spo74p and the MOP component Mpc70p are mutually dependent for recruitment to SPBs during meiosis. In contrast, both Ady4p and Spo74p are present at SPBs, albeit at reduced levels, in cells that lack the MOP component Mpc54p. Our findings suggest a model for the assembled MOP in which Mpc54p, Mpc70p, and Spo74p make up a core structural unit of the scaffold that initiates synthesis of the prospore membrane, and Ady4p is an auxiliary component that stabilizes the plaque.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Sporulation in Saccharomyces cerevisiae is a specialized form of cell division in which a single diploid cell produces four haploid spores within the cytoplasm of the original mother cell (10). The limiting membranes of the spores are synthesized de novo during meiotic segregation of nuclear DNA via the redirection of the secretory pathway (23). Golgi-derived vesicles that are destined for the plasma membrane during vegetative growth become targeted to the four spindle pole bodies (SPBs), the functional equivalents of centrosomes in higher eukaryotes, during sporulation. Fusion of these vesicles creates four discrete membrane compartments, the prospore membranes, which surround the nuclei upon the completion of meiosis to generate spores.

Prospore membrane synthesis is initiated during the transition from the first to the second meiotic division. In yeast, both meiotic divisions occur within a single, continuous nuclear envelope, in which the SPBs are embedded. The cytoplasmic face of each SPB, termed the outer plaque, expands in early meiosis II and becomes a site for docking and fusion of secretory vesicles (20). Formation of the prospore membrane occurs via the fusion of these vesicles to form a flattened sac that abuts the meiotic outer plaque (MOP). Each prospore membrane grows toward the center of the spindle during anaphase II and engulfs the adjacent lobe of the nucleus. As nuclear division occurs at the end of meiosis II, each prospore membrane fuses with itself to enclose a haploid nucleus within two continuous membranes. Spore wall material is deposited into the lumen between the two new membranes to produce a mature spore (19).

Regulation of SPB function during meiosis is critical for spore formation. The morphological expansion of the outer plaque during sporulation reflects a shift in its primary role from the anchoring of cytoplasmic microtubules to the initiation of prospore membrane synthesis. This modification of the outer plaque is required for prospore membrane formation (1, 5, 16). The change in morphology and function of the outer plaque during sporulation is due to an alteration in its protein composition. Spc72p, a component of the outer plaque during mitotic growth that binds to the gamma-tubulin complex of cytoplasmic microtubules, disappears from SPBs during meiosis and is replaced by the meiosis-specific components Mpc54p and Mpc70p/Spo21p (15, 16). Mpc54p, Mpc70p, and the constitutive outer plaque components Cnm67p and Nud1p/Spc94p are the four known proteins of the MOP (1, 16). Cnm67p, Mpc54p, and Mpc70p are predicted to have extended conformations with amino- and carboxy-terminal globular domains separated by central coiled-coil regions and are required for expansion of the outer plaque and formation of prospore membranes (1, 16, 31). The role of Nud1p in assembly of the MOP is more difficult to assess because this protein is essential for vegetative growth, but the interaction of Nud1p with Cnm67p, Mpc54p, and Mpc70p in the two-hybrid assay suggests that it may function as a docking protein for other outer plaque components (8, 16, 41).

Certain environmental stimuli or genetic perturbations that affect assembly of the MOP result in formation of nonsister dyads, two-spore asci in which one haploid nucleus from each meiosis II spindle has been packaged. For example, the removal of the carbon source after initiation but prior to completion of meiosis leads to modification of only two outer plaques and the production of nonsister dyads (5, 26). Nonsister dyads can also result from mutations that cause reduced levels of structural components of the MOP or affect their recruitment to the SPB (1, 7, 13, 40). In contrast, dyads caused by mutations that affect subsequent steps in sporulation, such as growth of prospore membranes or synthesis of spore wall components, contain spores in which two meiotic products have been packaged at random (9, 21, 25).

The identification of proteins with potential roles in assembly of the MOP and formation of the prospore membrane has been facilitated by genome-wide analyses (4, 14, 28, 29, 39). The results from such large-scale analyses have been used to elucidate the specific functions of several proteins involved in these processes (1, 21, 25). Other genes that have null phenotypes or transcription profiles comparable to those of components that mediate modification of the outer plaque and synthesis of the prospore membrane have yet to be analyzed intensively.

In this study, we have characterized the products of two meiotically induced genes, ADY4 and SPO74, involved in sporulation. We find that Ady4p and Spo74p are components of the MOP that are required for normal prospore membrane formation. MOPs and prospore membranes are absent in spo74{Delta}/spo74{Delta} cells and display heterogeneous defects in ady4{Delta}/ady4{Delta} cells. Quantitative analysis of fluorescence from green fluorescent protein (GFP) fusions during meiosis revealed that loss of either Mpc70p or Spo74p abolishes recruitment of the other to SPBs and that Ady4p is present at SPBs at reduced levels in cells that lack either Mpc54p, Mpc70p, or Spo74p. Our findings suggest that Mpc54p, Mpc70p, and Spo74p assemble at the SPB during meiosis into the ordered structure that initiates formation of the prospore membrane and that Ady4p is an ancillary component of the MOP that stabilizes the plaque.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Yeast strains and methods. Standard S. cerevisiae genetic methods and media were used (30). Strains are listed in Table 1. Diploid strains with AN, MND, and NY designations were derived from SK1 haploids AN117-4B and AN117-16D and are isogenic with each other, diploid strains with MYA designation are derived from a separate SK1 stock strain and are isogenic with each other, and YCJ4 is not of SK1 ancestry. AN117-4B, AN117-16D, AN120, YCJ4, and MYA strains have been described previously (12, 24, 29). The following chromosomal insertions were made in AN117-4B and AN117-16D, and the resulting strains were mated to create AN279, AN282, MND57, MND58, and NY541, respectively: GFP-HIS3MX6 at the 3' end of the ADY4 open reading frame (ORF), GFP-HIS3MX6 at the 3' end of the SPO74 ORF, HIS3MX6 replacing the ADY4 ORF, HIS3MX6 replacing the SPO74 ORF, and HIS3MX6 replacing the MPC54 ORF. SnaBI-cut pRS304 was integrated into one allele of trp1 in MND57 to create MND59 and into an unknown site in AN117-4B, which was then mated to the spo74::HIS3MX6 derivative of AN117-16D to create MND61.


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  		TABLE 1. S. cerevisiae strains used in this study

 
Gene insertions and replacements were performed by transformation of strains with PCR-generated DNA cassettes and verified by PCR (18). The sequences of oligonucleotide primers used to amplify insertion cassettes by PCR are listed in Table 2. The cassettes used to replace ADY4, MPC54, and SPO74 in MND57, NY541, and MND58, respectively, were amplified from pFA6a-HIS3MX6 with the following pairs of primers: ANO299-ANO283, HT76-HT77, and ANO297-ANO292. The cassettes used to insert the GFP gene into the 3' ends of ADY4 and SPO74 in AN279 and AN282, respectively, were amplified from pFA6a-yEGFP(S65T)-HIS3MX6 with the primer pairs ANO282-ANO283 and ANO291-ANO292.


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  		TABLE 2. Oligonucleotides used in this study

 
Cells were induced to sporulate in liquid medium as described previously (25). Cells were cultured to saturation in either yeast extract-peptone-dextrose or synthetic complete medium lacking the appropriate nutrient, cultured overnight to mid-log phase in YPA, and transferred to 2% potassium acetate at a concentration of 3 x 107 cells/ml (optical density at 600 nm of ~2.0).

Plasmids. Plasmids made for this study are listed in Table 3. All plasmids created in this study except pRS424-ADY4-GFP were made in three steps: PCR amplification of the sequence of interest from a plasmid or genomic DNA template, digestion of the PCR product at restriction sites within the PCR primers, and subcloning of the digested PCR product into restriction sites in the polylinker of a standard yeast vector. To make pRS424-ADY4-GFP, two partially overlapping PCR products with a common PacI site were amplified, each product was digested at the PacI site and at a second restriction site within the primer away from the region of overlap, and the two PCR products were simultaneously subcloned into pRS424. Oligonucleotide primers, template DNAs, and restriction sites used for subcloning are listed in Table 3.


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  		TABLE 3. PCR primers, templates, and restriction sites used for plasmid construction

 
The following plasmids used in this study have been described elsewhere: 1-66 (38); pFA6a-HIS3MX6 (18); pFA6a-yEGFP-HIS3MX6 and pGADGH-MPC70 (25); pMK169, pMK183, pMK184, pSM613, and pSM614 (8); and pRS316-SPO21 (1). The following plasmids were used to express the indicated fusion proteins for two-hybrid assays: 1-66, GAD-Mpc70p133-609; pMK169, GAD-Spc42p1-363; pMK183, GAD-Cnm67p386-580; pMK184, LexA-Cnm67p386-580; pSM613, GAD-Nud1p405-852; and pSM614, LexA-Nud1p405-852. pRS424-G20 contains a chimeric gene that consists of a fragment of SPO20 fused to the GFP gene (H. Nikanishi, unpublished data.)

Fluorescence microscopy. Preparation of cells for fluorescence microscopy and collection and analysis of images were performed essentially as described elsewhere (25). For visualization of naturally fluorescent proteins only, cells were fixed in 4% formaldehyde for 5 min, washed with phosphate-buffered saline (PBS) (130 mM NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4), and mounted with Vectashield mounting medium with DAPI (4',6'-diamidino-2-phenylindole) (Vector, Burlingame, Calif.). For simultaneous visualization of tubulin and naturally fluorescent proteins, cells were fixed for 10 to 15 min in 4% formaldehyde and processed for immunofluorescence as described elsewhere (25). Tubulin was visualized by using a 1:10 dilution of MAS 078b primary antibody (Harlan, Sussex, United Kingdom) and a 1:400 dilution of goat anti-mouse Alexa 546 (Molecular Probes, Eugene, Oreg.) secondary antibody, which cross-reacts with the MAS 078 rat monoclonal antibody. Fluorescence images were collected with a Zeiss Axioplan 2 microscope and Zeiss AxioCam HRm digital camera, and Axiovision 3.0.6 software was used for deconvolution and quantitation. Figures were prepared by using Photoshop 6.0 (Adobe Software, San Jose, Calif.) and Canvas 5.0.2 (Deneba Software).

Analysis of fluorescence intensity of GFP fusions for SPB components was performed as follows. Cells transformed with plasmids encoding GFP fusions were fixed after 6 to 8 h in 2% potassium acetate as described above, and the number of GFP foci at the spindle poles were analyzed in 50 meiosis II cells from each culture. Quantification of fluorescence intensity was performed on Z-series collections of raw (i.e., not deconvolved) images. A circle corresponding to a radius of 0.3 µm was drawn around each SPB, and the average pixel intensity in that circle from the brightest Z section was taken as the total value. The average pixel intensity of a same-sized circle that did not overlap an SPB was taken for each cell as the background value, and the specific signal was calculated as the background signal subtracted from the total signal for each SPB. The specific signal of >=15 SPBs was quantified for each combination of strain (including the wild type) and GFP fusion. The mean specific signal for a given GFP fusion in each mutant was compared to the mean specific signal for that GFP fusion in wild-type cells, which was defined as 100%, to calculate the relative signal intensity. The following strains and plasmids were used: strains AN120 (wild type), MND57 (ady4{Delta}/ady4{Delta}), AN161 (cnm67{Delta}/cnm67{Delta}), NY541 (mpc54{Delta}/mpc54{Delta}), AN180 (mpc70{Delta}/mpc70{Delta}), and MND58 (spo74{Delta}/spo74{Delta}) and plasmids pRS424-ADY4-GFP, pRS314-MPC54-GFP, pSB33 (MPC70-GFP), and pRS314-SPO74-GFP.

Electron microscopy. Cells were prepared for standard transmission electron microscopy as described elsewhere (37). Cells were prepared for transmission immunoelectron microscopy essentially as described previously (11) with the following slight modifications. Cells from sporulating cultures were harvested, fixed for 1 h in PBS containing 2% paraformaldehyde and 0.1% glutaraldehyde, washed in PBS, resuspended in PBS containing 8% sucrose, and stored overnight on ice. The cells were then rapidly frozen by high-pressure freezing (BAL-TEC HPM-010; Technotrade International, Manchester, N.H.) and freeze-substituted at -90°C in 0.2% glutaraldehyde plus 0.01% uranyl acetate in acetone for 96 h in an EM AFS device (Leica, Vienna, Austria). The cells were warmed over 22.5 h to -45°C and then infiltrated with HM20 resin over a period of 4 days. The cells were flat embedded under UV light at -45°C in HM20 for 3 days and then warmed to room temperature over a 24 h period. Embedded cells were sectioned and immunostained as follows: sections were (i) floated on blocking buffer (0.02% Tween 20, 0.8% bovine serum albumin, and 0.1% fish gelatin in 1x PBS) for 1 h, (ii) immunostained with anti-GFP overnight at 4°C as described previously (42), (iii) rinsed with PBS plus 0.1% Tween 20 three times, (iv) incubated with 10-nm-diameter colloidal gold (BB International, Cardiff, United Kingdom) for 2 h at room temperature, (v) rinsed and fixed with 1.0% glutaraldehyde for 5 min, (vi), stained with aqueous uranyl acetate and lead citrate, and (vii) imaged in a Philips CM-10 electron microscope.

ß-Galactosidase assays. ß-Galactosidase assays were performed as described previously (25). Transformants of YCJ4 were cultured overnight at 30°C on a Whatman 50 filter on the surface of a plate of synthetic complete medium lacking His and Leu, and the filter was immersed in liquid N2 for 10 s and then incubated at 30°C in Z buffer containing 0.1% 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal) and 0.027% ß-mercaptoethanol. At least three independent transformants for each combination of constructs were analyzed. None of GAD fusions interacted with LexA alone, but LexA-Spo74p1-413 gave moderate induction of ß-galactosidase expression in the absence of a GAD fusion. The following LexA fusions were used as positive controls for interactions with GAD fusions: Cnm67p386-580 for Spc42p1-363 and Nud1p405-852 for Mpc70p and Nud1p405-852.

Analysis of segregation of centromere-linked markers in dyads. Centromere linkage values of 11 and 9.5 centimorgans for ARG4 and RME1, respectively, in strain AN120 were calculated by analysis of 253 nonsister dyads formed in response to environmental conditions, and these values were used to calculate expected frequencies for dyad classes resulting from packaging of nonsister meiotic nuclei. All segregants from dyads were competent to mate with either MATa or MAT{alpha} strains and thus were inferred to be haploid.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ady4p and Spo74p are localized to SPBs during meiosis. To identify novel proteins with potential roles in prospore membrane formation, the subcellular localization of the products of genes that are induced during meiosis and required for normal ascus formation was analyzed (29). Strains with GFP fused to the 3' ends of ADY4, AMA1, SPO73, SPO74, and SPO77 were individually constructed and examined by fluorescence microscopy. The two of these gene products that produced functional, visible GFP fusions, Ady4p and Spo74p, formed foci at the ends of the meiotic spindles (Fig. 1). Spo74p-GFP could be easily detected and showed the same distribution when expressed homozygously from its natural locus or from a centromeric plasmid. Ady4p-GFP could be visualized consistently only when expressed from a multicopy plasmid but showed a similar distribution when expressed homozygously from its natural locus or from a centromeric plasmid. Analysis of Ady4p and Spo74p by immunoelectron microscopy revealed that both proteins were localized to the meiotic outer plaque of the SPB (Fig. 2). The localization patterns of Ady4p and Spo74p are similar to those that have been described for the MOP components Mpc54p and Mpc70p (1, 16).



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  		FIG. 1. Localization of Ady4p-GFP and Spo74p-GFP by fluorescence microscopy. Sporulating AN120 cells transformed with either pRS424-ADY4-GFP (top panels) or pRS314-SPO74-GFP (bottom panels) were processed for immunofluorescence microscopy with an antibody to tubulin and DAPI to visualize DNA. Natural fluorescence from GFP fusions is shown in green, immunofluorescent signal from antitubulin antibody is shown in red, and fluorescent signal from DAPI-stained DNA is shown in gray. Merged column shows overlaid images of GFP and tubulin fluorescences. Images of two cells from each strain are shown.

 


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  		FIG. 2. Localization of Ady4p-GFP and Spo74p-GFP by electron microscopy. Sporulating cells from strains AN279 (ADY4-GFP/ADY4-GFP) (A) and AN282 (SPO74-GFP/SPO74-GFP) (B) were processed for electron microscopy, and sections were stained with primary antibodies to GFP and gold-conjugated secondary antibodies. Arrows point to MOPs, and arrowheads point to selected gold particles. Bars, 100 nm.

 
Ady4p and Spo74p interact with components of the MOP in the two-hybrid assay. To examine whether Ady4p and Spo74p interact with known components of the SPB, these proteins were tested for the ability to bind various fusions of SPB proteins in the two-hybrid assay. A LexA fusion of Ady4p interacted with GAD fusions containing sequences from Cnm67p, Mpc70p, and Nud1p (Table 4). A LexA fusion of Spo74p interacted with GAD fusions containing sequences from Mpc70p and itself, and a GAD fusion of Spo74p interacted with a LexA fusion of Mpc54p (Table 4). The regions of Mpc70p that interact with Ady4p and Spo74p were mapped by using a series of fusions that contain the Gal4p activation domain and different regions of Mpc70p. Residues 100 to 300 of Mpc70p were required for maximal binding to Ady4p, whereas residues 401 to 609 were sufficient to mediate the interaction with Spo74p (Fig. 3). These results indicate that Ady4p and Spo74p interact with components of the MOP.


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  		TABLE 4. Two-hybrid interactions with Ady4p and Spo74p

 


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  		FIG. 3. Schematic of GAD-Mpc70p fusions and their ability to interact with LexA-Ady4p and LexA-Spo74p. Residues of Mpc70p included in fusion proteins are indicated at the left, and stippled regions represent coiled-coil domains of Mpc70p.

 
ady4{Delta}/ady4{Delta} and SPO74/spo74{Delta} mutants accumulate random and nonsister dyads, respectively. To characterize more thoroughly the roles of ADY4 and SPO74 in sporulation, the effects of null mutations in these genes on the distribution of ascal types was analyzed. In wild-type cultures, the majority of cells formed asci with three or four spores (Fig. 4). The spo74{Delta}/spo74{Delta} mutant, as reported previously, failed to sporulate (29; unpublished data), but SPO74/spo74{Delta} cells sporulated efficiently and formed mostly dyads and triads (Fig. 4). The accumulation of dyads due to heterozygosity of SPO74 is similar to the gene dosage effect observed for the outer plaque components MPC70 and MPC54 (1, 40; unpublished data). In ady4{Delta}/ady4{Delta} cultures, most cells formed spores, but the predominant ascal type was a dyad, which is consistent with the previous description of this mutant (Fig. 4) (29).



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  		FIG. 4. Distribution of ascal types in wild-type, ady4{Delta}/ady4{Delta}, and SPO74/spo74{Delta} cells. Cells from strains AN120 (wild type), MND57 (ady4{Delta}/ady4{Delta}), and MND61 (SPO74/spo74{Delta}) were cultured overnight in 2% potassium acetate, and 200 cells per culture were analyzed. The data shown are averages and standard deviations from at least three independent experiments.

 
To determine whether the dyads produced in ady4{Delta}/ady4{Delta} and SPO74/spo74{Delta} cells are nonsisters, segregation of the centromere-linked markers ARG4 and RME1 was analyzed. In the SPO74/spo74{Delta} mutant, the observed distributions of dyad types for both ARG4 and RME1 were close to the values predicted for nonsister dyads and were significantly different (P < 0.001) from the expected frequencies for random dyads (Table 5). This haplo-insufficient phenotype of SPO74 is also similar to that caused by loss of one copy of MPC70, in which dyads that are formed are exclusively nonsisters (1, 40). In contrast, the observed distributions of dyad types for both ARG4 and RME1 in the ady4{Delta}/ady4{Delta} mutant were significantly different (P < 0.001) from the values predicted for nonsister dyads and were close to the expected frequencies for random dyads (Table 5). The production of nonsister dyads in SPO74/spo74{Delta} cells suggests that Spo74p is a MOP component required for formation of prospore membranes, whereas the accumulation of random dyads in ady4{Delta}/ady4{Delta} cells represents a novel mutant phenotype for a sporulation-specific SPB component.


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  		TABLE 5. Segregation of ARG4 and RME1 in ady4{Delta}/ady4{Delta} and SPO74/spo74{Delta} dyads

 
Prospore membrane formation is defective in ady4{Delta}/ady4{Delta} and spo74{Delta}/spo74{Delta} cells. To determine whether Ady4p and Spo74p promote prospore membrane formation, the number and morphology of prospore membranes in mutants that lack these proteins were analyzed. Cells that express a prospore membrane marker, a fusion of GFP and an amino-terminal fragment of Spo20p (H. Nakanishi and A. M. Neiman, unpublished data), were induced to undergo meiosis and analyzed by fluorescence microscopy. In wild-type cultures, most cells in meiosis II had three or four prospore membranes of comparable size and shape, and nearly all prospore membranes in postmeiotic cells (>99%) surrounded a nucleus (Fig. 5 and Table 6). In spo74{Delta}/spo74{Delta} cultures, the GFP fusion could be detected in some cells as cytoplasmic spots, but no prospore membranes were observed in cells at any stage of meiosis (Fig. 5). The absence of prospore membranes in spo74{Delta}/spo74{Delta} cells is similar to the phenotype caused by loss of either MPC54 or MPC70 and demonstrates that Spo74p is essential for initiation of prospore membrane synthesis (1, 16).



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  		FIG. 5. Prospore membrane morphology in wild-type, ady4{Delta}/ady4{Delta}, and spo74{Delta}/spo74{Delta} cells. Cells from strains AN120 (wild type), MND57 (ady4{Delta}/ady4{Delta}), and MND58 (spo74{Delta}/spo74{Delta}) that had been transformed with pRS424-G20 were induced to enter meiosis, fixed, stained with DAPI, and analyzed by fluorescence microscopy. Prospore membranes (PrMs) were visualized by using the fusion protein of GFP and an amino-terminal fragment of Spo20p, and DNA was visualized with DAPI. PrM and DNA images from one meiosis II cell (left two columns) and PrM, DNA, and merged images from one postmeiotic cell (right three columns) from each strain are shown.

 

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  		TABLE 6. Prospore membrane formation in ady4{Delta}/ady4{Delta} cells

 
Prospore membranes were present in ady4{Delta}/ady4{Delta} cells but varied in morphology and number per cell. Among ady4{Delta}/ady4{Delta} cells in meiosis II, cells with two prospore membranes represented the predominant class and cells with one or three prospore membranes each made up about a quarter of the population (Table 6). In the ady4{Delta}/ady4{Delta} mutant, cells in meiosis II often displayed heterogeneity in the size and morphology of individual prospore membranes, and 16% of prospore membranes in postmeiotic cells had failed to capture nuclei (Fig. 5). These results suggest that Ady4p may facilitate growth of prospore membranes.

MOPs are defective in ady4{Delta}/ady4{Delta} and spo74{Delta}/spo74{Delta} cells. To determine whether modification of the outer plaque and anchoring of the prospore membrane occur normally during meiosis in ady4{Delta}/ady4{Delta} and spo74{Delta}/spo74{Delta} mutants, SPBs in these cells were examined by electron microscopy. In wild-type cells, the MOP appeared as an arc composed of two electron-dense layers of equal thickness (Fig. 6A). The prospore membrane in wild-type cells was closely apposed to the outer layer of the MOP and extended laterally from the MOP parallel to the nuclear envelope (Fig. 6A). In spo74{Delta}/spo74{Delta} cells, only a trace amount of MOP material could be detected in meiosis II, and prospore membranes were absent (Fig. 6C and D). The absence of prospore membranes and most outer plaque material from the SPBs of spo74{Delta}/spo74{Delta} cells is similar to the phenotype caused by loss of either MPC54 or MPC70 and provides further evidence that Spo74p is a MOP component required for prospore membrane synthesis (Fig. 6B) (1, 16).



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  		FIG. 6. SPB morphology in spo74{Delta}/spo74{Delta} cells. Cells from strains MYA-2089 (wild type) (A), MYA-2047 (mpc70{Delta}/mpc70{Delta}) (B), and MYA-1898 (spo74{Delta}/spo74{Delta}) (C and D) were induced to enter meiosis and processed for electron microscopy. Open arrow, prospore membrane in wild-type cell; closed arrow, MOP in wild-type cell; arrowheads, central plaques. Bars, 50 nm.

 
In ady4{Delta}/ady4{Delta} cells, SPBs in meiosis II displayed heterogeneous defects. Of 17 SPBs in ady4{Delta}/ady4{Delta} cells that were analyzed, 13 had normal-looking MOPs and prospore membranes, 2 displayed defects in attachment of the prospore membrane to the MOP, and 2 showed deficiencies in MOP material. Significantly, morphological differences could be detected among SPBs within the same ady4{Delta}/ady4{Delta} cell (Fig. 7). Of the three SPBs that could be identified in a single ady4{Delta}/ady4{Delta} cell in meiosis II, one appeared to be normal (Fig. 7D), another displayed detachment of the prospore membrane from the MOP (Fig. 7C), and the third entirely lacked a prospore membrane and the electron-dense layers of the MOP (Fig. 7E). These findings suggest that Ady4p may facilitate growth of the prospore membrane by stabilizing the MOP.



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  		FIG. 7. SPB morphology in an ady4{Delta}/ady4{Delta} cell. Cells from strain MYA-1993 (ady4{Delta}/ady4{Delta}) were induced to sporulate and processed for electron microscopy. (A and B) Low-magnification images from different sections of the same cell. (C) High-magnification image of the boxed region on the left side of panel A. (D) High-magnification image of the boxed region on the right side of panel A. (E) High-magnification image of the boxed region of panel B. Open arrows, prospore membranes; closed arrows, MOPs; arrowheads, central plaques. Bars, 500 nm (A and B) and 100 nm (C, D, and E).

 
Components of the MOP are interdependent for localization to the SPB. To elucidate the molecular basis of MOP assembly, recruitment to SPBs was individually analyzed for each of the meiosis-specific components of the outer plaque in mutants that lack one of the other components. Strains with homozygous deletions of ADY4, MPC54, MPC70, or SPO74 were transformed with plasmids that contained functional, GFP-tagged versions of the same genes, and plasmids with ADY4-GFP and SPO74-GFP were also introduced into a strain that lacks the constitutive outer plaque component CNM67. Cells were induced to enter meiosis, and the intensity of fluorescent foci at SPBs of cells in meiosis II was analyzed. The results are shown in Table 7. To verify that quantification of the fluorescent signal from SPB foci reflects the amount of a given GFP fusion present at SPBs, fluorescent foci at SPBs during meiosis II were also analyzed in cells in which MPC70-GFP was expressed either homozygously (MPC70-GFP/MPC70-GFP) or heterozygously (MPC70-GFP/MPC70) from its natural locus. The mean fluorescent signal from SPBs in MPC70-GFP/MPC70 cells was 30% of the value from MPC70-GFP/MPC70-GFP cells (data not shown), indicating that the intensity of the fluorescent signal from SPB foci is an approximate representation of the amount of a given GFP fusion present at the SPB.


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  		TABLE 7. SPB fluorescence of GFP fusion proteins in MOP mutants

 
Analysis of recruitment of MOP components to SPBs in outer plaque mutants revealed various interdependencies of these proteins for assembly of the MOP. First, both Ady4p and Spo74p were absent from SPBs in cnm67{Delta}/cnm67{Delta} cells, providing additional evidence that these proteins are components of the outer plaque. Another functional relationship revealed by this analysis is that localization of Mpc70p and Spo74p to SPBs was mutually dependent; the deletion of one eliminated recruitment of the other to SPBs. A third set of interactions that emerged from this analysis is that loss of either Mpc54p, Mp70p, or Spo74p caused a decrease in the recruitment of the other three MOP components to SPBs. Ady4p was most affected by deletion of these components; the loss of any one of these proteins resulted in a >=84% decrease in the amount of Ady4p present at SPBs. The amount of Mpc54p present at SPBs was also decreased significantly by the loss of either Mpc70p or Spo74p, and reciprocally, the loss of Mpc54p caused a significant decrease in the amount of Mpc70p and Spo74p at SPBs. In contrast, the loss of Ady4p had no discernible effect on the recruitment to SPBs of Mpc54p, Mpc70p, or Spo74p. These results indicate that the MOP is formed by the independent recruitment to the SPB of Ady4p and Mpc54p and the coordinated recruitment of Mpc70p and Spo74p and that the latter two recruitment events are required for MOP assembly.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
During meiosis in S. cerevisiae, the outer plaque of the SPB becomes a site of de novo membrane synthesis. Expansion of the outer plaque via an alteration in its molecular composition creates a protein scaffold on which formation of the prospore membrane occurs. In this study we have identified two proteins, Ady4p and Spo74p, that are involved in the meiosis-specific modification of the outer plaque and formation of the prospore membrane.

Functions of Ady4p and Spo74p. Our analysis of SPO74 reveals many similarities between the product of this gene and the meiosis-specific outer plaque components Mpc54p and Mpc70p. First, each of these proteins is localized to spindle poles during meiosis II, and the dependence of this localization on Cnm67p has been demonstrated for Mpc70p and Spo74p (1) (Table 7). Second, heterozygous loss of MPC54, MPC70, or SPO74 leads to accumulation of nonsister dyads (1, 40; M. E. Nickas and A. M. Neiman, unpublished data) (Fig. 4; Table 5). Third, homozygous loss of each of these genes prevents expansion of the outer plaque during meiosis and abolishes formation of the prospore membrane (1, 16) (Fig. 5 and 6). These findings suggest that Spo74p, like Mpc54p and Mpc70p, is a primary structural component of the MOP.

Our analysis of ADY4 demonstrates that it encodes a component of the MOP that is not essential for assembly of this structure. Ady4p is localized to SPBs during meiosis II in a Cnm67p-dependent manner, but expansion of outer plaques and formation of prospore membranes can occur in the absence of Ady4p (Fig. 1, 2, 5, and 7; Tables 6 and 7). Mutations of components involved in assembly of the MOP that incompletely block sporulation result in formation of nonsister dyads, which is thought to be a consequence of the failure to modify daughter SPBs (1, 40). Dyads formed by ady4{Delta}/ady4{Delta} cells, however, are random, indicating that the role of Ady4p in MOP structure and/or function is distinct from those of Mpc54p, Mpc70p, and Spo74p.

The formation of prospore membranes that fail to capture nuclei, observed in ady4{Delta}/ady4{Delta} cells, has also been described for cells that lack Cnm67p, another outer plaque component (1). Despite this similarity, however, the phenotypic defects in ady4{Delta}/ady4{Delta} and cnm67{Delta}/cnm67{Delta} cells are distinct. The majority of SPBs in ady4{Delta}/ady4{Delta} cells have recognizable MOPs that give rise to prospore membranes (Fig. 7 and Table 6). In contrast, the SPBs in cnm67{Delta}/cnm67{Delta} cells lack MOPs entirely, and no prospore membranes can be detected in the majority of these cells (1). When prospore membranes do form in cnm67{Delta}/cnm67{Delta} cells, they are synthesized in an Mpc70p-dependent manner in the cytoplasm, unassociated with nuclei (1). Thus, it has been proposed that prospore membranes in cnm67{Delta}/cnm67{Delta} cells are synthesized on MOP-like structures that are not bound to SPBs.

Another mutant in which prospore membranes fail to capture nuclei that may shed more light on the function of Ady4p is the spo20{Delta} mutant. As in ady4{Delta}/ady4{Delta} cells, prospore membranes in spo20{Delta}/spo20{Delta} cells are initially synthesized on MOPs associated with SPBs in early meiosis II but become detached from SPBs at later stages (23). ady4{Delta}/ady4{Delta} and spo20{Delta}/spo20{Delta} cells also display a similar distribution in the number of prospore membranes per cell (24) (Table 6). SPO20 encodes a sporulation-specific protein required for fusion of vesicles to promote growth of the prospore membrane (23). The similarity of ady4{Delta}/ady4{Delta} and spo20{Delta}/spo20{Delta} mutant phenotypes raises the possibility that Ady4p may play a role in vesicle fusion at the SPB during prospore membrane synthesis.

An alternate possibility for the function of Ady4p is that it preserves the structural integrity of the assembled MOP. A role for Ady4p in stabilizing the MOP is consistent with the defects in the morphology of MOPs and prospore membranes observed in ady4{Delta}/ady4{Delta} cells. The absence of Ady4p could result in a stochastic probability of MOP disassembly during meiosis II and produce the variable abnormalities observed in ady4{Delta}/ady4{Delta} cells. For example, MOPs that disassemble shortly after formation would not form prospore membranes at all, whereas MOPs that disassemble later in meiosis II would produce prospore membranes that become detached from SPBs and fail to capture nuclei. Those MOPs that remain intact in ady4{Delta}/ady4{Delta} cells could give rise to viable spores. Moreover, the observation that dyads formed in ady4{Delta}/ady4{Delta} cells are random is consistent with the idea that MOP disassembly occurs by chance. Further support for a role for Ady4p in promoting MOP stability comes from preliminary three-dimensional tomographic analysis of meiotic SPBs in ady4{Delta}/ady4{Delta} cells, which reveals ultrastructural defects in the MOP that are not readily apparent in standard thin-section transmission electron micrographs (C. Schwartz and A. M. Neiman, unpublished data).

Model of meiotic outer plaque assembly. The results presented here and those of previous studies suggest that the four meiosis-specific components of the outer plaque are recruited to the SPB via the independent binding to Nud1p of Ady4p, Mpc54p, and Mpc70p. First, Ady4p, Mpc54p, and Mpc70p are localized to SPBs during meiosis II in the absence of either of the other two (16) (Table 7). Further support for this idea is the finding that Ady4p, Mpc54p, and Mpc70p interact with Nud1p in the two-hybrid assay (16) (Table 4). Finally, the observation that Mpc70p and Spo74p are mutually dependent for localization to SPBs suggests that recruitment of Spo74p is mediated by Mpc70p (Table 7). The failure of Mpc70p to localize to SPBs in the absence of Spo74p may indicate that Spo74p regulates the function of Mpc70p or forms a subcomplex with it prior to recruitment to the SPB.

The findings presented here and elsewhere can be incorporated into a structural model of the MOP at the SPB (Fig. 8). Several observations suggest that Mpc54p, Mpc70p, and Spo74p assemble into a repeating unit at the SPB that forms the ordered matrix upon which prospore membrane synthesis occurs. First, the phenotypes described above caused by deletion of MPC54, MPC70, and SPO74 are similar. Second, each of these proteins interacts with itself and with the other two in the two-hybrid assay (16) (Fig. 3 and Table 4). Third, the loss of any one of these proteins leads to a decrease in the amount of the other two and of Ady4p present at SPBs (Table 7). In contrast, the phenotypes of ady4{Delta}/ady4{Delta} cells suggest that Ady4p is not a primary structural component of the ordered array that promotes synthesis of the prospore membrane. The observation that Ady4p interacts specifically with Mpc70p and Nud1p in the two-hybrid assay suggests that Ady4p may localize to the interface between the constitutive components of the outer plaque and the meiosis-specific matrix, consistent with a potential role for this protein in maintaining the stability of the MOP, as discussed above.



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  		FIG. 8. Model of the assembled MOP of the SPB. Six polypeptide subunits of the MOP are shown in ordered array between the central plaque and the prospore membrane. Arrows to the electron micrograph of the MOP on the left show layers that correspond to structures in the model.

 
Our model of MOP assembly gives rise to specific questions about the structure and function of this protein matrix. One important issue is the stoichiometry and orientation of Mpc54p, Mpc70p, and Spo74p in the basic structural unit of the MOP. In other regions of the SPB, coiled-coil proteins such as Spc42p and Cnm67p form tightly packed, organized arrays of parallel (i.e., head-to-head) subunits that create asymmetric layers (3, 31). Thus, a parallel arrangement seems likely for subunits of Mpc54p and Mpc70p within the meiosis-specific layers of the outer plaque. Another critical question is how the MOP promotes homotypic fusion of secretory vesicles to initiate prospore membrane synthesis. Formation of prospore membranes requires Mpc54p, Mpc70p, and Spo74p but can occur, albeit abnormally, in the absence of either Cnm67p or Ady4p (1, 16) (Fig. 5 to 7). Thus, oligomerization of Mpc54p, Mpc70p, and Spo74p during assembly of the MOP matrix may trigger vesicle fusion and drive prospore membrane formation. Finally, how does the MOP anchor the prospore membrane? Ady4p appears to facilitate anchoring of the membrane to the plaque, but neither Ady4p nor any of the other MOP components yet identified has a recognizable membrane-binding domain. Future studies will be focused on addressing these questions.

Biogenesis of membranes on protein scaffolds. Insight into the molecular mechanism by which the MOP promotes synthesis of the prospore membrane in S. cerevisiae may come from the study of the biogenesis of other membranous organelles that are assembled on protein scaffolds. One such process is formation of cell walls in the syncytial endosperm of Arabidopsis thaliana. During endosperm development, cellularization of the multinucleate endosperm occurs via the de novo synthesis of cell walls between adjacent nuclei (6). These new cell walls are synthesized by the expansion of tubular membranes that are surrounded by a filamentous coat, which may act as a membrane-organizing center in a manner similar to that of the MOP during formation of the prospore membrane (27).

A better-characterized example of de novo membrane synthesis on a protein scaffold is assembly of the Golgi complex. Upon exit from mitosis, the Golgi complex assembles by concurrent formation of membrane cisternae and a matrix composed largely of coiled-coil proteins termed Golgins (17). Under conditions that disrupt transport of Golgi membranes, the Golgi matrix remains intact during interphase and is equally partitioned during mitosis (32, 33) It has thus been proposed that the Golgi matrix acts as a substratum that maintains the structural organization of the membrane cisternae. The Golgi matrix plays two roles in the formation of Golgi stacks that may be analogous to specific functions of the MOP in assembly of the prospore membrane. First, the proteins of the Golgi matrix and associated coiled-coil proteins act as tethers to dock precursor vesicles in proximity to one another, a requisite step for fusion of vesicles into cisternae (22, 35). Second, these proteins anchor Golgi membranes to the matrix to stabilize the cisternae in the stacked arrangement characteristic of this organelle (2, 34, 36).

Assembly of the MOP and synthesis of the prospore membrane may be similar in mechanism to formation of the Golgi complex. It has been proposed that vesicle precursors to the prospore membrane accumulate at the SPB prior to assembly of the MOP (21). Thus, oligomerizaton of the coiled-coil proteins Mpc54p and Mpc70p may trigger fusion of such precursor vesicles by bringing them into proximity in a Golgin-like manner. Subsequently, Mpc54p and Mpc70p may assume another Golgin-like role by anchoring the growing prospore membrane to the assembled MOP. A more precise understanding of the early events of assembly of the MOP and prospore membrane will reveal the extent to which the similarities between this process and biogenesis of the Golgi complex reflect common molecular mechanisms of de novo synthesis of membranes on protein scaffolds.


   ACKNOWLEDGMENTS
 
We thank Hideki Nakanishi for providing pRS424-G20 prior to publication; Hiroyuki Tachikawa, Elmar Schiebel, and Jeremy Thorner for providing strains and plasmids; J. Kahana and P. Silver for providing antibodies to GFP; Eileen O'Toole for technical assistance with electron microscopy; and Andrew Staehelin and members of the Neiman lab for helpful discussion.

This work was supported by National Institutes of Health grant GM62154 to A.M.N. and National Institutes of Health Biotechnology Resources grant RR00592 to J. R. McIntosh.


   FOOTNOTES
 
* Corresponding author. Mailing address: 332 Life Sciences, Department of Biochemistry and Cell Biology, SUNY Stony Brook, Stony Brook, NY 11794-5215. Phone: (631) 632-1543. Fax: (631) 632-8575. E-mail: Aaron.Neiman{at}sunysb.edu. Back


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 Materials and Methods
 Results
 Discussion
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Eukaryotic Cell, June 2003, p. 431-445, Vol. 2, No. 3
1535-9778/03/$08.00+0     DOI: 10.1128/EC.2.3.431-445.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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