Modification of the SPB.
The SPB is the sole microtubule-organizing center in an S. cerevisiae
cell and spans the nuclear envelope so that it has distinct nuclear and cytoplasmic faces (67
). The SPB is a multilaminar structure with a nuclear face, or inner plaque, from which spindle microtubules are nucleated; a central plaque, which contains the Spc42 protein, spanning the nuclear envelope; and a cytoplasmic face, or outer plaque, which is the organizing center for cytoplasmic microtubules (Fig. ). In vegetative cells, the outer plaque consists primarily of three proteins, Cnm67p, Nud1p, and Spc72p (183
). Spc72p acts a receptor for the gamma-tubulin complex, anchoring cytoplasmic microtubules to the SPB (75
FIG. 3. Organization of proteins within the meiosis II outer plaque. The changes in organization and composition between a mitotic/meiosis I outer plaque and a meiosis II outer plaque are shown in the cartoon. The Spc72p and γ-tubulin complex proteins (more ...)
At the onset of meiosis II, Spc72p is lost from the cytoplasmic face of the SPB and replaced by a several meiosis-specific proteins, which together make up the meiosis II outer plaque (76
) (Fig. ). This change in composition converts the function of the cytoplasmic face of the SPB from microtubule nucleation to membrane nucleation. The meiosis II outer plaque is the site of formation of the prospore membrane (52
). Early work established a correlation between the number of SPBs displaying an outer plaque and the number of prospore membranes formed, suggesting that the meiosis II outer plaque is essential for prospore membrane formation (31
). More recently, several gene products that are components of this structure have been identified. The first of these was Cnm67p, a coiled-coil protein that is present on the vegetative SPB outer plaque (10
). The original study noted that cnm67
homozygous mutants fail to sporulate, suggesting that Cnm67p is also required for assembly of the meiosis II outer plaque, a prediction that was subsequently confirmed (4
). Of the other identified components of the vegetative outer plaque, only Nud1p is also present in outer plaques in meiosis II (76
). The remaining genes known to encode components of this structure are meiosis specific in their expression. These are MPC54
, and ADY4
Figure shows the proposed arrangement of these proteins within the meiosis II outer plaque (76
). The large electron density that is in contact with the prospore membrane is composed of the Mpc54, Spo21, and Spo74 proteins. Two of these proteins, Spo21p and Mpc54p, contain predicted coiled-coil domains, while Spo74p is globular (4
). By analogy to areas of lower electron density in other parts of the SPB (149
), the lower density in the middle of the Mpc54p/Spo21p/Spo74p region is thought to be the coiled-coil regions of the Mpc54 and Spo21 proteins (Fig. ). Individual deletion of any of these three genes produces a common phenotype: the outer plaque structure is ablated, and no prospore membrane forms (4
). Thus, assembly of the outermost layers of the plaque requires all three components, and, as inferred in the earlier studies, this structure is essential for the coalescence of vesicles into a prospore membrane.
The Nud1 and Ady4 proteins are thought to reside on the inner side of the Mpc54p/Spo21p/Spo74p complex (Fig. ) (113
). Nud1p is also present in vegetative outer plaques, where it serves as the SPB anchor for Spc72p and for components of the mitotic exit network (51
). The function of Nud1p at the SPB during sporulation has not been described. Ady4p appears to be a minor component of the meiotic SPB. Though not essential for formation of the meiosis II outer plaque, it is important for stability of the structure. In ady4
mutants a fraction of the outer plaques appear to disassemble during meiosis II, leading to the failure of some prospore membranes to package nuclei and the production of asci with fewer than four spores (113
The large area of low density between the outer plaque and the central plaque has been demonstrated in vegetative cells to correspond to the coiled-coil domain of Cnm67p (149
) (Fig. ). It is thought that Cnm67p also links the central plaque to the outer plaque in the meiotic SPB (4
). The particular importance of the meiosis II outer plaque in allowing membrane formation is shown by the distinct phenotype of cnm67
mutants during sporulation (4
). In cnm67
cells, no outer plaque structures are seen at the SPB; in fact, even less residual electron density is present than in mpc54
, or spo74
mutants. Unlike these other mutants, a fraction of cnm67
cells do form prospore membranes; however, these membranes are not anchored to the SPB and fail to capture nuclei. This ectopic formation of prospore membranes is proposed to result from the formation of MPC54
complexes that are not anchored to the rest of the SPB, an interpretation supported by the observation that ectopic membranes are absent in a cnm67 spo21
double mutant (4
The identification of multiple components of the meiosis II outer plaque has led to the demonstration that this structure is essential both for the coalescence of vesicles into a prospore membrane and to anchor that membrane to the nucleus and facilitate nuclear capture. The mechanisms by which this structure binds to the membrane and promotes fusion remain unknown. However, as described in the next section, the critical role of the meiosis II outer plaque in membrane formation allows the cell to regulate spore formation by regulating this modification of the SPB.
(i) Regulation of SPB assembly by carbon availability.
Sporulation of diploid cells occurs in response to starvation conditions, specifically the absence of nitrogen and the presence of a nonfermentable carbon source such as acetate. In a seminal study, Davidow et al. (31
) defined a secondary response to the depletion of the carbon source during the process of sporulation, the formation of a particular form of two-spored ascus or dyad. Using a strain background that was temperature sensitive for progression out of meiotic prophase, the authors found that the strain could be held in prophase by incubation at 37°C and that when released by transfer to lower temperature, it proceeded through meiosis and sporulated. If the strain was held at 37°C for up to 24 h, it formed tetrads upon a shift to lower temperature. However, when the temperature was lowered after arrest for greater than 24 h, the cells predominantly formed dyads. Dissection and analysis of marker genes segregating in the dyads revealed that the spores were viable and haploid. Moreover, for heterozygous markers that were tightly centromere linked, there was always one spore of each genotype. That is, the chromosomes present in the two spores were homologues, not sister chromatids. This segregation pattern distinguished these dyads from other mutants that form dyads of two haploid spores in which centromeric markers segregate randomly or dyads which contain two diploid spores (73
). Because of the chromosome segregation pattern, these dyads were termed nonsister dyads (NSDs). An outline of NSD formation is shown in Fig. .
FIG. 4. Nonsister dyad formation. (A to C) During the first meiotic division, homologous chromosomes segregate to opposite poles, and at the second meiotic division, sister chromatids are separated. (C) When sporulated under acetate-depleted conditions, only (more ...)
Two critical features of NSD formation were elucidated in this study. First, it was shown that the switch from tetrads to NSDs is triggered by the depletion of acetate from the medium. This suggests that NSD formation is a regulated response to carbon depletion. In fact, a subsequent study found that transfer of cells from sporulation medium to water prior to meiosis I could trigger the same phenomenon (158
). Second, in cells forming NSDs, the basis for the formation of two spores was shown to be that only two of the four SPBs, one on each of the two meiosis II spindles, are modified on their outer plaque. As a consequence, only two prospore membranes form and, therefore, only two spores. Because one pole from each spindle is modified, the resulting spores will contain centromeres from homologous chromosomes, which separate from each other at the first meiotic division (Fig. ). Thus, NSD formation is a conservation mechanism that allows a cell that is committed to sporulation to save resources by reducing the number of daughter cells it will form. Moreover, this reduction results from the regulation of SPB modification in response to carbon availability. This regulated change in SPB modification will be referred to as the NSD response.
Subsequent studies identified two mutants that formed NSDs at high frequency even under conditions where acetate was plentiful (33
). The first of these mutant genes, hfd1
, has been lost (T. Iino, personal communication). The second, ady1
, encodes a nucleus-localized protein that triggers NSD formation by causing only two of the four SPBs to be modified, similar to the metabolic response (33
). Although the molecular function of ADY1
remains unknown, it may play a role in SPB modification in response to carbon limitation.
A third mutant condition that causes NSD formation is haplo-insufficiency of outer plaque components. Strains heterozygous for a deletion of SPO21
, or MPC54
form a high percentage of NSDs under conditions where wild-type cells would form tetrads (4
; A. M. Neiman, unpublished observations). Additionally, a particular allele of the central plaque component gene SPC42
has been reported to increase NSD formation (62
). Not all mutations affecting the SPB lead to this phenotype, however, as cnm67
heterozygotes form tetrads and ady4
homozygotes form random, rather than nonsister, dyads (A. M. Neiman and M. Nickas, unpublished observations) (113
). The consistent pattern that emerges from these studies is that decreasing the abundance of any one of the outermost components of the meiosis II outer plaque leads to formation of NSDs.
The studies described above suggested that SPO21
, and/or MPC54
might be a regulatory target of the NSD response. Direct observation of the localization of these three proteins in cells undergoing the NSD response revealed that Spo74p and Spo21p are absent from two of the four spindle poles during meiosis II but that Mpc54p and the other outer plaque components are still found predominantly on all four SPBs (111
). Thus, localization of Spo74p and Spo21p to the SPB seems to be regulated in response to carbon availability. Under normal conditions, these two proteins are interdependent for localization to the SPB: Spo74p does not localize to spindle poles in a spo21
mutant and vice versa, so regulation of either one of the proteins would account for the effects seen (113
(ii) Asymmetric control of mother and daughter SPBs during sporulation.
How the localization of Spo21p and Spo74p to the pole is regulated during NSD response remains an open question, but some insight into this issue was afforded by the identification of which SPBs these proteins are recruited to (or excluded from) during the NSD response. At the meiosis I/meiosis II transition, the two SPBs present during meiosis I duplicate to form the two poles of each meiosis II spindle so that each meiosis II spindle contains a mother pole and daughter pole (Fig. ). During mitotic growth, the asymmetric localization of the Kar9 protein to the mother, but not daughter, poles is important for proper orientation of the mitotic spindle and for nuclear segregation into the bud (88
plays no obvious role in sporulation (36
), but, by analogy, formation of NSDs could be explained if the mother and daughter poles are distinct in their capacity to form meiosis II outer plaques under NSD conditions.
By using an Spc42 fusion protein that specifically marks the older, mother SPBs (127
), it was shown that during the NSD response the Spo21 and Spo74 proteins are found almost exclusively on the daughter SPBs in meiosis II, indicating that NSDs are formed by modification of only the daughter SPBs (Fig. ) (111
). Remarkably, when NSDs are triggered by haplo-insufficiency of spo21
, it is again only the daughter SPBs that get modified (M. Nickas and A. M. Neiman, unpublished observations). While the molecular mechanisms at work remain to be elucidated, these results allow a more precise description of the phenomenon: the depletion of the carbon source during the process of sporulation leads to a failure to recruit the Spo74 and Spo21 proteins to the mother SPBs. Any model describing these events must account for the fact that simply lowering the amount of these proteins is sufficient to generate the asymmetric use of the mother and daughter SPBs. One proposed explanation is that at the meiosis I/meiosis II transition the daughter SPBs are modified—that is, assemble membrane-organizing outer plaques—before the mother SPBs (111
). This may be because the mother SPBs have a preexisting microtubule-nucleating outer plaque that must be rearranged. If the outer plaque components are limiting, the daughter SPBs use up the available pool and so the mother SPBs are not modified. If assembly of the outer plaque structure was highly cooperative, then limitation of any one of the components would cause this effect. This model can account for the haplo-insufficient phenotype of mpc54
, or spo74
mutants and suggests that limiting expression of SPO21
and or SPO74
could be the basis for regulation by carbon depletion. Though appealingly simple, this proposal has yet to be rigorously tested. Much remains to be learned about the regulatory mechanisms governing assembly of this membrane-organizing center.
(iii) An intermediary metabolite regulates SPB modification.
The original description of the NSD response demonstrated that depletion of acetate from the medium triggered this response (31
). What remained unclear was whether the cell was responding directly to the disappearance of acetate or perhaps to the absence of some downstream product of acetate metabolism. In sporulation medium, acetate is used by the cell as both the energy source and the source of structural carbon for biosynthesis of macromolecules (39
). The metabolic pathways of acetate usage are summarized in Fig. . For ATP generation, acetate is converted to acetyl coenzyme A (acetyl-CoA) and oxidized in the tricarboxylic acid (TCA) cycle to generate reducing equivalents for oxidative phosphorylation (157
). For biosynthetic purposes, the acetyl-CoA must feed into synthesis of lipids, nucleotides, and polysaccharides (there is no net synthesis of amino acids during sporulation because of the absence of a nitrogen source) (129
). Carboxylation of acetyl-CoA to malonyl-CoA provides the building blocks for lipid biosynthesis. However, nucleotide synthesis for DNA replication and synthesis of the polysaccharide layers of the spore wall require that the acetate first be converted into glucose (129
). Gluconeogenesis consumes two molecules of the TCA cycle intermediate oxaloacetate for each glucose molecule produced. Oxaloacetate is also required as a catalytic intermediate in the TCA cycle. To prevent gluconeogenic consumption of oxaloacetate from interfering with oxidation of acetyl-CoA, the yeast cell generates oxaloacetate via the glyoxylate cycle (77
) (Fig. ). The glyoxylate cycle consumes two acetyl-CoA molecules and produces a molecule of succinate without donating electrons to the electron transport chain. Succinate is then oxidized to oxaloacetate and provides the starting point for gluconeogenesis. It should be noted that many of the glyoxylate cycle enzymes are located in different cellular compartments from the analogous TCA cycle enzymes, and it is a cytoplasmic pool of oxaloacetate that is used for gluconeogenesis as opposed to the oxaloacetate that functions in the TCA cycle, which is located in the mitochondrial matrix (59
FIG. 5. Pathways of acetate metabolism in sporulating cells. Carbon sources mentioned in the text are shown in italic, and whether cells form tetrads or NSDs when sporulated on those carbon sources is indicated. Positions at which mutations in the glyoxylate (more ...)
If a product of acetate metabolism is used by the cell to detect the availability of the environmental carbon source, then blocks in acetate metabolism might lead to the formation of NSDs even in the presence of acetate. This “sensor metabolite” could be an end product of acetate metabolism, such as glucose or ATP, or an intermediate in acetate usage. Strikingly, when mutants blocked in the glyoxylate cycle or the subsequent conversion of succinate to fumarate were sporulated in acetate, about 50% of the cells formed NSDs, indicating that these cells were undergoing the NSD response (111
). These mutations do not effect the TCA cycle or ATP generation, and therefore these results implicate a downstream product of the glyoxylate cycle as the sensor metabolite. By contrast, mutations that block the committed steps of gluconeogenesis did not cause NSD production (111
). These results suggest that the sensor metabolite is produced even when gluconeogenesis is blocked. Together these genetic tests map the sensor to one of three metabolites: fumarate, malate, or oxaloacetate (Fig. ).
Further support for the idea that one of these three molecules is used by the cell as a carbon sensor was provided by changing the carbon source for sporulation. Sporulation on pyruvate as the principal carbon source restored tetrad formation to the glyoxylate cycle mutants. This suggests that the sensor intermediate is produced from pyruvate. To feed gluconeogenesis, pyruvate is converted directly to oxaloacetate, bypassing the need for the glyoxylate cycle (Fig. ). Thus, these experiments suggest that a cytoplasmic pool of oxaloacetate (or some derivative) is the sensor intermediate. Consistent with this interpretation, when wild-type cells are sporulated on glycerol as the sole carbon source, they form almost exclusively NSDs (111
). The conversion of glycerol to glucose, or its conversion to acetyl-CoA for oxidation in the TCA cycle, never requires the generation of cytoplasmic oxaloacetate (Fig. ). Thus, under these conditions, even though the cell has ample carbon available to meet both its energy and biosynthetic needs, it behaves as though carbon were limiting.
In sum, the NSD response allows the cell to conserve resources when it has committed to meiosis and sporulation but environmental conditions may not support the formation of four daughter cells. As cells sporulate in acetate, increased flux through the glyoxylate pathway generates a increase in the cytoplasmic pool of oxaloacetate. By a mechanism yet to be explained, the presence of oxaloacetate is measured by the cell as an indicator of the available biosynthetic carbon pool. If sufficient oxaloacetate is present, the cell modifies all four spindle pole bodies and a tetrad is formed. If oxaloacetate is not present, then Spo21p and Spo74p are not recruited to the mother SPBs and NSDs are formed. It is interesting that the NSD/tetrad decision appears to be tuned to an intermediate in acetate metabolism. Studies of sporulation on different carbon sources demonstrated that acetate is the preferred carbon source for sporulation (95
). Acetic acid-producing bacteria and yeasts are major components of the microbial flora that grow on broken grapes (43
). It is tempting to speculate that in nature S. cerevisiae
grows in competition with acetate-producing bacteria and, therefore, that acetate is the carbon source most likely to be present at times of nitrogen depletion. Perhaps for this reason S. cerevisiae
has tuned its sporulation program to respond to acetate levels.
Initial formation of the prospore membrane.
Once the meiosis II outer plaque has assembled, it becomes a site for the docking of precursor vesicles for the prospore membrane (104
). These vesicles are post-Golgi vesicles that carry proteins destined for an extracellular compartment and are analogous to secretory vesicles in vegetative cells (106
). Furthermore, the fusion of these vesicles requires many of the same proteins necessary for fusion of secretory vesicles at the plasma membrane (106
). In contrast to secretory vesicles in vegetative cells, which are delivered to the periphery along actin cables (132
), in sporulating cells vesicles are delivered initially to the spindle poles and then to the growing prospore membrane. Several lines of evidence suggest that actin does not play a critical role in vesicle delivery during sporulation. Mutations of many actin-organizing proteins that disrupt vesicle delivery in vegetative cells, as well as mutations of the ACT1
gene itself, do not significantly affect sporulation efficiency (34
). Moreover, while treatment of sporulating cells with the actin-depolymerizing drug Latrunculin A blocks sporulation, it does not inhibit prospore membrane growth (A. Coluccio and A. M. Neiman, unpublished observations). Taken together, these observations suggest that actin-based transport does not play a critical role in this process. Delivery apparently does not require cytoplasmic microtubules, which are absent by this stage of sporulation due to the changes in the SPB. The mechanism of vesicle delivery to the prospore membrane is unknown and remains an important challenge in our understanding of the process.
In addition to this change in vesicle delivery, the fusion of vesicles with the prospore membrane has different genetic requirements than fusion of vesicles with the plasma membrane. After secretory vesicles are delivered to the plasma membrane, the fusion of the vesicle membrane and the plasma membrane is then mediated by a heterotrimeric SNARE complex (141
). Analogous SNARE complexes function for each fusion event in the secretory pathway, and much of the specificity of vesicle/target membrane interactions that is required for proper trafficking through the secretory pathway is thought to be based on cognate interactions between SNARE proteins (123
). SNARE complexes are based on the formation of a four-helix bundle between different subunits of the hetero-oligomer (162
). Three of the helices are provided by proteins associated with the target membrane (the t-SNAREs) and one is provided by a protein on the surface of the vesicle (the v-SNARE). The v-SNARE and at least one of the t-SNARE proteins are transmembrane proteins, and assembly of the bundle drives these transmembrane domains together, possibly leading directly to the fusion of the two lipid bilayers (180
For fusion of vesicles with the plasma membrane, the t-SNARE consists of the Sso1 or Sso2 protein, which are redundant for this purpose, in partnership with the Sec9 protein (1
). Sso1p or Sso2p provides the transmembrane domain and one helix of the oligomer, whereas Sec9p is a peripheral membrane protein that provides two helices. The v-SNARE is encoded by a second redundant pair of genes, SNC1
). The first genetic distinction between vesicle fusion at the plasma membrane or prospore membrane was the finding that the t-SNARE SEC9
was dispensable for sporulation (106
). During sporulation, Sec9p is replaced by the related, sporulation-specific Spo20 protein. Although Spo20p and Sec9p are homologous, they are largely distinct in their sites of action. Ectopic expression of SPO20
in vegetative cells cannot rescue the growth defect of a sec9
-ts mutant, nor can expression of SEC9
under the SPO20
promoter rescue the sporulation defect in a spo20
). However, there is some overlap of function in sporulation, as a spo20
single mutant produces abnormal prospore membranes whereas a spo20 sec9
-ts mutant displays no prospore membranes at all, suggesting that SEC9
is capable of supporting at least abortive prospore membrane assembly (106
). In chimera studies, the major determinant of sporulation function of Spo20p mapped to an amino-terminal region outside of the domain in which it is homologous to Sec9p (107
Also indicative of the close relationship between Sec9p and Spo20p, both bind in vitro to the Sso1 protein and can form ternary complexes with Sso1p and Snc2p with similar efficiency (107
). Indirect immunofluorescence using polyclonal antibodies to the Sso and Snc proteins demonstrated that these SNAREs localize to the prospore membrane. Based on these observations, the change in fusion functions between the plasma membrane and the prospore membrane was proposed to be a switch in one subunit of the SNARE complex, an exchange between Spo20p and Sec9p (107
However, the requirements for vesicle fusion at the prospore membrane are more complicated than this initial picture, as there is further specialization of the SNARE complex (Fig. ). A study of sso1
mutants revealed that an sso1
single mutant, which has no strong vegetative phenotype, is completely defective in sporulation (66
). The sso1
phenotype could not be rescued by overexpression of SSO2
. A subsequent chimera study suggested that the specificity of SSO1
for sporulation lay partly in its amino-terminal region (the only region of the proteins with significant sequence divergence) and, surprisingly, in the 3′ untranslated region of the SSO1
). The 3′ untranslated region did not seem to have a significant effect on the levels of the Sso1 protein, so the basis for this effect remains mysterious. An examination of the sporulation defect in sso1
cells revealed that prospore membranes are absent in the mutant, indicating that SSO1
is required for the fusion of vesicles to form a prospore membrane (H. Nakanishi and A. M. Neiman, unpublished data). Thus, Sso1p and Sso2p are redundant for fusion at the plasma membrane, but only Sso1p can function during fusion at the prospore membrane.
FIG. 6. Specific SNARE complexes mediate fusion with the plasma membrane and prospore membrane. Secretory vesicle fusion with the plasma membrane is mediated by a heterotrimer consisting of Sso1p or Sso2p, Sec9p, and Snc1p or Snc2p (left panel). At the prospore (more ...)
Studies of the lipid-modifying enzyme phospholipase D suggest that changes in lipid composition are also important for assembly of the prospore membrane (143
). This enzyme, which hydrolyzes the choline head group of phosphatidylcholine to produce phosphatidic acid, is encoded by the SPO14
gene, and the Spo14 protein is localized to the prospore membrane during sporulation (140
). Originally identified on the basis of its sporulation defect, this gene is transcribed in both vegetative and sporulating cells, but deletion of the gene has significant phenotypes only during sporulation. Deletion of SPO14
leads to a failure to form prospore membranes (143
). Prospore membranes are also absent in cells expressing a point mutation in SPO14
that encodes a catalytically inactive protein, indicating that enzyme activity is critical for function (143
). In the presence of 1-butanol, phospholipase D will convert phosphatidylcholine into choline and phosphatidylbutanol rather than phosphatidic acid. Addition of 1-butanol, but not 2-butanol, to sporulation medium blocks sporulation (144
). Together these results indicate that it is the production of phosphatidic acid rather than the turnover of phosphatidylcholine which is in some way critical for membrane assembly (144
One role for Spo14-generated phosphatidic acid in prospore membrane formation may be in enhancing the activity of Spo20p. As noted earlier, the amino-terminal domain of Spo20p contains a region crucial for the function of this protein at the prospore membrane (103
). This crucial “activating” function is a lipid binding domain that can bind to phosphatidic acid and is essential for localization of the Spo20p in vivo (103
). Moreover, translocation of Spo14p to the plasma membrane in vegetative cells, an arrangement analogous to that on the prospore membrane, allows Spo20p to replace Sec9p for fusion of vesicles at the plasma membrane (28
). This is true even for derivatives of Spo20p that lack the phosphatidic acid binding domain. However, the phenotype of a spo14
mutant is more extreme than that of a spo20
). Therefore, although SPO14
may be important for the function of Spo20p-containing SNAREs, phosphatidic acid may play additional roles in prospore membrane assembly as well. Deciphering the interaction between SNARE specificity and lipid composition during fusion at the prospore membrane is an important challenge that will shed light more generally on how the specificity of fusion is regulated in the secretory pathway.
Septins are a conserved family of filament-forming proteins (72
). In vegetative cells, five distinct septin proteins, Cdc3, Cdc10, Cdc11, Cdc12, and Sep7, form a ring structure at the bud neck (48
). The septin ring plays a variety of important roles as a scaffold for the organization of complexes involved in cytokinesis, cell wall deposition, and cell cycle regulation (48
). In addition, the septin ring appears to form a diffusion barrier that aids in differentiating the bud from the mother cell (5
). In sporulating cells, septin rings on the cell plasma membrane are disassembled and the septins relocalized to the forming prospore membrane (41
At least three and possibly all five of the septins found in septin rings are localized to the prospore membrane, and two of the genes, CDC3
, are strongly upregulated by Ndt80p in mid-sporulation (25
). In addition, two sporulation-specific septin proteins, Spr3p and Spr28p, are also found in the prospore membrane-associated septin structures. Several lines of evidence suggest that the organization of the septins on the prospore membrane is distinct from that in the septin ring. First, while the septin ring in vegetative cells is a relatively static structure, during sporulation the septins move as the prospore membrane expands (35
). Second, their arrangement on the prospore membrane is different than that at the bud neck. By immunofluorescence, the septins appear first to be organized as rings near the SPB when prospore membranes are small and then to expand into a pair of bars that run parallel to the long axis of the prospore membrane as the membrane expands (Fig. ) (41
). Three-dimensional reconstructions reveal these bars to be a pair of sheets running on opposite sides of the membrane (163
). Moreover, these sheets contain the Spr3 and Spr28 proteins, which cannot be incorporated into the septin ring when expressed in vegetative cells (41
; H. Tachikawa, personal communication). Finally, in vegetative cells mutation of any one septin (all except for SEP7
) leads to the disappearance of bud neck rings. By contrast, mutation of individual septins, or even an spr3 spr28 cdc10
triple mutation, has no strong effect on the ability of the other septins to organize on the prospore membrane (35
; A. M. Neiman, unpublished observations). Thus, the organization of these filaments may be distinct in sporulating cells.
What is the role of the septins in spore formation? Not only does mutation of one or multiple septins during sporulation not disrupt the localization of the other septins, the mutants do not have a strong sporulation phenotype (35
; A. M. Neiman, unpublished observations). This suggests that if, as implied by their localization pattern, the septins play an important role in spore formation, then they must function redundantly in this process. Analogous to their role in vegetative cells, the septins may help to localize other proteins to specific areas of the prospore membrane, for example, the Gip1 protein. GIP1
encodes a sporulation-specific regulatory subunit of the Glc7 protein phosphatase (170
). During sporulation, Gip1p and Glc7p colocalize with the septins throughout prospore membrane formation (163
). Mutation of gip1
or alleles of glc7
that cannot interact with Gip1p leads to the failure of septins to localize to the prospore membrane, indicating that these proteins both organize and may be part of the septin complex (163
mutants lack organized septins, the phenotype of the mutant may provide some insight into the role of septins in spore formation. In fact, prospore membrane formation is largely normal in gip1
mutants. Four membranes are formed per cell, and these membranes, though they appear slightly smaller than in wild-type cells, capture nuclei efficiently (163
). The most striking defect in gip1
mutants appears to be in triggering the assembly of the spore wall in response to closure of the prospore membrane. Whether this phenotype is due to the absence of organized septins or the absence of an independent function of GIP1
remains to be determined, but the phenotype of gip1
raises the possibility that septins may be involved in monitoring membrane growth and closure rather than in controlling membrane growth.
(ii) The leading-edge complex.
Electron micrographs reveal an electron-dense coat, termed the leading-edge complex, located at the lip of each growing prospore membrane (21
). Three components of this coat have been identified: Ssp1p, Ady3p, and Don1p (101
). Localization of these proteins by immunofluorescence and green fluorescent protein tagging confirms that they form a ring structure at the mouth of the growing prospore membrane (101
were originally identified on the basis of their sporulation-specific expression (101
, by contrast, was identified by its ability to bind to meiotic SPB components in both two-hybrid and copurification studies (63
). At early times of prospore membrane assembly, Ady3p is found on the SPB, whereas the other two subunits are localized in a punctate pattern in the cytosol, suggesting an association with transport vesicles (101
). As the vesicles coalesce into a membrane, all three proteins can be found at the SPB. It has been suggested that this pattern of localization indicates a role for these proteins in the recruitment of vesicles to the SPB (101
). As the prospore membrane expands, the localization patterns of the three proteins resolve into a ring structure that remains associated with the leading edge of the prospore membrane (Fig. ). This structure is distinct from the septin sheets described above. Double-labeling studies indicate that the septins run backwards from the leading edge towards the SPB (101
). However, the assemblies of the two structures appear to be independent: septin localization is retained in leading-edge complex mutants, and the leading-edge complex is unaffected in a gip1
mutant where septin localization is lost (163
The organization of Ssp1p, Don1p, and Ady3p within the leading-edge complex is not known, but localization dependence studies suggest a stratified arrangement (101
). Deletion of DON1
does not affect the localization of the other two proteins. Deletion of ADY3
causes the delocalization of Don1p, but Ssp1p remains in a ring at the prospore membrane lip. Deletion of SSP1
causes the disappearance of all three proteins. Therefore, Ssp1p may be the most membrane-proximal component, serving to connect Ady3p to the leading edge, and Ady3p in turn anchors Don1p.
Mirroring this stratified arrangement, the sporulation phenotypes of the three mutants are progressively severe. Mutation of DON1
produces no obvious sporulation defect (76
). As suggested by its name, loss of ADY3
ads) leads to an increase in asci with fewer than four spores (133
). The spores present in these dyads are haploid and are packaged randomly with respect to centromere-linked markers, unlike the nonsister dyads described above (101
). In fact, in addition to dyads, the culture contains significant numbers of asci with no spores, as well as monads and triads. Surprisingly, the defect in spore formation is caused by a failure of individual spores in the ady3
mutant to form mature spore walls. Cells lacking ADY3
show no apparent defects in prospore membrane formation or nuclear capture by the prospore membrane, but a significant fraction of these prospores fail to elaborate a spore wall (112
Mutation of SSP1
causes the most severe phenotype: a complete block to sporulation. In these cells, prospore membranes are still formed, but they are grossly abnormal (101
). The membranes are tubular rather than round and appear to be adherent to the nuclear envelope. The membranes also occasionally grow in the wrong direction, resulting in a failure to capture nuclei. As in wild-type cells, four membranes are formed; however, in ssp1
cells these membranes often fragment. The ssp1
phenotype demonstrates the necessity for the leading-edge complex (or at least Ssp1p) for proper membrane growth.
While the phenotype of ssp1
mutants is dramatic, it is unclear how loss of SSP1
results in these abnormalities. One proposed explanation is that in the absence of the leading-edge complex, secretory vesicles may fuse only with the outer membrane of the prospore membrane, creating a disproportionate force that pushes the prospore membrane onto the nuclear envelope (101
). An alternative possibility is that the leading-edge complex performs functions analogous to the septin ring in budding cells. For example, it could function as a diffusion barrier that distinguishes the inner prospore membrane, which will ultimately become the plasma membrane of the spore, from the outer prospore membrane, which will ultimately be degraded (see below). Although no protein whose localization is asymmetric between these two regions of the prospore membrane has been described, it is tempting to speculate that such proteins exist and that the loss of this asymmetry in ssp1
mutants might lead to some of the membrane defects seen in the mutant.
The position of the leading-edge complex at the mouth of the prospore membrane also raises the possibility that it could play a role in the segregation of organelles into the spore or even in the closure of the membrane. A failure in one of these processes could account for the subsequent spore wall defects seen in ady3
). With respect to closure, the leading-edge complex might function in a fashion similar to that of the septins at the bud neck in defining the site of closure. Alternatively, the leading-edge complex might take a more active role in causing the membrane to constrict and close. Whether the leading-edge complex is dynamic or static is an important area for future study.