The Yeast CDC37 Gene Interacts with MPS1 and Is Required for Proper Execution of Spindle Pole Body Duplication

The MPS1 gene from Saccharomyces cerevisiae encodes an essential protein kinase required for spindle pole body (SPB) duplication and for the mitotic spindle assembly checkpoint. Cells with the mps1-1 mutation fail early in SPB duplication and proceed through monopolar mitosis with lethal consequences. We identified CDC37 as a multicopy suppressor of mps1-1 temperature-sensitive growth. Suppression is allele specific, and synthetic lethal interactions occur between mps1 and cdc37 alleles. We examined the cdc37-1 phenotype for defects related to the SPB cycle. The cdc37-1 temperature-sensitive allele causes unbudded, G1 arrest at Start (Reed, S.I. 1980. Genetics. 95: 561–577). Reciprocal shifts demonstrate that cdc37-1 arrest is interdependent with α-factor arrest but is not a normal Start arrest. Although the cells are responsive to α-factor at the arrest, SPB duplication is uncoupled from other aspects of G1 progression and proceeds past the satellite-bearing SPB stage normally seen at Start. Electron microscopy reveals side-by-side SPBs at cdc37-1 arrest. The outer plaque of one SPB is missing or reduced, while the other is normal. Using the mps2-1 mutation to distinguish between the SPBs, we find that the outer plaque defect is specific to the new SPB. This phenotype may arise in part from reduced Mps1p function: although Mps1p protein levels are unaffected by the cdc37-1 mutation, kinase activity is markedly reduced. These data demonstrate a requirement for CDC37 in SPB duplication and suggest a role for this gene in G1 control. CDC37 may provide a chaperone function that promotes the activity of protein kinases.     

酵母纺锤体极体复制缺陷与染色体不稳定性的发生

纺锤体极体作为酵母细胞的微管组织中心,在功能上等同于高等真核细胞的中心体,它在细胞周期中的准确复制是两极纺锤体组装和染色体正确分离的前提。纺锤体极体复制缺陷会导致异倍体和多倍体的形成,造成染色体不稳定性的发生。以酿酒酵母细胞为模型,研究纺锤体极体复制过程相关蛋白质的突变,有助于揭示酵母细胞中染色体不稳定性发生的分子机制,并为动物细胞中心体复制的研究提供良好的借鉴。     

Changes in the Nuclear Envelope Environment Affect Spindle Pole Body Duplication in Saccharomyces cerevisiae

The Saccharomyces cerevisiae nuclear membrane is part of a complex nuclear envelope environment also containing chromatin, integral and peripheral membrane proteins, and large structures such as nuclear pore complexes (NPCs) and the spindle pole body. To study how properties of the nuclear membrane affect nuclear envelope processes, we altered the nuclear membrane by deleting the SPO7 gene. We found that spo7Δ cells were sickened by the mutation of genes coding for spindle pole body components and that spo7Δ was synthetically lethal with mutations in the SUN domain gene MPS3. Mps3p is required for spindle pole body duplication and for a variety of other nuclear envelope processes. In spo7Δ cells, the spindle pole body defect of mps3 mutants was exacerbated, suggesting that nuclear membrane composition affects spindle pole body function. The synthetic lethality between spo7Δ and mps3 mutants was suppressed by deletion of specific nucleoporin genes. In fact, these gene deletions bypassed the requirement for Mps3p entirely, suggesting that under certain conditions spindle pole body duplication can occur via an Mps3p-independent pathway. These data point to an antagonistic relationship between nuclear pore complexes and the spindle pole body. We propose a model whereby nuclear pore complexes either compete with the spindle pole body for insertion into the nuclear membrane or affect spindle pole body duplication by altering the nuclear envelope environment.THE nuclear envelope is composed of distinct outer and inner nuclear membranes. The outer nuclear membrane is continuous with the endoplasmic reticulum. The inner nuclear membrane is associated with a unique set of proteins, some of which mediate interactions between the nuclear envelope and chromatin (reviewed in Zhao et al. 2009). Nuclear pore complexes traverse both membranes and allow transport of proteins and solutes between the cytoplasm and the nucleus. The inner and outer nuclear membranes fuse in the region surrounding each nuclear pore complex.In animal cells, the nuclear envelope disassembles as cells enter mitosis and reassembles upon mitotic exit. Nuclear envelope breakdown allows the association of chromosomes with spindle microtubules, which are nucleated from centrosomes that reside in the cytoplasm. In contrast, certain types of fungi, such as the budding yeast Saccharomyces cerevisiae, undergo closed mitosis, where the nuclear envelope remains intact throughout the entire cell cycle. Closed mitosis is possible because the yeast centrosome-equivalent, the spindle pole body (SPB), is embedded in the nuclear envelope, allowing the SPB to nucleate both cytoplasmic and nuclear microtubules.SPB duplication requires a mechanism for inserting the new SPB into the nuclear envelope (reviewed in Jaspersen and Winey 2004). The new SPB begins to form in late G₁/early S phase as satellite material deposited on the cytoplasmic face of an electron-dense region of the nuclear envelope, called the half-bridge. The satellite material matures into a duplication plaque, which is then inserted into the nuclear membrane and becomes the daughter SPB. Many genes are known to be required for SPB duplication, and this process has been carefully examined cytologically (Rose and Fink 1987; Winey et al. 1991, 1993; Spang et al. 1995; Bullitt et al. 1997; Adams and Kilmartin 1999; Elliott et al. 1999; Schramm et al. 2000; Jaspersen et al. 2002; Nishikawa et al. 2003; Araki et al. 2006). However, the exact mechanisms by which SPB duplication and insertion occur remain a mystery.Equally unclear is how nuclear pore complexes are inserted into an intact nuclear envelope (reviewed in Hetzer and Wente 2009). For both the SPB and nuclear pore complexes, the inner and outer nuclear membranes must fuse to allow insertion into the nuclear envelope. Yeast and vertebrate nuclear pore complexes each have four pore membrane (POM) nucleoporins containing transmembrane domains. Other nucleoporins have motifs with potential for bending membranes or sensing membrane curvature. Thus, certain nuclear pore complex components may have the ability to alter the nuclear membrane or stabilize particular membrane conformations (Devos et al. 2004, 2006; Alber et al. 2007; Drin et al. 2007). It is interesting to note that, in S. cerevisiae, nuclear pore complexes are enriched in the vicinity of the SPB (Heath et al. 1995; Winey et al. 1997; Adams and Kilmartin 1999), but the significance of this phenomenon is not known. The SPB and nuclear pore complexes share at least two common components, the integral membrane protein Ndc1p and the small calcium-binding protein Cdc31p (Chial et al. 1998; Fischer et al. 2004). Ndc1p is thought to play a role in insertion of both SPBs and nuclear pore complexes into the nuclear membrane.SUN domain proteins are a conserved family of inner nuclear membrane proteins that interact with specific outer nuclear membrane proteins to form a physical bridge across the nuclear envelope (reviewed in Hiraoka and Dernburg 2009; Razafsky and Hodzic 2009). One of the components of the S. cerevisiae SPB is the SUN domain protein Mps3p. The N terminus of Mps3p is in the nucleoplasm, while the C terminus, containing the SUN domain, is found in the space between the inner and outer nuclear membranes. In addition to the SPB, Mps3p localizes to multiple foci at the nuclear periphery, and these two pools of Mps3p have distinct functions (Jaspersen et al. 2002, 2006; Nishikawa et al. 2003). At the SPB, Mps3p is required for half-bridge formation and early steps of SPB duplication, and cells compromised for Mps3p function accumulate in mitosis with a single SPB and a monopolar spindle (Jaspersen et al. 2002; Nishikawa et al. 2003). At the nuclear periphery, Mps3p is involved in tethering telomeres to the nuclear envelope in mitosis and meiosis, sequestering DNA double-strand breaks away from recombination factors, and associating with soluble chromatin proteins (Antoniacci et al. 2004, 2007; Bupp et al. 2007; Conrad et al. 2007, 2008; Oza et al. 2009; Schober et al. 2009).While many structural features of the yeast nucleus have been identified, little is known about how the physical properties of the nuclear membrane contribute to processes that occur at the nuclear envelope. As noted above, resident proteins of the nuclear envelope may affect nuclear membrane properties. In addition, the nuclear membrane is affected by altering lipid biosynthesis, for example, by inactivating the phosphatidic acid (PA) phosphohydrolase Pah1p or by inactivating the phosphates complex, made of Spo7p and Nem1p, which activates Pah1p. In the absence of Spo7p, Nem1p, or Pah1p, cells exhibit nuclear envelope extensions and extensive ER membrane sheets, and they also have altered membrane lipid composition, including a decrease in phosphatidylcholine and an increase in PA, phosphatidylethanolamine, and phosphatidylinositol (Siniossoglou et al. 1998; Santos-Rosa et al. 2005; Campbell et al. 2006; Han et al. 2006). These three proteins are unique among phospholipid biosynthesis proteins in their ability to affect nuclear morphology upon gene disruption (Han et al. 2008). A similar phenotype was seen upon overexpression of DGK1, which counteracts the activity of Pah1p by converting diacylglycerol to PA, leading to an increase in PA levels at the nuclear envelope (Han et al. 2008). Consistent with a conserved role for Pah1p in regulating nuclear envelope processes, deletion of either NEM1 or SPO7 is synthetically lethal with deletions of certain nucleoporin genes (Siniossoglou et al. 1998), and inactivation of the PAH1 homolog in Caenorhabditis elegans, LPIN-1, results in defects in nuclear envelope disassembly and reassembly (Golden et al. 2009; Gorjanacz and Mattaj 2009).To identify processes that are affected by altered nuclear membrane properties, we screened for pathways that are compromised in spo7Δ cells. We found that SPO7 inactivation strongly influences the SPB. By screening for proteins that could alleviate spo7Δ-induced SPB defects, we uncovered an unexpected inhibitory role for nucleoporins in SPB function, revealing that nuclear pore complexes, or components thereof, act antagonistically to the SPB in the nuclear envelope. Taken together, our findings indicate that the nuclear envelope environment is important for the function of protein complexes and biological processes occurring at the nuclear periphery.     

SPO21 Is Required for Meiosis-specific Modification of the Spindle Pole Body in Yeast

During meiosis II in the yeast Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body changes from a site of microtubule initiation to a site of de novo membrane formation. These membranes are required to package the haploid meiotic products into spores. This functional change in the spindle pole body involves the expansion and modification of its cytoplasmic face, termed the outer plaque. We report here that SPO21 is required for this modification. The Spo21 protein localizes to the spindle pole in meiotic cells. In the absence of SPO21 the structure of the outer plaque is abnormal, and prospore membranes do not form. Further, decreased dosage of SPO21 leaves only two of the four spindle pole bodies competent to generate membranes. Mutation of CNM67, encoding a known component of the mitotic outer plaque, also results in a meiotic outer plaque defect but does not block membrane formation, suggesting that Spo21p may play a direct role in initiating membrane formation.     

Measuring Affinities of Fission Yeast Spindle Pole Body Proteins in Live Cells across the Cell Cycle

Characterizing protein-protein interactions is essential for understanding molecular mechanisms, although reproducing cellular conditions in vitro is challenging and some proteins are difficult to purify. We developed a method to measure binding to cellular structures using fission yeast cells as reaction vessels. We varied the concentrations of Sid2p and Mob1p (proteins of the septation initiation network) and measured their binding to spindle pole bodies (SPBs), the centrosome equivalent of yeast. From our measurements we infer that Sid2p and Mob1p both exist as monomeric, heterodimeric, and homodimeric species throughout the cell cycle. During interphase these species have widely different affinities for their common receptor Cdc11p on the SPB. The data support a model with a subset of Cdc11p binding the heterodimeric species with a K_d < 0.1 μM when Sid2p binds Mob1p-Cdc11p and K_d in the micromolar range when Mob1p binds Sid2p-Cdc11p. During mitosis an additional species presumed to be the phosphorylated Sid2p−Mob1p heterodimer binds SPBs with a lower affinity. Homodimers of Sid2p or Mob1p bind to the rest of Cdc11p at SPBs with lower affinity: K_ds > 10 μM during interphase and somewhat stronger during mitosis. These measurements allowed us to account for the fluctuations in Sid2p binding to SPBs throughout the cell cycle.     

Measuring Affinities of Fission Yeast Spindle Pole Body Proteins in Live Cells across the Cell Cycle

Characterizing protein-protein interactions is essential for understanding molecular mechanisms, although reproducing cellular conditions in vitro is challenging and some proteins are difficult to purify. We developed a method to measure binding to cellular structures using fission yeast cells as reaction vessels. We varied the concentrations of Sid2p and Mob1p (proteins of the septation initiation network) and measured their binding to spindle pole bodies (SPBs), the centrosome equivalent of yeast. From our measurements we infer that Sid2p and Mob1p both exist as monomeric, heterodimeric, and homodimeric species throughout the cell cycle. During interphase these species have widely different affinities for their common receptor Cdc11p on the SPB. The data support a model with a subset of Cdc11p binding the heterodimeric species with a K_d < 0.1 μM when Sid2p binds Mob1p-Cdc11p and K_d in the micromolar range when Mob1p binds Sid2p-Cdc11p. During mitosis an additional species presumed to be the phosphorylated Sid2p−Mob1p heterodimer binds SPBs with a lower affinity. Homodimers of Sid2p or Mob1p bind to the rest of Cdc11p at SPBs with lower affinity: K_ds > 10 μM during interphase and somewhat stronger during mitosis. These measurements allowed us to account for the fluctuations in Sid2p binding to SPBs throughout the cell cycle.     

Stu2p: A Microtubule-Binding Protein that Is an Essential Component of the Yeast Spindle Pole Body

Previously we isolated tub2-423, a cold-sensitive allele of the Saccharomyces cerevisiae gene encoding β-tubulin that confers a defect in mitotic spindle function. In an attempt to identify additional proteins that are important for spindle function, we screened for suppressors of the cold sensitivity of tub2-423 and obtained two alleles of a novel gene, STU2. STU2 is an essential gene and encodes a protein whose sequence is similar to proteins identified in a variety of organisms. Stu2p localizes primarily to the spindle pole body (SPB) and to a lesser extent along spindle microtubules. Localization to the SPB is not dependent on the presence of microtubules, indicating that Stu2p is an integral component of the SPB. Stu2p also binds microtubules in vitro. We have localized the microtubule-binding domain of Stu2p to a highly basic 100-amino acid region. This region contains two imperfect repeats; both repeats appear to contribute to microtubule binding to similar extents. These results suggest that Stu2p may play a role in the attachment, organization, and/or dynamics of microtubule ends at the SPB.     

Dominant Negative Alleles of SEC10 Reveal Distinct Domains Involved in Secretion and Morphogenesis in Yeast

The accurate targeting of secretory vesicles to distinct sites on the plasma membrane is necessary to achieve polarized growth and to establish specialized domains at the surface of eukaryotic cells. Members of a protein complex required for exocytosis, the exocyst, have been localized to regions of active secretion in the budding yeast Saccharomyces cerevisiae where they may function to specify sites on the plasma membrane for vesicle docking and fusion. In this study we have addressed the function of one member of the exocyst complex, Sec10p. We have identified two functional domains of Sec10p that act in a dominant-negative manner to inhibit cell growth upon overexpression. Phenotypic and biochemical analysis of the dominant-negative mutants points to a bifunctional role for Sec10p. One domain, consisting of the amino-terminal two-thirds of Sec10p directly interacts with Sec15p, another exocyst component. Overexpression of this domain displaces the full-length Sec10 from the exocyst complex, resulting in a block in exocytosis and an accumulation of secretory vesicles. The carboxy-terminal domain of Sec10p does not interact with other members of the exocyst complex and expression of this domain does not cause a secretory defect. Rather, this mutant results in the formation of elongated cells, suggesting that the second domain of Sec10p is required for morphogenesis, perhaps regulating the reorientation of the secretory pathway from the tip of the emerging daughter cell toward the mother–daughter connection during cell cycle progression.     

High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the Yeast Saccharomyces cerevisiae

The spindle pole body (SPB) is the major microtubule-organizing center of budding yeast and is the functional equivalent of the centrosome in higher eukaryotic cells. We used fast-frozen, freeze-substituted cells in conjunction with high-voltage electron tomography to study the fine structure of the SPB and the events of early spindle formation. Individual structures were imaged at 5-10 nm resolution in three dimensions, significantly better than can be achieved by serial section electron microscopy. The SPB is organized in distinct but coupled layers, two of which show ordered two-dimensional packing. The SPB central plaque is anchored in the nuclear envelope with hook-like structures. The minus ends of nuclear microtubules (MTs) are capped and are tethered to the SPB inner plaque, whereas the majority of MT plus ends show a distinct flaring. Unbudded cells containing a single SPB retain 16 MTs, enough to attach to each of the expected 16 chromosomes. Their median length is approximately 150 nm. MTs growing from duplicated but not separated SPBs have a median length of approximately 130 nm and interdigitate over the bridge that connects the SPBs. As a bipolar spindle is formed, the median MT length increases to approximately 300 nm and then decreases to approximately 30 nm in late anaphase. Three-dimensional models confirm that there is no conventional metaphase and that anaphase A occurs. These studies complement and extend what is known about the three-dimensional structure of the yeast mitotic spindle and further our understanding of the organization of the SPB in intact cells.     

Multiple Gene Duplication and Rapid Evolution in the groEL Gene: Functional Implications

The chaperonins, GroEL and GroES, are present ubiquitously and provide a paradigm in the understanding of assisted protein folding. Due to its essentiality of function, GroEL exhibits high sequence conservation across species. Complete genome sequencing has shown the occurrence of duplicate or multiple copies of groEL genes in bacteria such as Mycobacterium tuberculosis and Corynebacterium glutamicum. Monophyly of each bacterial clade in the phylogenetic tree generated for the GroEL protein suggests a lineage-specific duplication. The duplicated groEL gene in Actinobacteria is not accompanied by the operonic groES despite the presence of upstream regulatory elements. Our analysis suggests that in these bacteria the duplicated groEL genes have undergone rapid evolution and divergence to function in a GroES-independent manner. Evaluation of multiple sequence alignment demonstrates that the duplicated genes have acquired mutations at functionally significant positions including those involved in substrate binding, ATP binding, and GroES binding and those involved in inter-ring and intra-ring interactions. We propose that the duplicate groEL genes in different bacterial clades have evolved independently to meet specific requirements of each clade. We also propose that the groEL gene, although essential and conserved, accumulates nonconservative substitutions to exhibit structural and functional variations. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Debashish Bhattacharya]     

The Yeast Spindle Pole Body Component Spc72p Interacts with Stu2p and Is Required for Proper Microtubule Assembly

We have previously shown that Stu2p is a microtubule-binding protein and a component of the Saccharomyces cerevisiae spindle pole body (SPB). Here we report the identification of Spc72p, a protein that interacts with Stu2p. Stu2p and Spc72p associate in the two-hybrid system and can be coimmunoprecipitated from yeast extracts. Stu2p and Spc72p also interact with themselves, suggesting the possibility of a multimeric Stu2p-Spc72p complex. Spc72p is an essential component of the SPB and is able to associate with a preexisting SPB, indicating that there is a dynamic exchange between soluble and SPB forms of Spc72p. Unlike Stu2p, Spc72p does not bind microtubules in vitro, and was not observed to localize along microtubules in vivo. A temperature-sensitive spc72 mutation causes defects in SPB morphology. In addition, most spc72 mutant cells lack cytoplasmic microtubules; the few cytoplasmic microtubules that are observed are excessively long, and some of these are unattached to the SPB. spc72 cells are able to duplicate and separate their SPBs to form a bipolar spindle, but spindle elongation and chromosome segregation rarely occur. The chromosome segregation block does not arrest the cell cycle; instead, spc72 cells undergo cytokinesis, producing aploid cells and polyploid cells that contain multiple SPBs.     

Spontaneous Mitotic Recombination in Yeast: The Hyper-Recombinational Rem1 Mutations Are Alleles of the Rad3 Gene

The RAD3 gene of Saccharomyces cerevisiae is required for UV excision-repair and is essential for cell viability. We have identified the rem1 mutations (enhanced spontaneous mitotic recombination and mutation) of Saccharomyces cerevisiae as alleles of RAD3 by genetic mapping, complementation with the cloned wild-type gene, and DNA hybridization. The high levels of spontaneous mitotic gene conversion, crossing over, and mutation conferred upon cells by the rem1 mutations are distinct from the effects of all other alleles of RAD3. We present preliminary data on the localization of the rem1 mutations within the RAD3 gene. The interaction of the rem1 mutant alleles with a number of radiation-sensitive mutations is also different than the interactions reported for previously described (UV-sensitive) alleles of RAD3. Double mutants of rem1 and a defect in the recombination-repair pathway are inviable, while double mutants containing UV-sensitive alleles of RAD3 are viable. The data presented here demonstrate that: (1) rem1 strains containing additional mutations in other excision-repair genes do not exhibit elevated gene conversion; (2) triple mutants containing rem1 and mutations in both excision-repair and recombination-repair are viable; (3) such triple mutants containing rad52 have reduced levels of gene conversion but wild-type frequencies of crossing over. We have interpreted these observations in a model to explain the effects of rem1. Consistent with the predictions of the model, we find that the size of DNA from rem1 strains, as measured by neutral sucrose gradients, is smaller than wild type.     

Interactions between N-terminal Modules in MPS1 Enable Spindle Checkpoint Silencing

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Sec66-Dependent Regulation of Yeast Spindle-Pole Body Duplication Through Pom152

In closed mitotic systems such as Saccharomyces cerevisiae, the nuclear envelope (NE) does not break down during mitosis, so microtubule-organizing centers such as the spindle-pole body (SPB) must be inserted into the NE to facilitate bipolar spindle formation and chromosome segregation. The mechanism of SPB insertion has been linked to NE insertion of nuclear pore complexes (NPCs) through a series of genetic and physical interactions between NPCs and SPB components. To identify new genes involved in SPB duplication and NE insertion, we carried out genome-wide screens for suppressors of deletion alleles of SPB components, including Mps3 and Mps2. In addition to the nucleoporins POM152 and POM34, we found that elimination of SEC66/SEC71/KAR7 suppressed lethality of cells lacking MPS2 or MPS3. Sec66 is a nonessential subunit of the Sec63 complex that functions together with the Sec61 complex in import of proteins into the endoplasmic reticulum (ER). Cells lacking Sec66 have reduced levels of Pom152 protein but not Pom34 or Ndc1, a shared component of the NPC and SPB. The fact that Sec66 but not other subunits of the ER translocon bypass deletion mutants in SPB genes suggests a specific role for Sec66 in the control of Pom152 levels. Based on the observation that sec66∆ does not affect the distribution of Ndc1 on the NE or Ndc1 binding to the SPB, we propose that Sec66-mediated regulation of Pom152 plays an NPC-independent role in the control of SPB duplication.     

cut11+: A Gene Required for Cell Cycle-dependent Spindle Pole Body Anchoring in the Nuclear Envelope and Bipolar Spindle Formation in Schizosaccharomyces pombe

The “cut” mutants of Schizosaccharomyces pombe are defective in spindle formation and/or chromosome segregation, but they proceed through the cell cycle, resulting in lethality. Analysis of temperature-sensitive alleles of cut11⁺ suggests that this gene is required for the formation of a functional bipolar spindle. Defective spindle structure was revealed with fluorescent probes for tubulin and DNA. Three-dimensional reconstruction of mutant spindles by serial sectioning and electron microscopy showed that the spindle pole bodies (SPBs) either failed to complete normal duplication or were free floating in the nucleoplasm. Localization of Cut11p tagged with the green fluorescent protein showed punctate nuclear envelope staining throughout the cell cycle and SPBs staining from early prophase to mid anaphase. This SPB localization correlates with the time in the cell cycle when SPBs are inserted into the nuclear envelope. Immunoelectron microscopy confirmed the localization of Cut11p to mitotic SPBs and nuclear pore complexes. Cloning and sequencing showed that cut11⁺ encodes a novel protein with seven putative membrane-spanning domains and homology to the Saccharomyces cerevisiae gene NDC1. These data suggest that Cut11p associates with nuclear pore complexes and mitotic SPBs as an anchor in the nuclear envelope; this role is essential for mitosis.     

Origin of the Yeast Whole-Genome Duplication

Whole-genome duplications (WGDs) are rare evolutionary events with profound consequences. They double an organism’s genetic content, immediately creating a reproductive barrier between it and its ancestors and providing raw material for the divergence of gene functions between paralogs. Almost all eukaryotic genome sequences bear evidence of ancient WGDs, but the causes of these events and the timing of intermediate steps have been difficult to discern. One of the best-characterized WGDs occurred in the lineage leading to the baker’s yeast Saccharomyces cerevisiae. Marcet-Houben and Gabaldón now show that, rather than simply doubling the DNA of a single ancestor, the yeast WGD likely involved mating between two different ancestral species followed by a doubling of the genome to restore fertility.The unicellular baker’s yeast Saccharomyces cerevisiae was the first eukaryote to have its genome sequenced, using the first generation of automated sequencing machines and before the advent of the whole-genome shotgun approach. The sequencing was done during the period between 1990 and 1996 by an international consortium that included many small European laboratories, one of which was mine. Each laboratory was given a “tranche” of about 30 kb to sequence, and when you had completed that chunk, you could apply for another one. We were paid €2 per base pair. Progress meetings, chaired energetically by André Goffeau [1], were held every six months to ensure that the project remained on track. At these meetings, each group would make a 5-minute presentation about the genes they had found in their current chunk. The presentations were often tedious, enlivened only by the occasional exigency for André to reassign pieces of DNA from the sequencing tortoises to the hares. But as the project progressed, a pattern began to emerge: many of the chunks were similar to other chunks. The first clone that I sequenced happened to contain the centromere of chromosome II, and I noticed that a gene beside it had a paralog beside the centromere of chromosome IV [2]. My second chunk, from chromosome XV, contained four genes that had four paralogs, in the same order, on chromosome I [3].When the complete genome was released in April 1996, we were able to identify 55 large duplicated blocks of this type, ranging in size from three to 18 duplicated genes (Fig 1) [4]. Two observations indicated that the duplications were quite old: the average amino acid sequence identity between the gene pairs was only 63%, and within each block only about 25% of the genes were actually duplicated, the others being single copy. This pattern suggested that the whole block was initially duplicated, and then many individual genes were deleted. Two other observations suggested that the blocks were remnants of duplicated chromosomes that had become rearranged during evolution: there were almost no overlaps between the blocks, and the orientation of each pair of blocks was conserved relative to the centromeres and telomeres. This layout of blocks was consistent with duplication of the whole genome followed by both extensive deletion of single genes and genome rearrangement solely by the process of reciprocal translocation between chromosomes [4]. Under this hypothesis, there had been an ancient whole-genome duplication (WGD), and the 55 blocks that we could identify were simply the most duplicate-dense regions that still survived without evolutionary rearrangement.Open in a separate windowFig 1A simple model of WGD, gene loss, and synteny relationships.The upper panel shows how duplicated blocks were initially identified using only genes that remain in duplicate in S. cerevisiae [4]. The lower panel shows how additional data from non-WGD yeasts such as Lachancea waltii [5] allowed the parts of the genome that were not initially allocated to blocks to be placed into pairs, providing a duplication map that covered the whole S. cerevisiae genome. Letters A–W represent genes, and dots represent centromeres. Only two chromosomes (yellow and brown) are shown.The hypothesis of a WGD in S. cerevisiae was confirmed in 2004 when three groups sequenced the genomes of species that had branched off from this lineage before the WGD occurred [5–7]. These non-WGD genomes had a “double conserved synteny” relationship with the S. cerevisiae genome—that is, instead of each pair of duplicated regions, they had a single region containing all the genes in a merged order (Fig 1). This discovery allowed the entire genome of S. cerevisiae to be mapped into pairs of regions via their double conserved synteny with the non-WGD species, even if the pairs retain no duplicated genes, thus filling the gaps between the initial map of 55 duplicated blocks. These analyses proved that the WGD encompassed the entire genome of S. cerevisiae and showed that its 16 centromeres fall into eight ancestral pairs that are syntenic with centromeres of the non-WGD species. It therefore appeared that the WGD turned an eight-chromosome ancestor into a 16-chromosome descendant. From this complete map, we now know that among the 5,774 protein-coding genes of S. cerevisiae, there are 551 pairs of duplicated genes (ohnologs) that were formed by the WGD and that about 144 chromosomal rearrangements scrambled the genome after the WGD [8,9]. We also know that the WGD is not confined to Saccharomyces but occurred in the common ancestor of six genera, some of which diverged from each at an early stage when more than 4,000 genes were still duplicated, leading to later losses of different gene copies in different lineages [10].What were the molecular events that caused the WGD? It is relatively easy to draw a diagram summarizing the history of each chromosomal region (Fig 2), but it is much more difficult to specify the provenance of the intermediate molecules and the timescales involved. Two alternative scenarios can describe the steps in Fig 2. In both scenarios, event 1 is a DNA replication, and cells W and Z are each capable of mating (they are respectively a non-WGD haploid and a post-WGD haploid). The key question is whether the DNA molecules labeled X and Y existed in (1) two different cells of the same species or (2) two cells of two different species. Scenario 1 is called autopolyploidization, in which case event 1 corresponds to a simple cell division and event 2 is a mating between gametes from the same species or some other form of cell fusion. Scenario 2 is called allopolyploidization or hybridization, in which case event 1 is a speciation and event 2 is an interspecies mating or cell fusion. If event 2 was a mating, then an additional step such as deletion of one allele at the MAT locus is necessary to convert cell Z from a nonmating zygote to a mating gamete—but it is not essential that this additional step occurred immediately after event 2. In fact, a long delay in which cell Z replicated mitotically for many generations could be useful because it could allow reproductive isolation from cells of type W to build up. Eventually (event 3), mating between two post-WGD haploid cells of type Z can produce a post-WGD diploid like cell ZZ, which is the state in which S. cerevisiae is normally found in nature.Open in a separate windowFig 2Tracing the history of a single chromosomal region.See text for details. In an allopolyploidization, the red and blue chromosomes are called homeologs.The major difference between these two scenarios is the amount of time (T) that elapsed between events 1 and 2: was it a few generations or millions of years? In scenario 1, molecules X and Y must be identical, whereas in scenario 2 they could have any level of sequence divergence from minimal to extensive, and they could also differ by chromosomal rearrangements. It has been difficult to design tests that could differentiate between these scenarios, but an analysis of the inferred order of genes along molecules X and Y did not find any rearrangements and so did not rule out scenario 1 [9]. However, it has been frustrating that we could not pin down the details of this crucial phase of yeast evolution, which gave birth to many pairs of genes with substantially divergent functions [11–15].In this issue of PLOS Biology, Marcet-Houben and Gabaldón now report strong evidence in support of interspecies hybridization (scenario 2) as the source of the two subgenomes in post-WGD species [16]. By phylogenetic analysis using state-of-the-art methods, they show that molecules X and Y have phylogenetic affinities to two different non-WGD lineages that they call the KLE and ZT clades. The KLE clade (Kluyveromyces, Lachancea, and Eremothecium) is the group of non-WGD species that was sequenced in 2004 [5–7]. The ZT clade (Zygosaccharomyces and Torulaspora) is a separate, more recently studied non-WGD lineage [17,18]. Previous phylogenetic studies using supertrees or concatenated data suggested that the ZT clade is sister to the post-WGD clade, with the KLE clade being an out-group to them both [17,19,20]. The new analysis [16] made trees for each gene individually and found that, although the majority of genes in post-WGD species do cluster phylogenetically with the ZT clade as expected, a significant minority (about 30%) instead either cluster with the KLE clade or form an outgroup to a KLE + ZT clade. This phylogenetic heterogeneity was not noticed before because the KLE signal is only present in a minority of genes, and it is swamped by the ZT signal in methods that try to place the post-WGD clade at a single point on the tree.Marcet-Houben and Gabaldón interpret this phylogenetic heterogeneity as evidence that the two post-WGD subgenomes have separate origins, one from the ZT clade and the other from an unidentified lineage that is an outgroup to KLE + ZT. Under the simplest hypothesis of hybridization, we might then expect that phylogenetic trees constructed from ohnolog pairs should show one S. cerevisiae gene grouping with the ZT clade and the other grouping with the KLE clade, but in fact, most ohnolog pairs group with each other, with the ZT clade as their closest relative [16]. The authors’ explanation for these two results—an excess of ZT-like ohnolog pairs, and an excess of ZT-like genes in the whole genome (which is mostly singletons)—is that the post-WGD genomes have been affected by biased gene conversion that preferentially replaced some KLE-derived sequences with copies of the homeologous ZT-derived sequences, homogenizing these regions and obliterating their signal of KLE ancestry.The hybridization proposed by Marcet-Houben and Gabaldón makes a lot of sense in terms of what we know about the biology of yeast interspecies hybrids. Many yeast strains, most notably those used in commercial settings where stress tolerance is important, have turned out to be interspecies hybrids. For instance, the yeast used to brew lager (S. pastorianus) is a hybrid between S. cerevisiae and S. eubayanus [21,22], and many other combinations of genomes from different species of Saccharomyces have been found in nature [23]. These interspecies hybrids are usually infertile (unable to sporulate) because the two copies (homeologs) of each chromosome that they contain are too dissimilar to pair properly during meiosis [24–26]. One simple way to restore fertility is to double the genome, allowing each chromosome to pair with an identical partner instead of trying to pair with the homeolog. In this model, cell Z changes from being a nonmater (effectively diploid) to a mater (effectively haploid—perhaps by deletion of a MAT allele), then two cells of type Z mate to produce cell ZZ (diploid), and cell ZZ is able to go through meiosis and make spores with twice the DNA content of cell W. Thus, one hypothesis that Marcet-Houben and Gabaldón propose is that event 2 was an interspecies mating and event 3 was a restoration of fertility by genome doubling, with a possible interval of many mitotic generations between these two events. Alternatively, they hypothesize that event 2 may have been an interspecies fusion of diploid cells, obviating the need for a separate event 3.The obscuring of the phylogenetic signal of hybridization by subsequent gene conversions [16] is consistent with the known genome structures of some interspecies hybrids. The yeasts Pichia sorbitophila [27] and Candida orthopsilosis [28] are both interspecies hybrids, but in each case extensive homogenization of parts of the genome has occurred. This process of homogenization has been called overwriting, loss of heterozygosity, or gene conversion by different groups. It leaves the number of chromosomes unchanged (equal to the sum of the numbers of chromosomes in the two incoming subgenomes) but involves the replacement of sequences in one subgenome by sequences copied from the other subgenome (cell H in Fig 2). Homogenization could occur on scales as small as a few hundred base pairs (gene conversion) or as large as whole chromosome arms (break-induced replication). In the latter case, even differences in gene order such as inversions between the parental species could be ironed out.The discovery that the yeast WGD was an allopolyploidization adds complexity to what initially seemed to be a simple story of duplication. If an interspecies hybrid such as P. sorbitophila with a partly homogenized genome developed two mating types and these could mate to form a diploid that could sporulate efficiently, the result would be a species with a genome resembling the inferred progenitor of the post-WGD clade (cell HH in Fig 2). Allopolyploidy answers some old questions about why genes were retained in duplicate if their sequences were identical (answer: they weren’t identical), what the immediate selective advantage of the post-WGD cell was (answer: hybrid vigor), and how the post-WGD lineage became reproductively isolated from the pre-WGD lineage (answer: delay between events 2 and 3). But it also raises new questions about homogenization (how much of the genome? how often? why is it biased?) and about the mechanism of restoration of fertility (why is event 3 so rare, apparently happening only once in the budding yeast family even though event 2 happened quite often?).Ancient WGDs have been detected right across the eukaryotic tree of life, including in animals, ciliates, fungi, and, most prominently, plants [29–32]. If extensive gene conversion can obscure the traces of allopolyploidization in yeast genomes, one might wonder how many of these other ancient WGDs also began as interspecies hybridizations. In fact, there is evidence from plants that gene conversion acts continually to homogenize ohnolog pairs [32,33] and that hybrid plants can show preferential retention of DNA from one parent over the other [34] similar to the situation in P. sorbitophila [27]. Detecting the yeast hybridization in the presence of these obscuring factors required both good luck and good timing: good luck that a reference species closer to one parent than to the other had been sequenced and good timing that the hybrid was sampled before all traces of its hybrid origin had faded away. These fortunate circumstances may not hold for ancient hybridizations in other eukaryotes, but as a famous golfer once said, “The harder I practice, the luckier I get.” Detecting that they are hybridizations may become possible with exhaustive sampling of possible parental lineages and the use of sensitive phylogenomic methods of the type introduced by the authors [16].     

The Mammalian γ-Tubulin Complex Contains Homologues of the Yeast Spindle Pole Body Components Spc97p and Spc98p

γ-Tubulin is a universal component of microtubule organizing centers where it is believed to play an important role in the nucleation of microtubule polymerization. γ-Tubulin also exists as part of a cytoplasmic complex whose size and complexity varies in different organisms. To investigate the composition of the cytoplasmic γ-tubulin complex in mammalian cells, cell lines stably expressing epitope-tagged versions of human γ-tubulin were made. The epitope-tagged γ-tubulins expressed in these cells localize to the centrosome and are incorporated into the cytoplasmic γ-tubulin complex. Immunoprecipitation of this complex identifies at least seven proteins, with calculated molecular weights of 48, 71, 76, 100, 101, 128, and 211 kD. We have identified the 100- and 101-kD components of the γ-tubulin complex as homologues of the yeast spindle pole body proteins Spc97p and Spc98p, and named the corresponding human proteins hGCP2 and hGCP3. Sequence analysis revealed that these proteins are not only related to their respective homologues, but are also related to each other. GCP2 and GCP3 colocalize with γ-tubulin at the centrosome, cosediment with γ-tubulin in sucrose gradients, and coimmunoprecipitate with γ-tubulin, indicating that they are part of the γ-tubulin complex. The conservation of a complex involving γ-tubulin, GCP2, and GCP3 from yeast to mammals suggests that structurally diverse microtubule organizing centers such as the yeast spindle pole body and the animal centrosome share a common molecular mechanism for microtubule nucleation.