Kar4 is required for the normal pattern of meiotic gene expression.

Kar4p, the yeast homolog of the mammalian methyltransferase subunit METTL14, is required for efficient mRNA m6A methylation, which regulates meiotic entry. Kar4p is also required for a second seemingly non-catalytic function during meiosis. Overexpression of the early meiotic transcription factor, IME1, can bypass the requirement for Kar4p in meiotic entry but the additional overexpression of the translational regulator, RIM4, is required to permit sporulation in kar4Δ/Δ. Using microarray analysis and RNA sequencing, we sought to determine the impact of removing Kar4p and consequently mRNA methylation on the early meiotic transcriptome in a strain background (S288c) that is sensitive to the loss of early meiotic regulators. We found that kar4Δ/Δ mutants have a largely wild type transcriptional profile with the exception of two groups of genes that show delayed and reduced expression: (1) a set of Ime1p-dependent early genes as well as IME1, and (2) a set of late genes dependent on the mid-meiotic transcription factor, Ndt80p. The early gene expression defect is likely the result of the loss of mRNA methylation and is rescued by overexpressing IME1, but the late defect is only suppressed by overexpression of both IME1 and RIM4. The requirement for RIM4 led us to predict that the non-catalytic function of Kar4p, like methyltransferase complex orthologs in other systems, may function at the level of translation. Mass spectrometry analysis identified several genes involved in meiotic recombination with strongly reduced protein levels, but with little to no reduction in transcript levels in kar4Δ/Δ after IME1 overexpression. The low levels of these proteins were rescued by overexpression of RIM4 and IME1, but not by the overexpression of IME1 alone. These data expand our understanding of the role of Kar4p in regulating meiosis and provide key insights into a potential mechanism of Kar4p's later meiotic function that is independent of mRNA methylation.

Kar4, the yeast homolog of METTL14, is required for mRNA m6A methylation and meiosis.

KAR4, the yeast homolog of the mammalian mRNA N6A-methyltransferase complex component METTL14, is required for two disparate developmental programs in Saccharomyces cerevisiae: mating and meiosis. To understand KAR4's role in yeast mating and meiosis, we used a genetic screen to isolate 25 function-specific mutant alleles, which map to non-overlapping surfaces on a predicted structure of the Kar4 protein (Kar4p). Most of the mating-specific alleles (Mat-) abolish Kar4p's interaction with the transcription factor Ste12p, indicating that Kar4p's mating function is through Ste12p. In yeast, the mRNA methyltransferase complex was previously defined as comprising Ime4p (Kar4p's paralog and the homolog of mammalian METTL3), Mum2p (homolog of mammalian WTAP), and Slz1p (MIS), but not Kar4p. During meiosis, Kar4p interacts with Ime4p, Mum2p, and Slz1p. Moreover, cells lacking Kar4p have highly reduced levels of mRNA methylation during meiosis indicating that Kar4p is a key member of the methyltransferase complex, as it is in humans. Analysis of kar4Δ/Δ and 7 meiosis-specific alleles (Mei-) revealed that Kar4p is required early in meiosis, before initiation of S-phase and meiotic recombination. High copy expression of the meiotic transcriptional activator IME1 rescued the defect of these Mei- alleles. Surprisingly, Kar4p was also found to be required at a second step for the completion of meiosis and sporulation. Over-expression of IME1 in kar4Δ/Δ permits pre-meiotic S-phase, but most cells remained arrested with a monopolar spindle. Analysis of the function-specific mutants revealed that roughly half became blocked after premeiotic DNA synthesis and did not sporulate (Spo-). Loss of Kar4p's Spo function was suppressed by overexpression of RIM4, a meiotic translational regulator. Overexpression of IME1 and RIM4 together allowed sporulation of kar4Δ/Δ cells. Taken together, these data suggest that Kar4p regulates meiosis at multiple steps, presumably reflecting requirements for methylation in different stages of meiotic gene expression.

A mechanism for the coordination of proliferation and differentiation by spatial regulation of Fus2p in budding yeast.

Yeast cells induce the genes required for mating prior to the completion of mitosis. To ensure proper cell cycle progression prior to mating differentiation, a key cytoplasmic regulator of cell fusion, Fus2p, is sequestered in the nucleus by cyclin-dependent kinase (Cdk). In response to pheromone signaling, the mitogen-activated protein kinase Fus3p phosphorylates Ser 84 in Fus2p to drive nuclear export. We found that Fus3p becomes active and phosphorylates S84 as early as S phase, raising the question of how Cdk prevents inappropriate activation of Fus2p. Countering Fus3p, Cdk and a p21-activated kinase, Cla4p, maintain Fus2p's nuclear localization by phosphorylating Ser 67, which drives nuclear import and inhibits nuclear export. When Cdk and Cla4p activities drop after cell division, Fus3p promotes Fus2p export both via S84 phosphorylation and by down-regulating S67 phosphorylation. Thus, potential premature activation of Fus2p in mitosis is prevented by cell cycle-dependent phosphorylation that overrides the mating pheromone-induced phosphorylation that drives nuclear export.

Influence of the bud neck on nuclear envelope fission in Saccharomyces cerevisiae.

Studies have shown that nuclear envelope fission (karyokinesis) in budding yeast depends on cytokinesis, but not distinguished whether this was a direct requirement, indirect, because of cell cycle arrest, or due to bud neck-localized proteins impacting both processes. To determine the requirements for karyokinesis, we examined mutants conditionally defective for bud emergence and/or nuclear migration. The common mutant phenotype was completion of the nuclear division cycle within the mother cell, but karyokinesis did not occur. In the cdc24 swe1 mutant, at the non-permissive temperature, multiple nuclei accumulated within the unbudded cell, with connected nuclear envelopes. Upon return to the permissive temperature, the cdc24 swe1 mutant initiated bud emergence, but only the nucleus spanning the neck underwent fission suggesting that the bud neck region is important for fission initiation. The neck may be critical for either mechanical reasons, as the contractile ring might facilitate fission, or for regulatory reasons, as the site of a protein network regulating nuclear envelope fission, mitotic exit, and cytokinesis. We also found that 77-85% of pairs of septin mutant nuclei completed nuclear envelope fission. In addition, 27% of myo1Δ mutant nuclei completed karyokinesis. These data suggested that fission is not dependent on mechanical contraction at the bud neck, but was instead controlled by regulatory proteins there.

Stable Pseudohyphal Growth in Budding Yeast Induced by Synergism between Septin Defects and Altered MAP-kinase Signaling.

Upon nutrient limitation, budding yeasts like Saccharomyces cerevisiae can be induced to adopt alternate filament-like growth patterns called diploid pseudohyphal or invasive haploid growth. Here, we report a novel constitutive pseudohyphal growth state, sharing some characteristics with classic forms of filamentous growth, but differing in crucial aspects of morphology, growth conditions and genetic regulation. The constitutive pseudohyphal state is observed in fus3 mutants containing various septin assembly defects, which we refer to as sadF growth (septin assembly defect induced filamentation) to distinguish it from classic filamentation pathways. Similar to other filamentous states, sadF cultures comprise aggregated chains of highly elongated cells. Unlike the classic pathways, sadF growth occurs in liquid rich media, requiring neither starvation nor the key pseudohyphal proteins, Flo8p and Flo11p. Moreover sadF growth occurs in haploid strains of S288C genetic background, which normally cannot undergo pseudohyphal growth. The sadF cells undergo highly polarized bud growth during prolonged G2 delays dependent on Swe1p. They contain septin structures distinct from classical pseudo-hyphae and FM4-64 labeling at actively growing tips similar to the Spitzenkörper observed in true hyphal growth. The sadF growth state is induced by synergism between Kss1p-dependent signaling and septin assembly defects; mild disruption of mitotic septins activates Kss1p-dependent gene expression, which exacerbates the septin defects, leading to hyper-activation of Kss1p. Unlike classical pseudo-hyphal growth, sadF signaling requires Ste5, Ste4 and Ste18, the scaffold protein and G-protein ß and γ subunits from the pheromone response pathway, respectively. A swe1 mutation largely abolished signaling, breaking the positive feedback that leads to amplification of sadF signaling. Taken together, our findings show that budding yeast can access a stable constitutive pseudohyphal growth state with very few genetic and regulatory changes.

Kar4 is Required for the Normal Pattern of Meiotic Gene Expression.

Kar4p, the yeast homolog of the mammalian methyltransferase subunit METTL14, is required for the initiation of meiosis and has at least two distinct functions in regulating the meiotic program. Cells lacking Kar4p can be driven to sporulate by co-overexpressing the master meiotic transcription factor, IME1 , and the translational regulator, RIM4 , suggesting that Kar4p functions at both the transcriptional and translational level to regulate meiosis. Using microarray analysis and RNA sequencing, we found that kar4 Δ/Δ mutants have a largely wild type transcriptional profile with the exception of two groups of genes that show delayed and reduced expression: (1) a set of Ime1p-dependent early genes as well as IME1 , and (2) a set of late genes dependent on the mid-meiotic transcription factor, Ndt80p. The early gene expression defect is rescued by overexpressing IME1 , but the late defect is only suppressed by overexpression of both IME1 and RIM4 . Mass spectrometry analysis identified several genes involved in meiotic recombination with strongly reduced protein levels, but with little to no reduction in transcript levels in kar4 Δ/Δ after IME1 overexpression. The low levels of these proteins were rescued by overexpression of RIM4 and IME1 , but not by the overexpression of IME1 alone. These data expand our understanding of the role of Kar4p in regulating meiosis and provide key insights into a potential mechanism of Kar4p's later meiotic function that is independent of mRNA methylation. Author Summary: Kar4p is required at two stages during meiosis. Cells lacking Kar4p have a severe loss of mRNA methylation and arrest early in the meiotic program, failing to undergo either pre-meiotic DNA synthesis or meiotic recombination. The early block is rescued by overexpression of the meiotic transcription factor, IME1 . The kar4 Δ/Δ cells show delayed and reduced expression of a set of Ime1p-dependent genes expressed early in meiosis as well as a set of later genes that are largely Ndt80p-dependent. Overexpression of IME1 rescues the expression defect of these early genes and expedites the meiotic program in the wild type S288C strain background. However, IME1 overexpression is not sufficient to facilitate sporulation in kar4 Δ/Δ. Completion of meiosis and sporulation requires the additional overexpression of a translational regulator, RIM4 . Analysis of kar4 Δ/Δ's proteome during meiosis with IME1 overexpression revealed that proteins important for meiotic recombination have reduced levels that cannot be explained by equivalent reductions in transcript abundance. IME1 overexpression by itself rescues the defect associated with a catalytic mutant of Ime4p, implying that the early defect reflects mRNA methylation. The residual defects in protein levels likely reflect the loss of a non-catalytic function of Kar4p, and the methylation complex, which requires overexpression of RIM4 to suppress.

Vir1p, the yeast homolog of virilizer, is required for mRNA m6A methylation and meiosis.

N6-Methyladenosine (m6A) is among the most abundant modifications of eukaryotic mRNAs. mRNA methylation regulates many biological processes including playing an essential role in meiosis. During meiosis in the budding yeast, Saccharomyces cerevisiae, m6A levels peak early, before the initiation of the meiotic divisions. High-throughput studies suggested, and this work confirms that the uncharacterized protein Ygl036wp interacts with Kar4p, a component of the mRNA m6A-methyltransferase complex. Protein structure programs predict that Ygl036wp folds like VIRMA/Virilizer/VIR, which is involved in mRNA m6A-methylation in higher eukaryotes. In addition, Ygl036wp contains conserved motifs shared with VIRMA/Virilizer/VIR. Accordingly, we propose the name VIR1 for budding yeast ortholog of VIRMA/Virilizer/VIR 1. Vir1p interacts with all other members of the yeast methyltransferase complex and is itself required for mRNA m6A methylation and meiosis. In the absence of Vir1p proteins comprising the methyltransferase complex become unstable, suggesting that Vir1p acts as a scaffold for the complex. The vir1Δ/Δ mutant is defective for the premeiotic S-phase, which is suppressed by overexpression of the early meiotic transcription factor IME1; additional overexpression of the translational regulator RIM4 is required for sporulation. The vir1Δ/Δ mutant exhibits reduced levels of IME1 mRNA, as well as transcripts within Ime1p's regulon. Suppression by IME1 revealed an additional defect in the expression of the middle meiotic transcription factor, Ndt80p (and genes in its regulon), which is rescued by overexpression of RIM4. Together, these data suggest that Vir1p is required for cells to initiate the meiotic program and for progression through the meiotic divisions and spore formation.

Vir1p, the Yeast Homolog of Virilizer, is Required for mRNA m 6 A Methylation and Meiosis.

N 6 -Methyladenosine (m 6 A) is one of the most abundant modifications found on eukaryotic mRNAs. mRNA methylation regulates a host of biological processes including meiosis, a specialized diploid cell division program that results in the formation of haploid cells (gametes). During budding yeast meiosis, m 6 A levels peak early, before the initiation of the meiotic divisions. High-throughput studies and work from our lab showed that Ygl036wp, a previously uncharacterized protein interacts with Kar4p, a meiotic protein required for mRNA m 6 A-methylation. Ygl036wp has no discernable domains except for several intrinsically disordered regions. However, protein folding prediction tools showed that Ygl036wp folds like VIRMA/Virilizer/VIR, which is involved in mRNA m 6 A-methylation in higher eukaryotes. In addition, Ygl036wp has several conserved motifs shared with VIRMA/Virilizer/VIR proteins. Accordingly, we propose to call the gene VIR1 for budding yeast ortholog of VIR MA/Virilizer/VIR 1 . In support, Vir1p interacts with all other members of the yeast methyltransferase complex and is required for mRNA m 6 A methylation and meiosis. Vir1p is required for the stability of proteins comprising the methyltransferase complex, suggesting that Vir1p acts as a scaffold to stabilize the complex. The vir1 Δ/Δ mutant is defective for premeiotic S-phase, which is suppressed by overexpression of the early meiotic transcription factor IME1; additional overexpression of the translational regulator RIM4 is required for sporulation. Consistent with IME1 suppression, vir1 Δ/Δ exhibits a defect in the abundance of IME1 mRNA, as well as transcripts within Ime1p's regulon. Suppression by IME1 revealed a defect in the expression of the middle meiotic transcription factor, Ndt80p (and genes in its regulon), which is rescued by additional overexpression of RIM4 . Together, these data suggest that Vir1p is required for cells to initiate the meiotic program and for progression through the meiotic divisions and spore formation. Author Summary: Ygl036wp is a previously uncharacterized protein that we propose to name Vir1p (budding yeast ortholog of VIR MA/Virilizer/VIR 1 ). Work from our lab and others initially found an interaction between Vir1p and members of the yeast mRNA methyltransferase complex (Kar4p and Mum2p). We found that Vir1p interacts with all known members of the methyltransferase complex and is required for mRNA methylation. Vir1p is required early in meiosis; vir1 Δ/Δ mutants arrest due to the reduced expression of Ime1p. Lower levels of Ime1p cause severe disruption to the meiotic transcriptome in vir1 Δ/Δ. The vir1 Δ/Δ meiotic defect can be partially suppressed by the overexpression of IME1 ; full suppression requires overexpression of both IME1 and RIM4 . Using recent advances in protein folding predictions, we found that Vir1p is a remote homolog of VIRMA/Virilizer/VIR and shares conserved motifs with the protein from other organisms. Vir1p, like VIRMA/Virilizer/VIR, stabilizes the methyltransferase complex.

Kar4, the Yeast Homolog of METTL14, is Required for mRNA m 6 A Methylation and Meiosis.

KAR4 , the yeast homolog of the mammalian mRNA N 6 A-methyltransferase complex component METTL14 , is required for two disparate developmental programs in Saccharomyces cerevisiae : mating and meiosis. To understand KAR4 's role in yeast mating and meiosis, we used a genetic screen to isolate 25 function-specific mutant alleles, which map to non-overlapping surfaces on a predicted structure of the Kar4 protein (Kar4p). Most of the mating-specific alleles (Mat - ) abolish Kar4p's interaction with the transcription factor Ste12p, indicating that Kar4p's mating function is through Ste12p. In yeast, the mRNA methyltransferase complex was previously defined as comprising Ime4p (Kar4p's paralog and the homolog of mammalian METTL3), Mum2p (homolog of mammalian WTAP), and Slz1p (MIS), but not Kar4p. During meiosis, Kar4p interacts with Ime4p, Mum2p, and Slz1p. Moreover, cells lacking Kar4p have highly reduced levels of mRNA methylation during meiosis indicating that Kar4p is a key member of the methyltransferase complex, as it is in humans. Analysis of kar4 Δ/Δ and 7 meiosis-specific alleles (Mei - ) revealed that Kar4p is required early in meiosis, before initiation of S-phase and meiotic recombination. High copy expression of the meiotic transcriptional activator IME1 rescued the defect of these Mei- alleles. Surprisingly, Kar4p was also found to be required at a second step for the completion of meiosis and sporulation. Over-expression of IME1 in kar4 Δ/Δ permits pre-meiotic S-phase, but most cells remained arrested with a monopolar spindle. Analysis of the function-specific mutants revealed that roughly half became blocked after premeiotic DNA synthesis and did not sporulate (Spo - ). Loss of Kar4p's Spo function was suppressed by overexpression of RIM4 , a meiotic translational regulator. Overexpression of IME1 and RIM4 together allowed sporulation of kar4 Δ/Δ cells. Taken together, these data suggest that Kar4p regulates meiosis at multiple steps, presumably reflecting requirements for methylation in different stages of meiotic gene expression. Author Summary: In yeast, KAR4 is required for mating and meiosis. A genetic screen for function-specific mutations identified 25 alleles that map to different surfaces on a predicted structure of the Kar4 protein (Kar4p). The mating-specific alleles interfere with Kar4p's ability to interact with the transcription factor Ste12p, its known partner in mating. The meiosis-specific alleles revealed an independent function: Kar4p is required for entry into meiosis and initiation of S-phase. During meiosis, Kar4p interacts with all components of the mRNA methyltransferase complex and kar4 Δ/Δ mutants have greatly reduced levels of mRNA methylation. Thus, Kar4p is a member of the yeast methyltransferase complex. Overexpression of the meiotic transcriptional activator IME1 rescued the meiotic entry defect but did not lead to sporulation, implying that Kar4p has more than one meiotic function. Suppression by Ime1p overexpression led to arrest after premeiotic DNA synthesis, but before sporulation. Loss of Kar4's sporulation function can be suppressed by overexpression of a translation regulator, Rim4p. Overexpression of both IME1 and RIM4 allowed sporulation in kar4 Δ/Δ cells.

Cla4p Kinase Activity Is Down-Regulated by Fus3p during Yeast Mating.

In Saccharomyces cerevisiae, the p21-activated kinase Cla4p regulates polarized morphogenesis and cytokinesis. However, it remains unknown how Cla4p kinase activity is regulated. After pheromone exposure, yeast cells temporally separate the mitotic and mating programs by sequestering Fus2p in the nucleus until cell cycle completion, after which Fus2p exits to facilitate cell fusion. Previously, we showed that sequestration is regulated by two opposing protein kinases, Cla4p and Fus3p. Phosphorylation of Fus2p-S67 by Cla4p promotes nuclear localization by both activating nuclear import and blocking export. During mating, phosphorylation of Fus2p-S85 and Fus2p-S100 by Fus3p promotes nuclear export and blocks import. Here, we find that Cla4p kinase activity is itself down-regulated during mating. Pheromone exposure causes Cla4p hyper-phosphorylation and reduced Fus2p-S67 phosphorylation, dependent on Fus3p. Multiple phosphorylation sites in Cla4p are mating- and/or Fus3p-specific. Of these, Cla4p-S186 phosphorylation reduced the kinase activity of Cla4p, in vitro. A phosphomimetic cla4-S186E mutation caused a strong reduction in Fus2p-S67 phosphorylation and nuclear localization, in vivo. More generally, a non-phosphorylatable mutation, cla4-S186A, caused failure to maintain pheromone arrest and delayed formation of the mating-specific septin morphology. Thus, as cells enter the mating pathway, Fus3p counteracts Cla4p kinase activity to allow proper mating differentiation.

Differential Requirement for the Cell Wall Integrity Sensor Wsc1p in Diploids Versus Haploids.

The primary role of the Cell Wall Integrity Pathway (CWI) in Saccharomyces cerevisiae is monitoring the state of the cell wall in response to general life cycle stresses (growth and mating) and imposed stresses (temperature changes and chemicals). Of the five mechanosensor proteins monitoring cell wall stress, Wsc1p and Mid2p are the most important. We find that WSC1 has a stringent requirement in zygotes and diploids, unlike haploids, and differing from MID2's role in shmoos. Diploids lacking WSC1 die frequently, independent of mating type. Death is due to loss of cell wall and plasma membrane integrity, which is suppressed by osmotic support. Overexpression of several CWI pathway components suppress wsc1∆ zygotic death, including WSC2, WSC3, and BEM2, as well as the Rho-GAPS, BEM3 and RGD2. Microscopic observations and suppression by BEM2 and BEM3 suggest that wsc1∆ zygotes die during bud emergence. Downstream in the CWI pathway, overexpression of a hyperactive protein kinase C (Pkc1p-R398P) causes growth arrest, and blocks the pheromone response. With moderate levels of Pkc1p-R398P, cells form zygotes and the wsc1∆ defect is suppressed. This work highlights functional differences in the requirement for Wsc1p in diploids Versus haploids and between Mid2p and Wsc1p during mating.

MutSα mismatch repair protein stability is governed by subunit interaction, acetylation, and ubiquitination.

In eukaryotes, DNA mismatch recognition is accomplished by the highly conserved MutSα (Msh2/Msh6) and MutSß (Msh2/Msh3) complexes. Previously, in the yeast Saccharomyces cerevisiae, we determined that deleting MSH6 caused wild-type Msh2 levels to drop by ∼50%. In this work, we determined that Msh6 steady-state levels are coupled to increasing or decreasing levels of Msh2. Although Msh6 and Msh2 are reciprocally regulated, Msh3 and Msh2 are not. Msh2 missense variants that are able to interact with Msh6 were destabilized when Msh6 was deleted; in contrast, variants that fail to dimerize were not further destabilized in cells lacking Msh6. In the absence of Msh6, Msh2 is turned over at a faster rate and degradation is mediated by the ubiquitin-proteasome pathway. Mutagenesis of certain conserved lysines near the dimer interface restored the levels of Msh2 in the absence of Msh6, further supporting a dimer stabilization mechanism. We identified two alternative forms of regulation both with the potential to act via lysine residues, including acetylation by Gcn5 and ubiquitination by the Not4 ligase. In the absence of Gcn5, Msh2 levels were significantly decreased; in contrast, deleting Not4 stabilized Msh2 and Msh2 missense variants with partial function. The stabilizing effect on Msh2 by either the presence of Msh6 or the absence of Not4 are dependent on Gcn5. Taken together, the results suggest that the wild-type MutSα mismatch repair protein stability is governed by subunit interaction, acetylation, and ubiquitination.

Reciprocal regulation of nuclear import of the yeast MutSalpha DNA mismatch repair proteins Msh2 and Msh6.

DNA mismatch recognition is performed in eukaryotes by two heterodimers known as MutSalpha (Msh2/Msh6) and MutSbeta (Msh2/Msh3) that must reside in the nucleus to function. Two putative Msh2 nuclear localization sequences (NLS) were characterized by fusion to green fluorescent protein (GFP) and site-directed mutagenesis in the context of Msh2. One NLS functioned in GFP targeting assays and both acted redundantly within Msh2. We examined nuclear localization of each of the MutS monomers in the presence and absence of their partners. Msh2 translocated to the nucleus in cells lacking Msh3 and Msh6; however, cells lacking Msh6 showed significantly decreased levels of nuclear Msh2. Furthermore, the overall protein levels of Msh2 were significantly diminished in the absence of Msh6, particularly if Msh2 lacked a functional NLS. Msh3 localized in the absence of Msh2, but Msh6 localization depended on Msh2 expressing functional NLSs. Overall, the nuclear levels of Msh2 and Msh6 decline when the other partner is absent. The data suggest a stabilization mechanism to prevent free monomer accumulation in the cytoplasm.

Role of transcription factor Kar4 in regulating downstream events in the Saccharomyces cerevisiae pheromone response pathway.

Yeast Kar4 is a putative transcription factor required for karyogamy (the fusion of haploid nuclei during mating) and possibly other functions. Previously known to be required only for the transcriptional induction of KAR3 and CIK1, microarray experiments identified many genes regulated by Kar4 in both mating and mitosis. Several gene clusters are positively or negatively regulated by mating pheromone in a Kar4-dependent manner. Chromatin immunoprecipitation and gel shift assays confirmed that Kar4 binds to regulatory DNA sequences upstream of KAR3. Together with one-hybrid experiments, these data support a model in which both Kar4 and Ste12 bind jointly to the KAR3 promoter. Analysis of the upstream regions of Kar4-induced genes identified a DNA sequence motif that may be a binding site for Kar4. Mutation within the motif upstream of KAR3 eliminated pheromone induction. Genes regulated by Kar4, on average, are delayed in their temporal expression and exhibit a more stringent dose response to pheromone. Furthermore, the induction of Kar4 by pheromone is necessary for the delayed temporal induction of KAR3 and PRM2, genes required for efficient nuclear fusion during mating. Accordingly, we propose that Kar4 plays a critical role in the choreography of the mating response.

Pheromone-induced polarization is dependent on the Fus3p MAPK acting through the formin Bni1p.

During mating, budding yeast cells reorient growth toward the highest concentration of pheromone. Bni1p, a formin homologue, is required for this polarized growth by facilitating cortical actin cable assembly. Fus3p, a pheromone-activated MAP kinase, is required for pheromone signaling and cell fusion. We show that Fus3p phosphorylates Bni1p in vitro, and phosphorylation of Bni1p in vivo during the pheromone response is dependent on Fus3p. fus3 mutants exhibited multiple phenotypes similar to bni1 mutants, including defects in actin and cell polarization, as well as Kar9p and cytoplasmic microtubule localization. Disruption of the interaction between Fus3p and the receptor-associated Galpha subunit caused similar mutant phenotypes. After pheromone treatment, Bni1p-GFP and Spa2p failed to localize to the cortex of fus3 mutants, and cell wall growth became completely unpolarized. Bni1p overexpression suppressed the actin assembly, cell polarization, and cell fusion defects. These data suggest a model wherein activated Fus3p is recruited back to the cortex, where it activates Bni1p to promote polarization and cell fusion.

Arp10p is a pointed-end-associated component of yeast dynactin.

In metazoans, dynein-dependent vesicle transport is mediated by dynactin, containing an actin-related protein, Arp1p, together with a cargo-selection complex containing a second actin-related protein, Arp11. Paradoxically, in budding yeast, models of dynactin function imply an interaction with membranes, whereas the lack of microtubule-based vesicle transport implies the absence of a cargo-selection complex. Using both genetic and biochemical approaches, we demonstrate that Arp10p is the functional yeast homologue of Arp11, suggesting the possible existence of a pointed-end complex in yeast. Specifically, Arp10p interacts with Arp1p and other dynactin subunits and is dependent on Arp1p for stability. Conversely, Arp10p stabilizes the dynactin complex by association with the Arp1p filament pointed end. Using a novel hRAS-Arp1p one-hybrid assay, we show that Arp1p associates with the plasma membrane dependent on dynactin subunits, but independent of dynein, and sensitive to cell wall damage. We directly show the association of Arp1p with not only the plasma membrane but also with a less dense membrane fraction. Based on the hRAS-Arp1p assay, loss of Arp10p enhances the apparent association of dynactin with the plasma membrane and suppresses the loss of signaling conferred by cell wall damage.

Cell fusion in yeast is negatively regulated by components of the cell wall integrity pathway.

During mating, Saccharomyces cerevisiae cells must degrade the intervening cell wall to allow fusion of the partners. Because improper timing or location of cell wall degradation would cause lysis, the initiation of cell fusion must be highly regulated. Here, we find that yeast cell fusion is negatively regulated by components of the cell wall integrity (CWI) pathway. Loss of the cell wall sensor, MID2, specifically causes "mating-induced death" after pheromone exposure. Mating-induced death is suppressed by mutations in cell fusion genes ( FUS1, FUS2, RVS161, CDC42), implying that mid2Δ cells die from premature fusion without a partner. Consistent with premature fusion, mid2Δ shmoos had thinner cell walls and lysed at the shmoo tip. Normally, Cdc42p colocalizes with Fus2p to form a focus only when mating cells are in contact (prezygotes) and colocalization is required for cell fusion. However, Cdc42p was aberrantly colocalized with Fus2p to form a focus in mid2Δ shmoos. A hyperactive allele of the CWI kinase Pkc1p ( PKC1*) caused decreased cell fusion and Cdc42p localization in prezygotes. In shmoos, PKC1* increased Cdc42p localization; however, it was not colocalized with Fus2p or associated with cell death. We conclude that Mid2p and Pkc1p negatively regulate cell fusion via Cdc42p and Fus2p.

Functional characterization of pathogenic human MSH2 missense mutations in Saccharomyces cerevisiae.

Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with defects in DNA mismatch repair. Mutations in either hMSH2 or hMLH1 underlie the majority of HNPCC cases. Approximately 25% of annotated hMSH2 disease alleles are missense mutations, resulting in a single change out of 934 amino acids. We engineered 54 missense mutations in the cognate positions in yeast MSH2 and tested for function. Of the human alleles, 55% conferred strong defects, 8% displayed intermediate defects, and 38% showed no defects in mismatch repair assays. Fifty percent of the defective alleles resulted in decreased steady-state levels of the variant Msh2 protein, and 49% of the Msh2 variants lost crucial protein-protein interactions. Finally, nine positions are predicted to influence the mismatch recognition complex ATPase activity. In summary, the missense mutations leading to loss of mismatch repair defined important structure-function relationships and the molecular analysis revealed the nature of the deficiency for Msh2 variants expressed in the tumors. Of medical relevance are 15 human alleles annotated as pathogenic in public databases that conferred no obvious defects in mismatch repair assays. This analysis underscores the importance of functional characterization of missense alleles to ensure that they are the causative factor for disease.

Yeast mating: a model system for studying cell and nuclear fusion.

Haploid yeast cells mate to form a zygote, whose progeny are diploid cells. A fundamentally sexual event, related to fertilization, yeast mating nevertheless exhibits cytological properties that appear similar to somatic cell fusion. A large collection of mutations that lead to defects in various stages of mating, including cell fusion, has allowed a detailed dissection of the overall pathway. Recent advances in imaging methods, together with powerful methods of genetic analysis, make yeast mating a superb platform for investigation of cell fusion. An understanding of yeast cell fusion will provide insight into fundamental mechanisms of cell signaling, cell polarization, and membrane fusion.

Alanine scanning of Arp1 delineates a putative binding site for Jnm1/dynamitin and Nip100/p150Glued.

Arp1p is the only actin-related protein (ARP) known to form actin-like filaments. Unlike actin, Arp1p functions with microtubules, as part of the dynein regulator, dynactin. Arp1p's dissimilar functions imply interactions with a distinct set of proteins. To distinguish surface features relating to Arp1p's core functions and to identify the footprint of protein interactions essential for dynactin function, we performed the first complete charge-cluster-to-alanine scanning mutagenesis of an ARP and compared the results with a similar study of actin. The Arp1p mutations revealed three nonoverlapping surfaces with distinct genetic properties. One of these surfaces encompassed a region unique to Arp1p that is crucial for Jnm1p (dynamitin/p50) and Nip100p (p150(Glued)) association as well as pointed-end associations. Unlike the actin mutations, none of the ARP1 alleles disrupt filament formation; however, one pointed-end allele delayed the elution of Arp1p on gel filtration, consistent with loss of additional subunits.