Stimulation of later functions of the yeast meiotic protein kinase Ime2p by the IDS2 gene product.

Ime2p is a protein kinase that is expressed only during meiosis in Saccharomyces cerevisiae. Ime2p stimulates early, middle, and late meiotic gene expression and down-regulates expression of IME1, which specifies an activator of early meiotic genes that acts independently of Ime2p. We have identified a new gene, IDS2 (for IME2-dependent signaling), which has a functional relationship to Ime2p. An ids2 null mutation delays down-regulation of IME1 and expression of middle and late meiotic genes. In an ime1 null mutant that express IME2 from the GAL1 promoter (ime1 delta PGAL1-IME2 mutant), early meiotic gene expression depends only upon Ime2p. In such strains, Ids2p is dispensable for expression of the early genes HOP1 and SPO13 but is essential for expression of the middle and late genes SPS1, SPS2, and SPS100. Ids2p is also essential for the autoregulatory pathway through which Ime2p activates its own expression via the IME2 upstream activation sequences (UAS). An PGAL1-IME2 derivative that produces a truncated Ime2p (lacking its C-terminal 174 residues) permits IME2 UAS activation in the absence of Ids2p. This observation suggests that Ids2p acts upstream of Ime2p or that Ids2p and Ime2p act in independent, convergent pathways to stimulate IME2 UAS activity. Accumulation of epitope-tagged Ids2p derivatives is greatest in growing cells and declines during meiosis. We propose that Ids2p acts indirectly to modify Ime2p activity, thus permitting Ime2p to carry out later meiotic functions.     

Stimulation of yeast meiotic gene expression by the glucose-repressible protein kinase Rim15p.

The Saccharomyces cerevisiae RIM15 gene was identified previously through a mutation that caused reduced ability to undergo meiosis. We report here an analysis of the cloned RIM15 gene, which specifies a 1,770-residue polypeptide with homology to serine/threonine protein kinases. Rim15p is most closely related to Schizosaccharomyces pombe cek1+. Analysis of epitope-tagged derivatives indicates that Rim15p has autophosphorylation activity. Deletion of RIM15 causes reduced expression of several early meiotic genes (IME2, SPO13, and HOP1) and of IME1, which specifies an activator of early meiotic genes. However, overexpression of IME1 does not permit full expression of early meiotic genes in a rim15delta mutant. Ime1p activates early meiotic genes through its interaction with Ume6p, and analysis of Rim15p-dependent regulatory sites at the IME2 promoter indicates that activation through Ume6p is defective. Two-hybrid interaction assays suggest that Ime1p-Ume6p interaction is diminished in a rim15 mutant. Glucose inhibits Ime1p-Ume6p interaction, and we find that Rim15p accumulation is repressed in glucose-grown cells. Thus, glucose repression of Rim15p may be responsible for glucose inhibition of Ime1p-Ume6p interaction.     

The C-terminal region of the meiosis-specific protein kinase Ime2 mediates protein instability and is required for normal spore formation in budding yeast

The cyclin-dependent kinase Cdk1 and the related kinase Ime2 act in concert to trigger progression of the meiotic cell cycle in the yeast Saccharomyces cerevisiae. These kinases share several functions and substrates during meiosis, but their regulation seems to be clearly different. In contrast to Cdk1, no cyclin seems to be involved in the regulation of Ime2 activity. Ime2 is a highly unstable protein, and we aimed to elucidate the relevance of Ime2 instability. We first determined the sequence elements required for Ime2 instability by constructing a set of deletions in the IME2 gene. None of the small deletions in Ime2 affected its instability, but deletion of a 241 amino acid C-terminal region resulted in a highly stabilized protein. Thus, the C-terminal domain of Ime2 is important for mediating protein instability. The stabilized, truncated Ime2 protein is highly active in vivo. Replacement of the IME2 gene with the truncated IME2ΔC241 in diploid strains did not interfere with meiotic nuclear divisions, but caused abnormalities in spore formation, as manifested by the appearance of many asci with a reduced spore number such as triads and dyads. The truncated Ime2 caused a reduction of spore number in a dominant manner. We conclude that downregulation of Ime2 kinase activity mediated by the C-terminal domain is required for the efficient production of normal four-spore asci. Our data suggest a role for Ime2 in spore number control in S. cerevisiae.     

The Cdk-activating kinase Cak1p promotes meiotic S phase through Ime2p

CAK1 encodes an essential protein kinase in Saccharomyces cerevisiae that is required for activation of the Cdc28p Cdk. CAK1 also has several CDC28-independent functions that are unique to meiosis. The earliest of these functions is to induce S phase, which is regulated differently in meiosis than in mitosis. In mitosis, Cdc28p controls its own S-phase-promoting activity by signaling the destruction of its inhibitor, Sic1p. In meiosis, Sic1p destruction is signaled by the meiosis-specific Ime2p protein kinase. Our data show that Cak1p is required to activate Ime2p through a mechanism that requires threonine 242 and tyrosine 244 in Ime2p's activation loop. This activation promotes autophosphorylation and accumulation of multiply phosphorylated forms of Ime2p during meiotic development. Consistent with Cak1p's role in activating Ime2p, cells lacking Cak1p are deficient in degrading Sic1p. Deletion of SIC1 or overexpression of IME2 can partially suppress the S-phase defect in cak1 mutant cells, suggesting that Ime2p is a key target of Cak1p regulation. These data show that Cak1p is required for the destruction of Sic1p in meiosis, as in mitosis, but in meiosis, it functions through a sporulation-specific kinase.     

The Ime2 protein kinase family in fungi: more duties than just meiosis

Ime2 of the budding yeast Saccharomyces cerevisiae belongs to a family of conserved protein kinases displaying sequence similarities to both cyclin-dependent kinases and mitogen-activated protein kinases. Ime2 has a pivotal role for meiosis and sporulation. The involvement of this protein kinase in the regulation of various key events in meiosis, such as the initiation of DNA replication, the expression of meiosis-specific genes and the passage through the two consecutive rounds of nuclear divisions has been characterized in detail. More than 20 years after the identification of the IME2 gene, a recent report has provided the first evidence for a function of this gene outside of meiosis, which is the regulation of pseudohyphal growth. In the last few years, Ime2-related protein kinases from various fungal species were studied. Remarkably, these homologues are not generally required for meiosis, but instead have other specific tasks. In filamentous ascomycete species, Ime2 homologues are involved in the inhibition of fruiting body formation in response to environmental signals. In the pathogenic basidiomycetes Ustilago maydis and Cryptococcus neoformans, members of this kinase family apparently have primary roles in regulating mating. Thus, Ime2-related kinases exhibit an amazing variety in controlling sexual developmental programs in fungi.     

The meiosis-specific protein kinase Ime2 directs phosphorylation of replication protein A

In Saccharomyces cerevisiae, the cellular single-stranded DNA-binding protein replication protein A (RPA) becomes phosphorylated during meiosis in two discrete reactions. The primary reaction is first observed shortly after cells enter the meiotic program and leads to phosphorylation of nearly all the detectable RPA. The secondary reaction, which requires the ATM/ATR homologue Mec1, is induced upon initiation of recombination and only modifies a fraction of the total RPA. We now report that correct timing of both RPA phosphorylation reactions requires Ime2, a meiosis-specific protein kinase that is critical for proper initiation of meiotic progression. Expression of Ime2 in vegetative cells leads to an unscheduled RPA phosphorylation reaction that does not require other tested meiosis-specific kinases and is distinct from the RPA phosphorylation reaction that normally occurs during mitotic growth. In addition, immunoprecipitated Ime2 catalyzes phosphorylation of purified RPA. Our data strongly suggest that Ime2 is an RPA kinase in vivo. We propose that Ime2 directly catalyzes RPA phosphorylation in the primary reaction and indirectly promotes the Mec1-dependent secondary reaction by advancing cells through meiotic progression. Our studies have identified a novel meiosis-specific reaction that targets a key protein required for DNA replication, repair, and recombination. This pathway could be important in differentiating mitotic and meiotic DNA metabolism.     

Purification and some properties of Saccharomyces cerevisiae meiosis-specific protein kinase Ime2

Ime2 is the founding member of a family of protein kinases that are required for effective progression through meiotic development. Ime2 is essential for the induction of meiosis-specific genes and for the activation of meiotic DNA replication in the budding yeast Saccharomyces cerevisiae. Aside from the fact that Ime2 is a protein kinase and shares several amino acid motifs with cyclin dependent kinases, virtually nothing is known about its enzymatic properties or substrates. Biochemical characterization of Ime2 has been hindered by its low abundance and short half-life. We have created baculovirus expression vectors to produce recombinant Ime2 in insect cells. In this report, we describe the overproduction of Ime2 and its purification using affinity chromatography. Using this procedure, we have been able to purify up to 2mg Ime2 from 1L of infected insect cells. The Ime2 isolated by this method displays properties similar to those of the native enzyme that has been immunoprecipitated from yeast. The high level expression of Ime2 in this system and its ease of purification will be beneficial for more extensive biochemical analysis of Ime2 and related meiosis-specific kinases.     

Phosphorylation of Ime2 regulates meiotic progression in Saccharomyces cerevisiae

Ime2p is a meiosis-specific protein kinase in Saccharomyces cerevisiae that controls multiple steps in meiosis. Although Ime2p is functionally related to the Cdc28p cyclin-dependent kinase (CDK), no cyclin binding partners that regulate its activities have been identified. The sequence of the Ime2p catalytic domain is similar to CDKs and mitogen-activated protein kinases (MAPKs). Ime2p is activated by phosphorylation of its activation loop in a Cak1p-dependent fashion and is subsequently phosphorylated on multiple residues as cells progress through meiosis. In this study, we show that Ime2p purified from meiotic cells is phosphorylated on Thr(242) and Tyr(244) in its activation loop and on Ser(520) and Ser(625) in its C terminus. Ime2p autophosphorylates on threonine in its activation loop in vitro consistent with autophosphorylation of Thr(242) playing a role in its activation. Moreover, autophosphorylation in cis is required for Ime2p to become hyperphosphorylated. Phosphorylation of the C-terminal serines is not essential to sporulation. However, Ime2p C-terminal phosphorylation site mutants genetically interact with components of the FEAR network that controls exit from meiosis I. These data suggest that Ime2p plays a role in controlling the exit from meiosis I and demonstrate that a phospho-modification pathway regulates Ime2p during the different phases of meiotic development.     

The role and regulation of the preRC component Cdc6 in the initiation of premeiotic DNA replication

In all eukaryotes, the initiation of DNA replication is regulated by the ordered assembly of DNA/protein complexes on origins of DNA replication. In this report, we examine the role of Cdc6, a component of the prereplication complex, in the initiation of premeiotic DNA replication in budding yeast. We show that in the meiotic cycle, Cdc6 is required for DNA synthesis and sporulation. Moreover, similarly to the regulation in the mitotic cell cycle, Cdc6 is specifically degraded upon entry into the meiotic S phase. By contrast, chromatin-immunoprecipitation analysis reveals that the origin-bound Cdc6 is stable throughout the meiotic cycle. Preliminary evidence suggests that this protection reflects a change in chromatin structure that occurs in meiosis. Using the cdc28-degron allele, we show that depletion of Cdc28 leads to stabilization of Cdc6 in the mitotic cycle, but not in the meiotic cycle. We show physical association between Cdc6 and the meiosis-specific hCDK2 homolog Ime2. These results suggest that under meiotic conditions, Ime2, rather than Cdc28, regulates the stability of Cdc6. Chromatin-immunoprecipitation analysis reveals that similarly to the mitotic cell cycle, Mcm2 binds origins in G1 and meiotic S phases, and at the end of the second meiotic division, it is gradually removed from chromatin.