The meiosis-specific Cdc20 family-member Ama1 promotes binding of the Ssp2 activator to the Smk1 MAP kinase.

Smk1 is a meiosis-specific MAP kinase (MAPK) in budding yeast that is required for spore formation. It is localized to prospore membranes (PSMs), the structures that engulf haploid cells during meiosis II (MII). Similar to canonically activated MAPKs, Smk1 is controlled by phosphorylation of its activation-loop threonine (T) and tyrosine (Y). However, activation loop phosphorylation occurs via a noncanonical two-step mechanism in which 1) the cyclin-dependent kinase activating kinase Cak1 phosphorylaytes T207 during MI, and 2) Smk1 autophosphorylates Y209 as MII draws to a close. Autophosphorylation of Y209 and catalytic activity for substrates require Ssp2, a meiosis-specific protein that is translationally repressed until anaphase of MII. Ama1 is a meiosis-specific targeting subunit of the anaphase-promoting complex/cyclosome that regulates multiple steps in meiotic development, including exit from MII. Here, we show that Ama1 activates autophosphorylation of Smk1 on Y209 by promoting formation of the Ssp2/Smk1 complex at PSMs. These findings link meiotic exit to Smk1 activation and spore wall assembly.

Differential phosphorylation of a regulatory subunit of protein kinase CK2 by target of rapamycin complex 1 signaling and the Cdc-like kinase Kns1.

Transcriptional regulation of ribosome and tRNA synthesis plays a central role in determining protein synthetic capacity and is tightly controlled in response to nutrient availability and cellular stress. In Saccharomyces cerevisiae, the regulation of ribosome and tRNA synthesis was recently shown to involve the Cdc-like kinase Kns1 and the GSK-3 kinase Mck1. In this study, we explored additional roles for these conserved kinases in processes connected to the target of rapamycin complex 1 (TORC1). We conducted a synthetic chemical-genetic screen in a kns1Δ mck1Δ strain and identified many novel rapamycin-hypersensitive genes. Gene ontology analysis showed enrichment for TORC1-regulated processes (vesicle-mediated transport, autophagy, and regulation of cell size) and identified new connections to protein complexes including the protein kinase CK2. CK2 is considered to be a constitutively active kinase and in budding yeast, the holoenzyme comprises two regulatory subunits, Ckb1 and Ckb2, and two catalytic subunits, Cka1 and Cka2. We show that Ckb1 is differentially phosphorylated in vivo and that Kns1 mediates this phosphorylation when nutrients are limiting and under all tested stress conditions. We determined that the phosphorylation of Ckb1 does not detectably affect the stability of the CK2 holoenzyme but correlates with the reduced occupancy of Ckb1 on tRNA genes after rapamycin treatment. Thus, the differential occupancy of tRNA genes by CK2 is likely to modulate its activation of RNA polymerase III transcription. Our data suggest that TORC1, via its effector kinase Kns1, may regulate the association of CK2 with some of its substrates by phosphorylating Ckb1.

The anaphase promoting complex targeting subunit Ama1 links meiotic exit to cytokinesis during sporulation in Saccharomyces cerevisiae.

Ascospore formation in yeast is accomplished through a cell division in which daughter nuclei are engulfed by newly formed plasma membranes, termed prospore membranes. Closure of the prospore membrane must be coordinated with the end of meiosis II to ensure proper cell division. AMA1 encodes a meiosis-specific activator of the anaphase promoting complex (APC). The activity of APC(Ama1) is inhibited before meiosis II, but the substrates specifically targeted for degradation by Ama1 at the end of meiosis are unknown. We show here that ama1Delta mutants are defective in prospore membrane closure. Ssp1, a protein found at the leading edge of the prospore membrane, is stabilized in ama1Delta mutants. Inactivation of a conditional form of Ssp1 can partially rescue the sporulation defect of the ama1Delta mutant, indicating that an essential function of Ama1 is to lead to the removal of Ssp1. Depletion of Cdc15 causes a defect in meiotic exit. We find that prospore membrane closure is also defective in Cdc15 and that this defect can be overcome by expression of a form of Ama1 in which multiple consensus cyclin-dependent kinase phosphorylation sites have been mutated. These results demonstrate that APC(Ama1) functions to coordinate the exit from meiosis II with cytokinesis.

Regulation of spindle pole function by an intermediary metabolite.

Spore formation in the yeast Saccharomyces cerevisiae depends on a modification of spindle pole bodies (SPBs) at the onset of meiosis II that allows them to promote de novo membrane formation. Depletion of the environmental carbon source during sporulation results in modification of only one SPB from each meiosis II spindle and formation of a two-spored ascus, called a nonsister dyad (NSD). We have found that mutants impaired in the glyoxylate pathway, which is required for the conversion of acetate to glucose, make NSDs when acetate is the primary carbon source. Wild-type cells make NSDs when the carbon source is glycerol, which is converted to glucose independently of the glyoxylate pathway. During NSD formation in glycerol, only the two SPBs created at the meiosis I/II transition ("daughters") are modified. In these conditions, the SPB components Mpc70p and Spo74p are not recruited to mother SPBs. Moreover, cooverexpression of Mpc70p and Spo74p suppresses NSD formation in glycerol. Our findings indicate that flux through the glyoxylate pathway during sporulation regulates modification of mother SPBs via recruitment of Mpc70p and Spo74p. These results define a cellular response in which the accumulation of an intermediary metabolite serves as a measure of biosynthetic capacity to regulate the number of daughter cells formed.