Red1p, a MEK1-dependent Phosphoprotein That Physically Interacts with Hop1p during Meiosis in Yeast

Abstract

The synaptonemal complex (SC) is a proteinaceous structure formed between pairs of homologous chromosomes during prophase I of meiosis. The proper assembly of axial elements (AEs), lateral components of the SC, during meiosis in the yeast, Saccharomyces cerevisiae, is essential for wild-type levels of recombination and for the accurate segregation of chromosomes at the first meiotic division. Genetic experiments have indicated that the stoichiometry between two meiosis-specific components of AEs in S. cerevisiae, HOP1 and RED1, is critical for proper assembly and function of the SC. A third meiosis-specific gene, MEK1, which encodes a putative serine/threonine protein kinase, is also important for proper AE function, suggesting that AE formation is regulated by phosphorylation. In this paper, we demonstrate that Mek1p is a functional kinase in vitro and that catalytic activity is an essential part of the meiotic function of Mek1 in vivo. Immunoblot analysis revealed that Red1p is a MEK1-dependent phosphoprotein. Co-immunoprecipitation experiments demonstrated that the interaction between Hop1p and Red1p is enhanced by the presence of MEK1. Thus, MEK1-dependent phosphorylation of Red1p facilitates the formation of Hop1p/Red1p hetero-oligomers, thereby enabling the formation of functional AEs.

Budding Yeast Sae2 is an In Vivo Target of the Mec1 and Tel1 Checkpoint Kinases During Meiosis

“…Consistently, we found that the meiotic checkpoint kinase Mek1 is phosphorylated in a Spo11-dependent manner concomitantly with DSB accumulation during an unperturbed meiosis. Because Mek1-dependent phosphorylation events are necessary to maintain the checkpoint-dependent arrest of meiotic recombination mutants, [21][22][23] this suggests that meiotic DSBs persist enough to be sensed as DNA lesions and to activate the recombination checkpoint even in meiotic recombination-competent cells.…”

“…18 Mek1 is the meiosis-specific paralog of the Rad53 protein kinase, and its kinase activity is necessary to maintain the checkpointdependent arrest of meiotic recombination mutants. [21][22][23] Mek1 also exhibits Mec1-dependent phosphorylation, 24 suggesting that it substitutes for Rad53 in the context of meiotic recombination. Interestingly, the phospho-specific FHA domain of Mek1 is involved in its binding to phosphorylated Red1, which is in turn required for Mek1 phosphorylation.…”

Role for the Silencing Protein Dot1 in Meiotic Checkpoint Control

“…Red1 is a component of the lateral elements of the SC (Smith and Roeder, 1997), and Mek1 is a kinase that phosphorylates Red1 (Bailis and Roeder, 1998;de los Santos and Hollingsworth, 1999). Mutation of DOT1 or RED1 has similar effects when combined with dmc1: both bypass meiotic arrest, and both channel the repair of DSBs into a Rad54-dependent pathway (this work; Xu et al, 1997;Bishop et al, 1999).…”

Chemical Inactivation of Cdc7 Kinase in Budding Yeast Results in a Reversible Arrest That Allows Efficient Cell Synchronization Prior to Meiotic Recombination

“…DSB formation was quantitated using a Molecular Dynamics Phosphoimager and ImageQuant 1.11 software. Hop1 immunoprecipitation and detection were performed as described in de los Santos and Hollingsworth (1999). Meiotic progression was monitored by fixing the cells in 3.7% formaldehyde and staining them with 49,6-diamidino-2-phenylinodole (DAPI).…”

“…Time courses: Cells were sporulated in 2% potassium acetate at 30°as described in de los Santos and Hollingsworth (1999). Flow cytometry was performed by fixing 3 ml of sporulating cells with 70% ethanol overnight at 4°.…”