Mutually dependent degradation of Ama1p and Cdc20p terminates APC/C ubiquitin ligase activity at the completion of meiotic development in yeast

Abstract

BackgroundThe execution of meiotic nuclear divisions in S. cerevisiae is regulated by protein degradation mediated by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase. The correct timing of APC/C activity is essential for normal chromosome segregation. During meiosis, the APC/C is activated by the association of either Cdc20p or the meiosis-specific factor Ama1p. Both Ama1p and Cdc20p are targeted for degradation as cells exit meiosis II with Cdc20p being destroyed by APC/CAma1. In this study we investigated how Ama1p is down regulated at the completion of meiosis.FindingsHere we show that Ama1p is a substrate of APC/CCdc20 but not APC/CCdh1 in meiotic cells. Cdc20p binds Ama1p in vivo and APC/CCdc20 ubiquitylates Ama1p in vitro. Ama1p ubiquitylation requires one of two degradation motifs, a D-box and a “KEN-box” like motif called GxEN. Finally, Ama1p degradation does not require its association with the APC/C via its conserved APC/C binding motifs (C-box and IR) and occurs simultaneously with APC/CAma1-mediated Cdc20p degradation.ConclusionsUnlike the cyclical nature of mitotic cell division, meiosis is a linear pathway leading to the production of quiescent spores. This raises the question of how the APC/C is reset prior to spore germination. This and a previous study revealed that Cdc20p and Ama1p direct each others degradation via APC/C-dependent degradation. These findings suggest a model that the APC/C is inactivated by mutual degradation of the activators. In addition, these results support a model in which Ama1p and Cdc20p relocate to the substrate address within the APC/C cavity prior to degradation.

High‐throughput measurement of recombination rates and genetic interference in Saccharomyces cerevisiae

“…We checked this extensively by (1) observing non‐fluorescent spores coming from hemizygous diploids using fluorescence microscopy (see example in Figure S1) – no putative remaining fluorescent proteins were ever observed in the spore cytoplasm, and (2) analysing the distributions of fluorescence intensity in spores by flow cytometry (see example in Figure S13) – no peak overlap was observed in these distributions, so our fluorescence classes are reliable. This finding indicates also that, during the meiotic process, fluorescent proteins expressed in the diploid parent cells are either not transferred to the cytoplasm of the spore or degraded during meiosis as previously observed in the case of cell cycle‐related proteins (Tan, Lewandowski, Mallory, Strich, & Cooper, ). Then spore expression is due to autonomous expression of proteins as soon as the four spores resulting from meiosis are isolated by a membrane as previously shown (Neiman, ).…”

Meiosis II spindle disassembly requires two distinct pathways

“…In meiosis, the APC/C is also important for spindle disassembly, and the completion of meiosis II utilizes Ama1 as the activator for the anaphase promoting complex instead of Cdh1 ( Cooper et al. , 2000 ; Tan et al. , 2013 ).…”

The Role of the Anaphase-Promoting Complex/Cyclosome in Plant Growth

“…The GxEN-box degron is also found in the Ama1 protein, which is an APC/C activator specific for meiotic progression. In yeast, mutation of the GxEN-box degron leads to the stabilization of Ama1 (Tan et al, 2013). To date, no GxEN-box motif was found in plants.…”