Abstract
The anaphase-promoting complex (APC) or cyclosome is a multisubunit ubiquitin-protein ligase that ubiquitinates and thereby promotes the destruction of the mitotic cyclins and the separase inhibitor, securin. The contributions of the APC to progression through the meiotic program are not clear. To clarify the function of the APC in meiosis, we screened several yeast meiotic proteins as APC substrates in vitro. We found that the meiotic regulator Spo13 is an APC substrate that is degraded during anaphase I. Spo13 is expressed only in meiotic cells, where it has multiple functions, including the promotion of monopolar chromosome attachment in the first division. Spo13 ubiquitination by the APC depends on an LxExxxN sequence (residues 26-32) that is distinct from previously described destruction sequences of APC substrates. Mutation of one residue, leucine 26, prevented Spo13 ubiquitination by the APC in vitro and stabilized the protein through the meiotic divisions. Analysis of meiotic progression and spore viability of yeast containing the stabilized Spo13 mutant revealed no significant defects, indicating that Spo13 destruction in anaphase I is not essential for meiosis. We propose that Spo13 destruction is one of multiple mechanisms underlying the switch from monopolar to bipolar chromosome attachment between the meiotic divisions.
Highlights
Cdh1 [1, 2, 4], which are thought to recruit substrates to the enzyme for ubiquitination
Identification of Spo13 as an anaphase-promoting complex (APC) Substrate—To identify additional APC functions in meiosis, we performed a screen for novel substrates
The APC is necessary for meiotic progression, as yeast with reduced APC function arrest in meiotic metaphase I with unsegregated chromosomes [13, 14]
Summary
General Yeast Methods—All strains were based on the W303 background. For galactose-inducible SPO13 expression, the SPO13 coding sequence was inserted by recombination in either a low copy CEN/ARS plasmid (pRS314.1234) or high copy 2-micron plasmid (pAB1234) in frame with a C-terminal TAP tag, under the control of the GAL1–10 promoter. Measurement of Protein Stability in Vivo—W303 MAT a yeast, carrying low copy plasmids with galactose-inducible SPO13-TAP or spo13-db-TAP, were arrested in raffinose (2%) media supplemented with either ␣ factor (G1) or nocodazole (M) for 3 h. Galactose (2%) was added to the media to induce production of Spo13-TAP or Spo13-db-TAP. In some experiments (Fig. 2F), Cdh and Cdc were produced by translation in vitro, and the lysate used directly in the APC reactions; NEM was not added in these experiments because it inactivates Cdc. Mitotic Experiments—To determine the effect of SPO13 overexpression in mitotic cells, W303 MAT a yeast (with plasmids carrying galactose-inducible SPO13-TAP or spo13-dbTAP) were arrested in raffinose (2%) media supplemented with ␣ factor (1 g mlϪ1) at 30 °C for 2 h. The antibodies used were ␣-tubulin clone YOL1/34 (Serotec), ␣-HA clone 16B12 (Babco), and PAP (Sigma)
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