Degradation of the separase-cleaved Rec8, a meiotic cohesin subunit, by the n-end rule pathway

The Ate1 arginyltransferase (R- Transferase) is a component of the N-end rule pathway, which recognizes proteins containing N- Terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. Ate1 arginylates N- Terminal Asp, Glu, or (oxidized) Cys. The resulting N- Terminal Arg is recognized by ubiquitin ligases of the N-end rule pathway. In the yeast Saccharomyces cerevisiae, the separase-mediated cleavage of the Scc1/Rad21/ Mcd1 cohesin subunit generates a C- Terminal fragment that bears N- Terminal Arg and is destroyed by the N-end rule pathway without a requirement for arginylation. In contrast, the separase-mediated cleavage of Rec8, the mammalian meiotic cohesin subunit, yields a fragment bearing N- Terminal Glu, a substrate of the Ate1 R- Transferase. Here we constructed and used a germ cell-confined Ate1^-/- mouse strain to analyze the separase-generated C- Terminal fragment of Rec8. We show that this fragment is a short-lived N-end rule substrate, that its degradation requires N- Terminal arginylation, and that male Ate1^-/- mice are nearly infertile, due to massive apoptotic death of Ate1^-/- spermatocytes during the metaphase of meiosis I. These effects of Ate1 ablation are inferred to be caused, at least in part, by the failure to destroy the C- Terminal fragment of Rec8 in the absence of N- Terminal arginylation.