The processivity of multiubiquitination by the APC determines the order of substrate degradation

Abstract

The anaphase-promoting complex (APC) coordinates mitosis and G1 by sequentially promoting the degradation of key cell-cycle regulators. Following the degradation of its substrates in G1, the APC catalyzes the autoubiquitination of its E2 UbcH10. This stabilizes cyclin A and allows it to inactivate APCCdh1. How the APC establishes this complex temporal sequence of ubiquitina-tions, referred to as substrate ordering, is not understood. Here we show that sub-strate ordering depends on the relative processivity of substrate multiubiquitina-tion by the APC. Processive substrates ob-tain ubiquitin chains in a single APC binding event. The multiubiquitination of distribu-tive substrates requires multiple rounds of APC binding, which render it sensitive to lower APC concentrations, competition by processive substrates, and deubiquitina-tion. Consequently, more processive sub-strates are preferentially multiubiquitinated in vitro and degraded earlier in vivo. The processivityofmultiubiquitination isstrongly influenced by the D box within the sub-strate, suggesting that substrate ordering is established by a mechanism intrinsic to APCand its substrates and similar to kinetic proofreading