Mechanism of Ubiquitin Chain Formation by the human Anaphase‐Promoting Complex

Abstract

The ubiquitin ligase Anaphase‐Promoting Complex (APC/C) is essential for proliferation in all eukaryotes. It orchestrates progression through mitosis by catalyzing the disassembly of spindle checkpoint complexes and by promoting the sequential degradation of mitotic regulators. The APC/C mediates its functions by decorating substrates with ubiquitin chains of unknown topology. Previously, we had found that the number of substrate binding events required for ubiquitin chain formation, referred to as the processivity of ubiquitination, can determine the timing of substrate degradation. However, the mechanism that enables the human APC/C to assemble ubiquitin chains within a single substrate binding event has remained elusive. Similar to many ubiquitin ligases, the APC/C nucleates ubiquitin chain formation by transferring ubiquitin to a lysine in its substrate, but elongates chains by modifying lysine residues in attached ubiquitin molecules. Here, we have determined the topology of the ubiquitin chains that are assembled by the human APC/C and its specific E2 UbcH10. This allowed us to identify a patch of charged amino acids on the surface of ubiquitin, the TEK‐box, which is required for the APC/C‐dependent elongation of ubiquitin chains. Strikingly, sequences highly related to the TEK‐box in ubiquitin are found in APC/C‐substrates, where they facilitate the modification of substrate lysine residues, and thus, ubiquitin chain nucleation. We propose a mechanism of ubiquitin chain formation by the human APC/C, in which recognition of TEK‐boxes first orients lysine residues within substrates and later in ubiquitin relative to the active site of the E2 to allow the rapid formation of specific ubiquitin chains.