LAUSR

Proper protein destruction by the ubiquitin (Ub)‐proteasome system is vital for a faithful cell cycle. Hence, the activity of Ub ligases is tightly controlled. The Anaphase‐Promoting Complex/Cyclosome (APC/C) is a 1.2 MDa Ub ligase responsible for mitotic progression and G1 maintenance. At the G1/S transition, the APC/C is inhibited by EMI1 to prevent APC/C‐dependent polyubiquitination of cell cycle effectors. EMI1 uses several interaction motifs to block the recruitment of APC/C substrates as well as the APC/C‐associated E2s, UBE2C, and UBE2S. Paradoxically, EMI1 is also an APC/C substrate during G1. Using a comprehensive set of enzyme assays, we determined the context‐dependent involvement of the EMI1 motifs in APC/C‐dependent ubiquitination of EMI1 and other substrates. Furthermore, we demonstrated that an isolated C‐terminal peptide fragment of EMI1 activates APC/C‐dependent substrate priming by UBE2C. Together, these findings reveal the multiple roles of the EMI1 C‐terminus for G1 maintenance and the G1/S transition.