Septation mutants are sensitive to inappropriate activation of cell separation. (A) Cytokinesis in budding yeast. i: Normal, rapid cytokinesis can be separated into two broad phases: septation and cell separation. Only after completion of septation are septum-destroying enzymes secreted. To ensure this temporal order, we predict cells actively inhibit separation until septation is complete. ii: Cells with septation defects generate a remedial septum. Cells forming a slow remedial septum are particularly sensitive to premature septum degradation, and we propose cells activate a checkpoint-like mechanism to enforce the strict temporal order of septation and separation. (B) Inappropriate CTS1 expression is detrimental when septation fails. WT and inn1-AID cells transformed with an empty vector or a galactose-inducible vector expressing CTS1 were spotted in fivefold serial dilutions to plates containing glucose (CTS1 OFF) or galactose (CTS1 ON). Additionally, the plates contained 0.5 mM auxin (+Auxin) or DMSO (−Auxin). Plates were incubated at 30°C for 3 d. All strains express the E3 ligase TIR1. (C) CTS1 deletion partially restores viability to cells with disrupted septum synthesis. The indicated strains were grown on YPD plates containing 0.5 mM auxin (+Auxin) or DMSO (−Auxin) and incubated at 30°C for 3 d. All strains express the E3 ligase TIR1.