Fir1 is stabilized at the bud neck when septation is disrupted. (A) Fir1 localizes to the bud neck in late mitosis. Asynchronous cells expressing Fir1-GFP and Myo1-mCherry were subject to time-lapse microscopy. Maximum projection of serial z-stacks are shown in the xy and yz planes at the times indicated after beginning image acquisition. Bar, 1 µm. A representative cell is shown. See also Video 1. (B) Fir1 remains at the site of septation after the completion of cytokinesis. An aliquot of cells from a synchronized population was removed every 10 min after release and was imaged. The percentage of cells with bud neck localization of the indicated protein is shown (n > 20 cells per time point). (C) Fir1 is degraded before Cts1 secretion. Fir1-myc was immunoprecipitated from normalized lysates (input Hxk2 blot) following release from a synchronized culture at the times indicated at 30°C and subject to immunoblot. From the same culture, secreted Cts1 was collected and subject to immunoblot. A representative blot is shown. The asterisk indicates when peak cell separation was observed (Fig. S3 A). (D) Fir1 is not degraded when septation is disrupted. Fir1-myc was immunoprecipitated from normalized lysates (Hxk2 input) in synchronized inn1-AID cells treated with or without auxin at 30°C and subject to immunoblot. The asterisk indicates when peak cell separation was observed. Since inn1-AID cells treated with auxin do not separate, no asterisk is shown (see Fig. S3B). (E) Fir1 remains localized to the bud neck when septation is disrupted. Synchronized inn1-AID cells treated with or without auxin were released from arrest, and representative images are shown from the indicated time after release. The septin mCherry-Cdc3 was used to mark the bud neck. Bar, 5 µm (see also Fig. S3, C–E).