Figure 8.
Figure 8. Cbk1 promotes Cts1 secretion independent of CTS1 transcription. (A) Schematic of the ACE2-GOF allele bypassing the necessity of Cbk1 to activate CTS1 transcription. The ACE2-GOF allele mimics Cbk1 phosphorylation at S122, S137, and S436 to promote CTS1 expression in the absence of CBK1. (B) Cts1 secretion is reduced in the absence of CBK1 despite normal Cts1 production. WT, cbk1Δ, ACE2-GOF, ACE2-GOF cbk1Δ cells expressing HA-tagged Cts1 were synchronized in mitosis. Protein was collected at the indicated times following mitotic release at 30°C and were processed as in Fig. 2 A (see also Fig. S5 A). (C) Western blot quantification (as in Fig. 2 B). Fold change relative to the maximum signal of WT cells is shown. (D) Fir1 primarily functions to inhibit Cbk1 to prevent Cts1 secretion. inn1-AID ACE2-GOF cells with the additional genotype indicated to the right of the panel were treated with DMSO (−Auxin) or 0.5 mM auxin (+Auxin). Secreted protein was collected at the indicated times following mitotic release at 30°C, and a representative Western blot of secreted Cts1 is shown (see also Fig. S5 B). (E) Cbk1 deletion restores viability upon septation failure despite Cts1 expression. Fivefold serial dilutions of the strains in D (all strains express PrGal-CDC20 inn1-AID ACE2-GOF TIR1) were spotted to YP galactose plates with and without the addition of 0.5 mM auxin. Plates were incubated for 2 d at 30°C. (F) ECO pathway. Upon incomplete septation, stabilized Fir1 at the bud neck inhibits Cbk1, blocking secretion of septum destroying enzymes. Activation of ECO protects cytokinesis by ensuring the strict temporal sequence of opposing processes: septation and cell separation.

Cbk1 promotes Cts1 secretion independent of CTS1 transcription. (A) Schematic of the ACE2-GOF allele bypassing the necessity of Cbk1 to activate CTS1 transcription. The ACE2-GOF allele mimics Cbk1 phosphorylation at S122, S137, and S436 to promote CTS1 expression in the absence of CBK1. (B) Cts1 secretion is reduced in the absence of CBK1 despite normal Cts1 production. WT, cbk1Δ, ACE2-GOF, ACE2-GOF cbk1Δ cells expressing HA-tagged Cts1 were synchronized in mitosis. Protein was collected at the indicated times following mitotic release at 30°C and were processed as in Fig. 2 A (see also Fig. S5 A). (C) Western blot quantification (as in Fig. 2 B). Fold change relative to the maximum signal of WT cells is shown. (D) Fir1 primarily functions to inhibit Cbk1 to prevent Cts1 secretion. inn1-AID ACE2-GOF cells with the additional genotype indicated to the right of the panel were treated with DMSO (−Auxin) or 0.5 mM auxin (+Auxin). Secreted protein was collected at the indicated times following mitotic release at 30°C, and a representative Western blot of secreted Cts1 is shown (see also Fig. S5 B). (E) Cbk1 deletion restores viability upon septation failure despite Cts1 expression. Fivefold serial dilutions of the strains in D (all strains express PrGal-CDC20 inn1-AID ACE2-GOF TIR1) were spotted to YP galactose plates with and without the addition of 0.5 mM auxin. Plates were incubated for 2 d at 30°C. (F) ECO pathway. Upon incomplete septation, stabilized Fir1 at the bud neck inhibits Cbk1, blocking secretion of septum destroying enzymes. Activation of ECO protects cytokinesis by ensuring the strict temporal sequence of opposing processes: septation and cell separation.

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