Mutation of Hof1 PEST motif phosphorylation sites leads to retention of Hof1 at the bud neck and a decrease in the rate of myosin contraction

Abstract

Regulation of actomyosin ring contraction is important for the coordination of cytokinesis with mitosis. Hof1, a member of the Pombe Cdc15 homology (PCH) family of proteins, is required for efficient cytokinesis in budding yeast. Phosphorylation of Hof1 depends on the mitotic exit network (MEN), and its degradation at the end of mitosis depends on its PEST motif and interaction with the E3 ligase Grr1. To test the hypothesis that targeted destruction of Hof1 temporally couples mitotic exit with contraction of the actomyosin ring, we mutated the Hof1 PEST motif to prevent phosphorylation and subsequent degradation. These mutations increased the amount of Hof1 at the bud neck during cytokinesis, resulted in smaller bud neck diameter, and slowed the rate of myosin contraction. However, Hof1 PEST motif phosphorylation site mutants did not have cytokinesis defects, indicating that regulation of Hof1 levels does not control the onset of actomyosin ring contraction as predicted.

Dephosphorylation of Iqg1 by Cdc14 regulates cytokinesis in budding yeast

“…Contraction of the actomyosin ring did not occur earlier in the cell cycle in iqg1-4A cells, as Tub1-mCherry showed that the spindle was completely elongated before contraction began ( Figure 6A ). In control cells, Myo1-GFP exhibited symmetric contraction to a single dot over an average of 8.1 ± 1.0 min, consistent with previous results ( N = 6; Figure 6B ; see Supplemental Figure 6B.mov for another example; Lippincott and Li, 1998b ; Bi, 2001 ; Stockstill et al ., 2013 ). However, Myo1-GFP failed to contract normally in iqg1-4A cells.…”

“…The bud necks of the iqg1-4A cells were significantly larger than control cells, 1.9 compared with 1.3 μm ( p = 0.0002). Effects on the size of the bud neck have been seen after perturbation of many other cytokinesis and septation genes, including Hof1, Cyk3, Inn1, and Chs2 ( Lippincott and Li, 1998a ; Korinek et al ., 2000 ; Stockstill et al ., 2013 ; Kuilman et al ., 2015 ). Mechanistically, the reason for the altered bud neck size is unknown, but it is likely due to the coupling between cytokinesis, septation, and selection of the future bud site in budding yeast.…”

Aim44p regulates phosphorylation of Hof1p to promote contractile ring closure during cytokinesis in budding yeast

“…Hof1p begins to be degraded late in mitosis; however, whether its degradation is related to the regulation of actomyosin ring closure is controversial ( Blondel et al. , 2005 ; Stockstill et al. , 2013 ).…”