Abstract
In eukaryotic cells, the growth rate is strictly regulated for proper progression of the cell cycle. In the budding yeast Saccharomyces cerevisiae, it was previously shown that cell growth dramatically slows down when the cells start budding at the G1/S transition. However, the molecular mechanism for this G1/S-associated growth arrest is unclear. In this study, using exocytic secretion, cyclin-dependent kinase (CDK) assay, immunoprecipitation, and microscopy, we demonstrate that the exocyst subunit Exo84, which is known to be phosphorylated in mitosis, can also be phosphorylated directly by Cdk1 in the late G1 phase. Of note, we found that the Cdk1-mediated Exo84 phosphorylation impairs exocytic secretion in the late G1 phase. Using conditional cdc mutants and phosphodeficient and phosphomimetic exo84 mutants, we further observed that Cdk1-phosphoryated Exo84 inhibits the exocyst complex assembly, exocytic secretion, and cell growth, which may be important for proper execution of the G1/S-phase transition before commitment to a complete cell cycle. Our results suggest that the direct Cdk1-mediated regulation of the exocyst complex critically contributes to the coordination of cell growth and cell cycle progression.
Highlights
In eukaryotic cells, the growth rate is strictly regulated for proper progression of the cell cycle
In this study, using exocytic secretion, cyclin-dependent kinase (CDK) assay, immunoprecipitation, and microscopy, we demonstrate that the exocyst subunit Exo84, which is known to be phosphorylated in mitosis, can be phosphorylated directly by Cdk1 in the late G1 phase
Membrane growth and surface expansion of eukaryotic cells are tightly controlled during cell cycle progression and mainly rely on the tight regulation of polarized exocytic secretion, by which proteins and lipids from post-Golgi are incorporated into the plasma membrane
Summary
A and B, the internal Bgl levels were elevated in cdc and cdc mutant cells but not in WT cells, indicating that Bgl secretion was inhibited in these two mutants This result is consistent with the observation that cell growth is largely reduced in late G1 phase [11]. Neither cdc nor cdc mutant cells had invertase secretion block (Fig. 1C) This result is consistent with previous observation that exocytic secretion of invertase is not compromised during cell cycle progression [33, 39]. Because Cdk plays a central role in cell cycle progression, we speculated that Cdk may regulate the reduction of exocytic secretion in late G1 cells To test this prediction, we assayed Bgl secretion in cdc cells that contain an analog-sensitive cdk allele (cdk1-as1), which encodes a mutant kinase that is inhibited by 1NM-PP1 [40]. We examined cdc cells via thin-section electron microscopy (EM) for secretory vesicle accumula-
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