UNDERSTANDING HOW THE TRAFFICKING OF CYTOKINETIC ENZYMES IS REGULATED DURING MITOTIC EXIT IN SACCHAROMYCES CEREVISIAE

Abstract

Cytokinesis is the final step of cell division cycle in which a dividing cell undergoes physical separation to form two progeny cells. The splitting of the cell is usually restrained until completion of nuclear division. In budding yeast, cytokinesis is accomplished by the spatio-temporal coordination of acto-myosin ring constriction (AMR) and septation. The primary septum in yeast is synthesized by a transmembrane protein, chitin synthase II (Chs2p). The roles of Chs2p in regulating primary septum formation and facilitating AMR constriction are well documented in many studies. Nevertheless, the underlying mechanism that is responsible for the timely Chs2p ER export is yet to be elucidated. Here, this study shows that Chs2p is a novel substrate for Cdc14p phosphatase (the ultimate effector of the MEN). At late telophase, Chs2p ER export requires the direct reversal of the inhibitory Cdk1 phosphorylation by cytoplasmic localization of Cdc14p. Timely MEN dependent Cdc14p cytoplasmic localization at the end of mitosis ensures Chs2p ER export and septum formation invariably occurs only after completion of chromosome segregation.

At late mitosis, it has been previously shown that the Chs2p localizes to the division site before spindle disassembly, and subsequent cytokinesis. The deposition of primary septum by Chs2p at the neck has been demonstrated to provide mechanical support for AMR constriction. Therefore, this raises the question of how cells coordinate the constriction of AMR at the division site to ensure spindle disassembly precedes AMR constriction. In this study, we establish that cells coordinate these two events by regulating the continuous internalization of cytokinetic enzymes - Chs2p, Chs3p and Fks1p at the division site via clathrin-mediated endocytosis. Failure to endocytose cytokinetic enzymes at late mitosis results in a thickened cell wall, aberrant septation and mitotic spindle breakage. Interestingly, defective endocytosis of cytokinetic enzymes causes premature AMR constriction that leads to spindle breakage. As a consequence of mitotic spindle breakage, the spindle fails to reassemble in the subsequent round of cell division.