Post-translational phosphorylation of proteins by cyclin-dependent kinases (Cdks) enables regulation of cell cycle events with a temporal and spatial precision that would be unattainable by control of protein synthesis and degradation alone. To understand how processes are regulated to operate reliably in every repetition of the cycle thus requires an understanding of where and when Cdk-dependent phosphates appear throughout the proteome, and the molecular effects of those phosphates on the proteins in question.
Cytokinesis, the process by which one cell divides and becomes two, involves the ordered assembly of several protein populations at the site of cell division. In the budding yeast Saccharomyces cerevisiae, this occurs at the bud neck and has the end result of coordinating the contraction of an actomyosin ring with the synthesis of the primary septum, a cell wall precursor. The assembly process occurs during a period or proteome-wide reversal of Cdk-dependent phosphorylation, and is known to be sensitive to perturbations of the Cdk regulatory system.
This study focuses on Iqg1, an essential protein required for recruiting actin to the bud neck. Iqg1 is phosphorylated by Cdk1 (S. cerevisiae's sole essential Cdk) at several sites, and we find that mutation of these sites to yield a nonphosphorylatable Iqg1 disrupts the timing of several aspects of the pre-cytokinesis assembly process. When expressed in place of wild type Iqg1, the phosphomutant promotes early arrival of filamentous actin at the bud neck at levels comparable to those observed in response to global inhibition of Cdk1 activity. In this mutant, we also observe early onset of the accumulation of Iqg1 itself, and of Hof1, a regulator of primary septum deposition that we reveal as a likely Iqg1 in vivo binding partner.
These results reveal a common point of regulation for the two processes that comprise cytokinesis and illuminate the relationships between proteins that govern the timing of assembly of a complex multi-protein apparatus. The quantitative fluorescence microscopy methods used also provide a system for the further study of the unique molecular consequences of phosphorylation for each of Cdk1's many protein substrates.