Phosphoregulation of actin cytoskeleton during endocytosis in the yeast Saccharomayces cerevisiae

Abstract
Endocytosis requires appropriately controlled actin assembly and disassembly at specific steps. Actin polymerization is initiated by the Arp2/3p complex. In budding yeast, the Arp2/3p complex is activated by several nucleation promoting factors including Pan1p. Phosphorylation of Pan1p and other components of the coat complex by the kinase Prk1p lead to termination of actin polymerization and disassembly of the coat complex. A homologous kinase, Ark1p, has also been implicated in this regulatory process. In this study, the distinct roles of Prk1p and Ark1p were investigated. We found that the non-kinase domains determined the functional specificity of the two kinases. A short region located adjacent to the kinase domain unique to Prk1p was found to be required for the kinase to interact with Arp2p. Further studies demonstrated that the Prk1p-Arp2p interaction is essential for down-regulation of Pan1p. These findings suggest an auto-inhibitory mechanism that coordinates actin assembly and disassembly during endocytosis.

Figure 5.2. The Arp2p binding region of Prk1p is required for its cortical localization.
Wild-type cells (W303-1A) were transformed with plasmids carrying pGAL-PRK1^D158Y-GFP, pGAL-PRK1^D158Y 1-319-GFP, pGAL-PRK1^D158Y 1-298-GFP, pGAL-PRK1^D158YAR-GFP, and pGAL-PRK1^D158Y AR ∆PP-GFP. After 1-h galactose induction, cells were fixed and stained with rhodamine (Rd)-phalloidin. Because overexpression of Prk1p could disturb actin cytoskeleton, an inactive kinase (D158Y) was used in this experiment. Either the Arp2p-interacting region (shown by Prk11-319) or the Abp1-interacting region (shown by Prk1AR ) is sufficient for Prk1p to be localized to cortical patches.