Dynamic, Rho1p-dependent localization of Pkc1p to sites of polarized growth

Abstract

In eukaryotes, the Rho GTPases and their effectors are key regulators of the actin cytoskeleton, membrane trafficking and secretion, cell growth, cell cycle progression and cytokinesis. Budding yeast Pkc1p, a protein kinase C-like enzyme involved in cell wall biosynthesis and cytoskeletal polarity, is structurally and functionally related to the Rho-associated kinases (PRK/ROCK) of mammalian cells. In this study, localization of Pkc1p was monitored in live cells using a GFP fusion (Pkc1p-GFP). Pkc1p-GFP showed dynamic spatial and temporal localization at sites of polarized growth. Early in the cell cycle, Pkc1p-GFP was found at the pre-bud site and bud tips, becoming delocalized as the cell progressed further and finally relocalizing around the mother-daughter bud neck in an incomplete ring, which persisted until cell separation. Bud localization was actin-dependent but stability of Pkc1p-GFP at the neck was actin-independent, although localization at both sites required functional Rho1p. In addition, Pkc1p-GFP showed rapid relocalization after cell wall damage. These results suggest that the roles of Pkc1p in both polarized growth and the response to cell wall stress are mediated by dynamic changes in its localization, and suggest an additional potential role in cytokinesis.