Abstract
Kinase-Mediated Regulation of P4-ATPases Understanding kinase-mediated regulation and designing novel tools to study regulatory proteins of P4-ATPases P4-ATPases play a critical role in the biogenesis of transport vesicles in the secretory and endocytic pathways, and P4-ATPase activity is held responsible for the creation and maintenance of membrane phospholipid asymmetry through phospholipid flipping. As P4-ATPases are involved in such important cellular processes, it is expected that their activity is tightly regulated. In the yeast Saccharomyces cerevisiae two kinases named Fpk1p and Fpk2p have earlier been found to regulate and activate two plasma membrane P4-ATPases, Dnf1p and Dnf2p. Here we show that another yeast P4-ATPase from the endomembrane system, Dnf3p, is also activated by Fpk1p (and Fpk2p). Our results indicate that Dnf3p is able to mediate transport of primarily phosphatidylserine, but also phosphatidylethanolamine and phosphatidylcholine, in an Fpk1p (and Fpk2p)-dependent manner. Taken together, this could point to a conserved kinase-mediated regulatory mechanism of P4-ATPases. How P4-ATPases from higher eukaryotes are regulated by kinases is currently not known. We have designed a fast and efficient screening strategy to identify novel regulator proteins of P4-ATPases. The system is based on heterologous expression in a specially designed yeast strain, and regulatory proteins can be identified via change in activity of the P4-ATPase of interest. Hereby the first steps towards unraveling the kinase-mediated regulatory systems of P4-ATPases in higher eukaryotes have been taken.