In the yeast plasma membrane, phospholipids are asymmetrically distributed between the inner and outer leaflets. The outer leaflet contains predominantly phosphatidylcholine (PtdCho) and sphingolipids, whereas the inner leaflet is enriched in phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEth), phosphatidylinositol (PtdIns), and a phosphoinositide (PtdIns4,5P2). Five genes encode type 4 P‐ATPase implicated in translocating PtdSer, PtdEth and PtdCho. In particular, Dnf1 and Dnf2 catalyze inward movement of PtdEth and PtdSer from the outer to the inner leaflet. The action of these flippases depends on activating phosphorylation by Fpk1, a protein kinase located at the cell cortex. Using an assay that monitors Fpk1 activity in vivo, we found that Fpk1 is hyper‐active in cells lacking the protein kinase Gin4 that had previously been implicated in assembly of the septin ring at the bud neck. We found, instead, that Gin4 phosphorylates Fpk1 at multiple sites, which we mapped. After mutating the phospho‐acceptor residue to Ala in these sites, we found by several biochemical and phenotypic criteria that such an Fpk111A mutant is hyper‐active. Thus, Gin4 acts as a negative regulator of Fpk1 and, thus, negatively regulates indirectly flippase function. These observations suggest that control of plasma membrane asymmetry may be the primary function of Gin4 and may be necessary for optimal cytokinesis.
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