Relationship between Drs2 and Kes1 controlling protein trafficking in <i>Saccharomyces cerevisiae</i>

Abstract

Dissertation under the direction of Professor Todd R. Graham

Drs2 is a type IV P-type ATPase that catalyzes a phospholipid flippase activity in the trans-Golgi network (TGN) in Saccharomyces cerevisiae. Strains harboring a DRS2 null allele (drs2∆) are viable, but exhibit a strong cold-sensitive growth defect. Under this dissertation, a drs2∆ bypass suppressor screen was completed to better understand the mechanism underlying the cold-sensitive growth defect of drs2∆.

SDK1 (suppressor of drs2 knockout 1), was cloned and shown to be identical to KES1, an oxysterol binding protein. Genetic studies suggested that Kes1p represses the activity of Drs2p and the closely related P4-ATPases. kes1∆ suppression of the drs2∆ cold-sensitive growth defect requires the closely related Dnf P4-ATPases. Indeed, a flippase assay performed with TGN membranes purified from kes1∆ cells revealed a two-fold increase in Drs2 activity. Addition of recombinant Kes1 repressed Drs2 activity back to wild type levels.

Surprisingly, Genetic analyses suggested that Kes1 is hyperactive in drs2∆ cells and is particularly toxic to drs2∆ cells blocked in late stages of ergosterol synthesis. Indeed, the influence of Kes1 on intracellular sterol transport was dramatically enhanced in drs2∆ cells. Thus, the presence of wild-type Drs2 represses Kes1 activity, providing the first evidence for an antagonist relationship between a P4-ATPase and oxysterol binding OSH protein.

Drs2 is required for budding of a specific class of exocytic vesicles from the TGN and for AP1/clathrin coated vesicle trafficking between the TGN and early endosome. In contrast, Drs2 and Dnfs act redundantly in transporting proteins from the TGN to the late endosome and vacuole. In the absence of Drs2p, Dnfs can support transport to the late endosome at 30C but not 15C. Deletion of KES1 suppresses the cold-sensitive TGN to late endosome transport defect. These findings lead to a conclusion that the cold-sensitive growth defect of drs2∆ is caused by simultaneous trafficking defects in the TGN to early endosome and TGN to late endosme pathways at low temperature. Moreover, P4-ATPases are a downstream target of the Kes1 repressive effect on vesicular transport.