Control of Protein and Sterol Trafficking by Antagonistic Activities of a Type IV P-type ATPase and Oxysterol Binding Protein Homologue

Abstract

The oxysterol binding protein homologue Kes1p has been implicated in nonvesicular sterol transport in Saccharomyces cerevisiae. Kes1p also represses formation of protein transport vesicles from the trans-Golgi network (TGN) through an unknown mechanism. Here, we show that potential phospholipid translocases in the Drs2/Dnf family (type IV P-type ATPases [P4-ATPases]) are downstream targets of Kes1p repression. Disruption of KES1 suppresses the cold-sensitive (cs) growth defect of drs2Delta, which correlates with an enhanced ability of Dnf P4-ATPases to functionally substitute for Drs2p. Loss of Kes1p also suppresses a drs2-ts allele in a strain deficient for Dnf P4-ATPases, suggesting that Kes1p antagonizes Drs2p activity in vivo. Indeed, Drs2-dependent phosphatidylserine translocase (flippase) activity is hyperactive in TGN membranes from kes1Delta cells and is potently attenuated by addition of recombinant Kes1p. Surprisingly, Drs2p also antagonizes Kes1p activity in vivo. Drs2p deficiency causes a markedly increased rate of cholesterol transport from the plasma membrane to the endoplasmic reticulum (ER) and redistribution of endogenous ergosterol to intracellular membranes, phenotypes that are Kes1p dependent. These data suggest a homeostatic feedback mechanism in which appropriately regulated flippase activity in the Golgi complex helps establish a plasma membrane phospholipid organization that resists sterol extraction by a sterol binding protein.

A Detour for Yeast Oxysterol Binding Proteins

“…Instead of a direct role in sterol transfer, these results suggest that Osh proteins affect the organization and sorting of sterols between bilayer leaflets and/or in membrane domains. Consistent with this reasoning, genetic studies indicate a functional interaction between OSH4 and DRS2, which encodes an ATP-dependent phospholipid flippase (34). This interaction might reflect a wider role of Osh proteins as regulators of enzymes and proteins that control the composition and organization of membrane bilayers.…”

Membrane Phospholipid Asymmetry Counters the Adverse Effects of Sterol Overloading in the Golgi Membrane of Drosophila

“…In addition, both kes1 and drs2 mutants exhibit defects in sterol distribution, and the defects are largely diminished in kes1;drs2 double mutants. These results suggest antagonistic activities or negative correlations between Drs2P and Kes1p (Muthusamy et al 2009). A recent study of C. elegans tat mutants also supports a causal link between phospholipid flippase and cholesterol metabolism (Lyssenko et al 2008).…”

“…First, the phospholipid flippase and OSBP may have different or even opposite relationships in different biological processes. Indeed, although Drs2P and Kes1p have antagonistic effects in many aspects, kes1 does not suppress the drs2 defect in secretory vesicle budding in the Golgi apparatus (Muthusamy et al 2009). Alternatively, it is possible that species-specific differences between the fly and yeast lead to different outcomes.…”

“…Interestingly, loss of Kes1p/Osh4p, a yeast ORP, suppresses the growth defect of a partial lossof-function mutant of Drs2p. On the other hand, Drs2p also antagonizes the activity of Kes1p in intracellular cholesterol trafficking (Muthusamy et al 2009). The exact mechanism behind this mutual antagonistic activity between Drs2p and Kes1p is unknown.…”

A P4-ATPase Protein Interaction Network Reveals a Link between Aminophospholipid Transport and Phosphoinositide Metabolism

“…51 However, our finding that overexpression of Sac1p partially suppresses the cold-sensitive growth defect in ∆drs2 cells suggests that the model of an antagonistic relationship between the two proteins is too simple. 38 …”