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

Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235-1634, USA.
Molecular biology of the cell (Impact Factor: 4.47). 05/2009; 20(12):2920-31. DOI: 10.1091/mbc.E08-10-1036
Source: PubMed


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.

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Available from: Will Prinz, Mar 21, 2014
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    • "; Alder- Baerens et al., 2006; Zhou and Graham, 2009; Muthusamy et al., 2009 "
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    ABSTRACT: The plasma membrane, trans-Golgi network and endosomal system of eukaryotic cells are populated with flippases that hydrolyze ATP to help establish asymmetric phospholipid distributions across the bilayer. Upholding phospholipid asymmetry is vital to a host of cellular processes, including membrane homeostasis, vesicle biogenesis, cell signaling, morphogenesis and migration. Consequently, defining the identity of flippases and their biological impact has been the subject of intense investigations. Recent work has revealed a remarkable degree of kinship between flippases and cation pumps. In this Commentary, we review emerging insights into how flippases work, how their activity is controlled according to cellular demands, and how disrupting flippase activity causes system failure of membrane function, culminating in membrane trafficking defects, aberrant signaling and disease. © 2015. Published by The Company of Biologists Ltd.
    Journal of Cell Science 04/2015; 128(11). DOI:10.1242/jcs.102715 · 5.43 Impact Factor
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    • "Kes1 (Osh4) is the most well studied member of this family and numerous genetic and cell biology studies suggest Kes1 inhibits vesicular trafficking at the trans-Golgi. The mechanism is not clear, although it is known that Kes1 inhibits vesicular trafficking in a phosphatidylinositol (PI) 4-phosphate (PI-4P) dependent manner [6], [7], [8], [9], [10], [11], [12]. In vitro, Kes1 and other Osh proteins have been demonstrated to transfer sterols between membranes and a role for these proteins as direct non-vesicular sterol transporters has been proposed [9], [11]. "
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    ABSTRACT: The oxysterol binding protein family are amphitropic proteins that bind oxysterols, sterols, and possibly phosphoinositides, in a conserved binding pocket. The Saccharomyces cerevisiae oxysterol binding protein family member Kes1 (also known as Osh4) also binds phosphoinositides on a distinct surface of the protein from the conserved binding pocket. In this study, we determine that the oxysterol binding protein family member Kes1 is required to maintain the ratio of complex sphingolipids and levels of ceramide, sphingosine-phosphate and sphingosine. This inability to maintain normal sphingolipid homeostasis resulted in misdistribution of Pma1, a protein that requires normal sphingolipid synthesis to occur to partition into membrane rafts at the Golgi for its trafficking to the plasma membrane.
    PLoS ONE 04/2013; 8(4):e60485. DOI:10.1371/journal.pone.0060485 · 3.23 Impact Factor
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    • "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. "
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    ABSTRACT: Cholesterol and phospholipids serve as structural and functional components of cellular membranes in all eukaryotes. Heterogeneity in cholesterol and phospholipid content both within and between different organelles is an important characteristic of eukaryotic membranes. How this heterogeneity is achieved and orchestrated to maintain proper cellular physiology remains poorly understood. We previously found that overexpression of the Drosophila oxysterol-binding protein (OSBP) leads to sterol accumulation in the Golgi apparatus. Here, we show that Osbp overexpression in a set of neuroendocrine neurons compromises the function of the Golgi apparatus. It impairs trafficking of the neuropeptide bursicon and results in post-eclosion behavior defects characterized by unexpanded wings. We performed a genetic screen to identify modifiers that suppress the unexpanded wing phenotype. A putative phospholipid flippase-encoding gene, CG33298, was validated, suggesting that a membrane-asymmetry-directed mechanism balances cholesterol chaos within the Golgi membranes. Since the functional connection between cholesterol metabolism and the activity of phospholipid flippase has been implicated in studies in yeast and worms, our findings here support an evolutionarily conserved causal link between cholesterol homeostasis and phospholipid asymmetry that maintains normal cellular physiology.
    Genetics 01/2012; 190(4):1299-308. DOI:10.1534/genetics.111.137687 · 5.96 Impact Factor
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