The defining feature of a cell is the incredibly thin sheet of membrane that demarcates the intracellular and extracellular milieus. This cell membrane, or plasma membrane, has a lipid component that provides a barrier to passage of polar or charged molecules, and protein component that allows passage of privileged molecules to make the membrane selectively permeable. Phospholipids are critical building blocks of the plasma membrane and these amphipathic molecules pack together side-by-side to form a two-layered sheet. The polar phospholipid headgroups in each leaflet face outward to interact with water and the hydrophobic fatty acyl chains face the interior of the bilayer structure. A remarkable characteristic of the eukaryotic cell membrane is that these two layers have a very different phospholipid composition, a phenomenon known as """"""""membrane asymmetry"""""""". The inner leaflet facing the cytosol of mammalian cells is enriched in phosphatidylserine (PS) and phosphatidylethanolamine (PE) while the extracellular leaflet is enriched in sphingolipids, glycosphingolipids and phosphatidylcholine (PC). At least two classes of active-transport proteins are capable of moving phospholipids across a membrane bilayer to establish membrane asymmetry, including the type IV P-type ATPases (P4-ATPases) and members of the ATP-binding cassette (ABC) transporter families. Amazingly, even with decades of study and x-ray crystal structures of some ABC transporters, how lipids are transported remains a mystery. With ion- transporting P-type ATPases (type I, II and III), x-ray crystal structure have also been solved highlighting a structurally conserved substrate binding site in the center of the transmembrane domain (a canonical site). Whether P4-ATPases evolved the ability to recognize the much larger phospholipid substrate in this canonical site, or evolved a unique transport mechanism has been the subject of debate. Preliminary data supporting this proposal strongly suggests that the P4-ATPases are using a noncanonical transport pathway to flip their phospholipid substrate. We propose that phospholipid is being selected at both an """"""""entry gate"""""""" near the extracellular leaflet and an """"""""exit gate"""""""" near the cytosolic leaflet. Studies in this projet will test this two-gate hypothesis through mutational studies and better define the mechanism of phospholipid transport by the P4- ATPases. The information obtained will help us understand how human diseases arise from defects in membrane asymmetry or P4-ATPase deficiency.

Public Health Relevance

Defects in human P4-ATPases cause mental retardation and familial intrahepatic cholestasis. Studies with mice have further implicated P4-ATPases in immune deficiency, type 2 diabetes, hearing loss and hepatic cancer. Defining the mechanism of P4-ATPase function will further our understanding of these diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM107978-01
Application #
8575204
Study Section
Special Emphasis Panel (ZRG1-CB-G (02))
Program Officer
Chin, Jean
Project Start
2013-09-01
Project End
2017-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
1
Fiscal Year
2013
Total Cost
$290,224
Indirect Cost
$100,224
Name
Vanderbilt University Medical Center
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
van Leeuwen, Jolanda; Pons, Carles; Mellor, Joseph C et al. (2016) Exploring genetic suppression interactions on a global scale. Science 354:
Roland, Bartholomew P; Graham, Todd R (2016) Directed evolution of a sphingomyelin flippase reveals mechanism of substrate backbone discrimination by a P4-ATPase. Proc Natl Acad Sci U S A 113:E4460-6
Takar, Mehmet; Wu, Yuantai; Graham, Todd R (2016) The Essential Neo1 Protein from Budding Yeast Plays a Role in Establishing Aminophospholipid Asymmetry of the Plasma Membrane. J Biol Chem 291:15727-39
Wu, Yuantai; Takar, Mehmet; Cuentas-Condori, Andrea A et al. (2016) Neo1 and phosphatidylethanolamine contribute to vacuole membrane fusion in Saccharomyces cerevisiae. Cell Logist 6:e1228791
Roland, Bartholomew P; Graham, Todd R (2016) Decoding P4-ATPase substrate interactions. Crit Rev Biochem Mol Biol 51:513-527
Hankins, Hannah M; Sere, Yves Y; Diab, Nicholas S et al. (2015) Phosphatidylserine translocation at the yeast trans-Golgi network regulates protein sorting into exocytic vesicles. Mol Biol Cell 26:4674-85
Hankins, Hannah M; Baldridge, Ryan D; Xu, Peng et al. (2015) Role of flippases, scramblases and transfer proteins in phosphatidylserine subcellular distribution. Traffic 16:35-47
Zhou, Xiaoming; Sebastian, Tessy T; Graham, Todd R (2013) Auto-inhibition of Drs2p, a yeast phospholipid flippase, by its carboxyl-terminal tail. J Biol Chem 288:31807-15
Xu, Peng; Baldridge, Ryan D; Chi, Richard J et al. (2013) Phosphatidylserine flipping enhances membrane curvature and negative charge required for vesicular transport. J Cell Biol 202:875-86
Baldridge, Ryan D; Xu, Peng; Graham, Todd R (2013) Type IV P-type ATPases distinguish mono- versus diacyl phosphatidylserine using a cytofacial exit gate in the membrane domain. J Biol Chem 288:19516-27

Showing the most recent 10 out of 11 publications