Detergent-resistant membranes (DRMs) isolated from mammalian cells have been implicated in a diverse set of functions, ranging from intracellular sorting in polarized epithelial cells, to cell-surface signal transduction, to the structure of non-clathrin-coated plasma membrane pits called caveolae. Each of these roles will be examined, and a model explaining he structure of these membranes will be tested. Cholesterol-rich model membranes contain distinct domains; a novel phase called the liquid-ordered phase (Io) phase in addition to the liquid-crystalline (Ic) phase. Io phase lipids are packed more tightly than those in the Ic phase, but have more mobility than gel phase lipids. DRMs isolated from cells appear to be present in a phase similar to the Io phase. The long-term goal of this work is to determine how these domains function in cells. Many proteins that can be isolated from cells in DRMs are modified with saturated acyl chains (myristate and/or palmitate), that fit well into ordered domains. Three such proteins will be purified and incorporated into model membranes that contain Io phase domains; the caveolar coat protein caveolin, the Src- family kinase Yes, and the G protein Gi. The ability of these proteins to associate with DRMs from these liposomes in the absence of other proteins will be determined, as will the dependence of this behavior on protein acylation. Possible effects of Io phase domains on the function of these proteins will also be explored. These studies could have important health- related implications, as signaling proteins such as Src-family kinases can be oncogenic. Previous studies suggest that DRMs can be isolated from intracellular membranes as well as plasma membranes. DRMs will be isolated from the Golgi apparatus. Proteins found to be enriched in Golgi-derived DRMs may be important in sorting and trafficking along the secretory pathway, and will be further characterized. Complementary studies using cells and motel membranes will test our model that DRMs exist in cells as discrete domains. Recent results suggest that lipids with high acyl chain melting temperatures, such as sphingolipids, promote the formation of the Io phase in the presence of cholesterol. The lipid composition of DRMs from cells depleted of sphingolipid and/or cholesterol, and of model membranes that form DRMs, will be examined to test the correlation between formation of an Io-like phase and detergent-insolubility. Additional methods for examining the Io-like phase in model membranes and possibly in cell membranes will be used. Finally, the ability of the sphingolipid-poor inner leaflet of the plasma membrane to form DRMs will be tested using inside-out erythrocyte ghosts.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM047897-08S1
Application #
6330981
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Program Officer
Chin, Jean
Project Start
1992-09-01
Project End
2001-02-28
Budget Start
2000-09-01
Budget End
2001-02-28
Support Year
8
Fiscal Year
2000
Total Cost
$74,725
Indirect Cost
Name
State University New York Stony Brook
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Brown, Deborah A (2015) Preparation of detergent-resistant membranes (DRMs) from cultured mammalian cells. Methods Mol Biol 1232:55-64
Gucwa, Azad L; Brown, Deborah A (2014) UIM domain-dependent recruitment of the endocytic adaptor protein Eps15 to ubiquitin-enriched endosomes. BMC Cell Biol 15:34
Verma, Prakhar; Ostermeyer-Fay, Anne G; Brown, Deborah A (2010) Caveolin-1 induces formation of membrane tubules that sense actomyosin tension and are inhibited by polymerase I and transcript release factor/cavin-1. Mol Biol Cell 21:2226-40
Barr, Daniel J; Ostermeyer-Fay, Anne G; Matundan, Rachel A et al. (2008) Clathrin-independent endocytosis of ErbB2 in geldanamycin-treated human breast cancer cells. J Cell Sci 121:3155-66
Kirkham, Matthew; Nixon, Susan J; Howes, Mark T et al. (2008) Evolutionary analysis and molecular dissection of caveola biogenesis. J Cell Sci 121:2075-86
Listenberger, Laura L; Ostermeyer-Fay, Anne G; Goldberg, Elysa B et al. (2007) Adipocyte differentiation-related protein reduces the lipid droplet association of adipose triglyceride lipase and slows triacylglycerol turnover. J Lipid Res 48:2751-61
Brown, Deborah A (2007) Analysis of raft affinity of membrane proteins by detergent-insolubility. Methods Mol Biol 398:9-20
Brown, Deborah A (2006) Lipid rafts, detergent-resistant membranes, and raft targeting signals. Physiology (Bethesda) 21:430-9
Shogomori, Hidehiko; Hammond, Adam T; Ostermeyer-Fay, Anne G et al. (2005) Palmitoylation and intracellular domain interactions both contribute to raft targeting of linker for activation of T cells. J Biol Chem 280:18931-42
Ostermeyer, Anne G; Ramcharan, Lynne T; Zeng, Youchun et al. (2004) Role of the hydrophobic domain in targeting caveolin-1 to lipid droplets. J Cell Biol 164:69-78

Showing the most recent 10 out of 31 publications