The differential protein and lipid compositions of noncaveolar lipid microdomains and caveolae

Abstract

Morphologically, caveolae and lipid rafts are two different membrane structures. They are often reported to share similar lipid and protein compositions, and are considered to be two subtypes of membrane lipid microdomains. By modifying sucrose density gradient flotation centrifugation, which is used to isolate lipid microdomains, we were able to separate caveolae and noncaveolar lipid microdomains into two distinct fractions.

CLN3 Loss Disturbs Membrane Microdomain Properties and Protein Transport in Brain Endothelial Cells

“…For fractionation of PM microdomains, MBECs from three confluent 160-mm-diameter dishes were harvested and PM isolated and subjected to detergent-free sodium carbonate extraction and centrifugation over a discontinuous sucrose density gradient as previously described (Yao et al, 2009). For fractionation of TGN microdomains, TGN-enriched fractions were isolated and subjected to carbonate extraction and discontinuous sucrose density centrifugation as previously described (Waugh et al, 2011).…”

“…We first took a biochemical approach to assess partitioning of caveolin-1 and syntaxin-6 to membrane domains isolated from cell lysates. To avoid artifacts related to lipid reassembly during detergent extraction (Lichtenberg et al, 2005), we used a detergent-free, carbonate-based extraction method for microdomain fractionation (Song et al, 1996;Yao et al, 2009). Golgi-or PM-enriched membranes from cell lysates were sonicated, extracted with carbonate to remove loosely associated proteins, and separated by centrifugation over a sucrose density gradient.…”

“…Golgi-or PM-enriched membranes from cell lysates were sonicated, extracted with carbonate to remove loosely associated proteins, and separated by centrifugation over a sucrose density gradient. With this method, separation of membrane fragments is based on buoyancy, which correlates with increased lipid-protein ratio and enrichment for cholesterol and sphingolipids (Ortegren et al, 2004;Yao et al, 2009). Fractions collected from top to bottom of the gradients were analyzed by Western blot for caveolin-1 and syntaxin-6, and by dot blot for the GM1, a microdomainenriched sphingolipid (Fig.…”

Essential role of flotillin-1 palmitoylation in the intracellular localization and signaling function of IGF-1 receptor

“…Four-step sucrose density gradient fractionation was performed as described previously (Kwon et al, 2013;Yao et al, 2009) to separate the HLDM from the LLDM plasma membrane fraction. The purified plasma membrane pellet from HEK293T cells was resuspended with 2 ml of 500 mM sodium carbonate, pH 11.0, homogenized using ten strokes of a Dounce homogenizer, and mixed with 2 ml of 90% sucrose in MBS buffer.…”

A retinoic acid receptor RARα pool present in membrane lipid rafts forms complexes with G protein αQ to activate p38MAPK

“…Therefore, we aimed at deciphering the underlying mechanism of these rapid nongenomic effects. Here, we showed that a fraction of the cellular RARa pool is present in membrane lipid rafts, which are microdomains of cell membranes enriched not only in cholesterol and sphingolipids but also in signal-transducing molecules, such as flotillins, G proteins-coupled receptors, heterotrimeric G proteins, Rho and Rac GTPases, and their effectors (Pike, 2003;de Laurentiis et al, 2007;Sugawara et al, 2007;Yao et al, 2009). In lipid rafts, G proteins consist of three subunits a, b and g, and transmit signals to downstream effector molecules such as phospholipase C, proteinkinase C and p38MAPK (Mizuno and Itoh, 2009).…”