Both raft- and non-raft proteins associate with CHAPS-insoluble complexes: some APP in large complexes

doi:10.1016/S0006-291X(03)01470-0

Biochemical and Biophysical Research Communications

Volume 308, Issue 4, 5 September 2003, Pages 750-758

https://doi.org/10.1016/S0006-291X(03)01470-0 Get rights and content

Abstract

Components of caveolae and lipid rafts are characterized by their buoyancy after detergent extraction. Using flotations in density gradients, we now show that non-raft membrane molecules are also associated with detergent-insoluble, buoyant assemblies. When Triton X-100 cellular extracts were spun to equilibrium in Nycodenz, only components of classical rafts floated. In contrast, with the zwitterionic detergent CHAPS, non-raft residents such as calnexin and APP also buoyed. When CHAPS extracts were spun in non-equilibrium (velocity) conditions, some raft components rapidly exited the input fractions while other raft markers and non-raft molecules remained relatively immobile. This pointed to size heterogeneities of CHAPS-insoluble complexes. Combined velocity/equilibrium gradients broadly divided CHAPS-insoluble membrane complexes into three size categories, which all contained cholesterol and the glycosphingolipid GM1. Large complexes were enriched in caveolin and ESA. Medium size complexes were enriched in PrP, whereas small complexes contained non-raft proteins, PrP, and some ESA. While Alzheimer’s APP was primarily confined to small assemblies, a portion of its glycosylated form did buoy with large complexes. Large CHAPS-insoluble complexes resemble, but are not equal to, classical rafts. These findings extend considerably the range of detergent-insoluble membranal domains.

Section snippets

Materials and methods

Materials. Cell culture reagents were purchased from Biological Industries (Beit Haemek, Israel). Tissue culture plates were from Miniplast (Ein Shemer, Israel). All other chemicals were from Sigma (St. Louis, MO).

Cell lines and constructs. PrP^CD4 is a non-raft mouse PrP-CD4 chimera [29], which reacts with 3F4 epitope [30]. C6-MHM2 stably overexpress mouse PrP labeled with the 3F4 epitope [30]. Cells were maintained at 37 °C, 7.5% CO₂, in DMEM16 (with 1 g/L glucose) supplemented with 10% fetal

Membrane proteins of diverse topologies buoy in equilibrium gradients following CHAPS extraction

We studied the flotation properties of a number of membrane and non-membrane proteins in CHAPS and TX-100 extracts of several cell types and tissues. Representative results, obtained with C6 rat glioma, N2a mouse neuroblastoma, and a mouse brain, are shown in Fig. 1.

We first studied molecules classically associated with rafts or caveolae (Fig. 1A). (i) Caveolin-1 [34], [35] and ESA [37], [38] have a hairpin membrane topology and are palmitoylated. They had similar flotation patterns with both

Discussion

The most important finding of this paper is that membrane proteins buoy in equilibrium density gradients of CHAPS extracts, irrespective of whether or not they are associated with rafts. All the CHAPS-insoluble complexes contain cholesterol and GM1, and their flotation was largely sensitive to saponin (not shown). Combined velocity and equilibrium gradients showed that CHAPS generates insoluble complexes of broadly the same density distribution, but with a continuum of sizes (Fig. 5). Whether

Acknowledgements

This work was supported by the Israel Center for the Study of Emerging Diseases. We thank Avihai Hovav and Yuval Tal for help with the animal tissues.

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^☆: Abbreviations: APP, amyloid precursor protein; CHAPS, (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate; DRM, detergent-resistant membrane; ESA, endothelial surface antigen; GPI, glycophosphatidylinositol; mAb, monoclonal antibody; NOG, n-octyl-β-d-glucopyranoside; PNS, post-nuclear supernatant; PrP, prion protein; TX-100, Triton X-100 (polyoxylethylene glycol (9-10) p-t-octylphenol).

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Both raft- and non-raft proteins associate with CHAPS-insoluble complexes: some APP in large complexes☆

Abstract

Section snippets

Materials and methods

Membrane proteins of diverse topologies buoy in equilibrium gradients following CHAPS extraction

Discussion

Acknowledgements

Biochim. Biophys. Acta

Cell

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Curr. Opin. Cell Biol.

J. Biol. Chem.

Blood

Biochem. Biophys. Res. Commun.

Curr. Biol.

J. Biol. Chem.

J. Biol. Chem.

Curr. Opin. Cell Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

Neurobiol. Dis.

Curr. Opin. Cell Biol.

Curr. Opin. Cell Biol.

Biophys. Chem.

VIP21, a 21-kD membrane protein is an integral component of trans-Golgi-network-derived transport vesicles

J. Cell Biol.

Glycosphingolipid-enriched, detergent-insoluble complexes in protein sorting in epithelial cells

Biochemistry

Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior

Proc. Natl. Acad. Sci. USA

Lipid polarity and sorting in epithelial cells

J. Cell. Biochem.

Potocytosis: sequestration and transport of small molecules by caveolae

Science

Functional rafts in cell membranes

Nature