Cell Research (2009) 19:497-506. © 2009 IBCB, SIBS, CAS All rights reserved 1001-0602/09 $ 30.00 npg ORIGINAL ARTICLE www.nature.com/cr The differential protein and lipid compositions of noncaveolar lipid microdomains and caveolae Yao Yao1, Shangyu Hong1, Hu Zhou2, Taichang Yuan1, Rong Zeng2, Kan Liao1 1State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; 2Research Center for Proteome Analysis, Institute of Biochemis- try and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China 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. The caveolar membranes are membrane vesicles of 100-nm diameter, enriched with caveolin-1 and flotillin-1. The noncaveolar lipid microdomains are amorphous membranes and most likely the coalescence of heterogeneous lipid rafts. They are depleted of caveo- lin-1 and are more enriched with cholesterol and sphingolipids than the caveolae. Many membrane proteins, such as insulin-like growth factor-1 receptor (membrane receptor), aquaporin-1 (membrane transporter), Thy-1 and N- cadherin (glycosylphosphatidylinositol-anchored membrane protein and membrane glycoprotein), are specifically as- sociated with noncaveolar lipid microdomains, but not with caveolae. These results indicate that the lipid and protein compositions of caveolae differ from those of noncaveolar lipid microdomains. The difference in their protein compo- sitions implies that these two membrane microdomains may have different cellular functions. Keywords: membrane lipid microdomains, caveolae, noncaveolar lipid microdomains, lipid rafts, sucrose density gradient, sodium carbonate extraction Cell Research (2009) 19:497-506. doi: 10.1038/cr.2009.27; published online 3 March 2009 Introduction without caveolins and caveolae [2, 5-8]. For this reason, broader terms, such as detergent-resistant membranes or Caveolae have been widely accepted as distinct plasma membrane lipid microdomains, are used to define these membrane structures [1, 2]. The formation of 50- to 100- membrane fractions. nm invaginations on the plasma membrane is associated In contrast to caveolae that are readily identifiable with the presence of the caveolin-1 protein, and caveolae membrane invaginations marked by caveolin-1, lipid cannot form in the absence of caveolin-1 [3-5]. Besides rafts cannot be easily distinguished from the surround- their morphological visibility on the plasma membrane [2], ing membrane [6-8]. The dynamic nature of lipid rafts caveolar membranes are biochemically defined by (a) resis- makes it impossible to physically define a specified set of tance to solubilization by Triton X-100 at 4 ºC, (b) a light, membranes as lipid rafts. Lipid rafts are now considered buoyant density and (c) enrichment with sphingolipids a spatiotemporally regulated dynamic lipid-dependent and cholesterol [2]. However, membrane fractions with segregation of specific membrane components [9-14]. these biochemical characteristics can be isolated in cells They range from unstable nanoscale clusters of proteins, cholesterol and sphingolipids, to relatively stable micro- domains formed from preexisting small clusters through Correspondence: Kan Liao protein-protein or protein-lipid interactions. Thus, any Tel: +86-21-54921113; Fax: +86-21-54921011 isolated noncaveolar lipid microdomains are essentially E-mail: [email protected] Abbreviation: GPI-APs (glycosylphosphatidylinositol-anchored proteins) an equilibrated mixture of heterogeneous dynamic mem- Received 11 June 2008; revised 5 September 2008; accepted 25 September brane lipid rafts. 2008; published online 3 March 2009 Biochemically, both caveolae and lipid rafts exhibit a npg Caveolae and noncaveolar lipid microdomains 498 resistance to detergent extraction and are co-isolated by chrome c) or nuclei (histone H1) (Figure 1B). The lyso- light-density flotation centrifugation [1, 2, 5, 8]. Char- some (cathepsin D) and Golgi (g-adaptin) membranes acterizing this kind of membrane lipid microdomains were dissolved by sodium carbonate extraction and were reflects the mixed properties of caveolae and lipid rafts. not present in the fractionated insoluble lipid microdo- For the biochemical analysis of caveolae or lipid rafts, it main fractions (Figure 1E). is essential to separate caveolae and lipid rafts from each Two extraction methods are often used to isolate other. It may be impossible to isolate a defined set of membrane lipid microdomains: low-concentration Triton lipid rafts for characterization, but the well-defined cave- X-100 solution or detergent-free, pH 11, sodium carbon- olae can at least be isolated from the rest of the heteroge- ate solution [16-18]. In combination with sucrose density neous lipid rafts. gradient flotation centrifugation, the insoluble mem- In our previous study with caveolin-1 knockdown brane lipid microdomains can be isolated by flotation 3T3-L1 cells, we found that suppressing caveolin-1 in a sucrose density gradient. Triton X-100 extraction through RNA interference ablated the caveolae [5]. How- completely dissolved the membrane lipid microdomains ever, the fractionation of membrane lipid microdomains from the isolated plasma membrane (Figure 1D). The and the association of the insulin-like growth factor-1 lipid microdomain-associated proteins caveolin-1, flotil- (IGF-1) receptor with lipid microdomains are not af- lin-1 [19] and IGF-1 receptor [5, 20] were all dissolved fected by the ablation of caveolae, nor is IGF-1 receptor by the Triton X-100 solution (Figure 1D). In contrast, the signaling affected during 3T3-L1 adipocyte differentia- sodium carbonate solution could extract lipid microdo- tion induction [5]. Although IGF-1 receptor signaling mains from the purified plasma membrane (Figure 1D). is sensitive to cholesterol depletion, it is independent Caveolin-1, flotillin-1 and IGF-1 receptor were all asso- of caveolae and caveolin-1 [5]. In the differentiated ca- ciated with low-density sphingolipid-enriched insoluble veolin-1 knockdown 3T3-L1 adipocyte, the absence of membranes (Figure 1D). Because Triton X-100 extrac- caveolae has no adverse effect on insulin receptor signal- tion was affected by the amount of cellular membrane ing, GLUT4 translocation or glucose uptake [15]. These mass (Supplementary information, Figures S1 and S2), results indicate that IGF-1 receptor and insulin receptor detergent-free sodium carbonate was used in subsequent signaling are membrane cholesterol sensitive, but cave- studies with purified plasma membrane. olae independent. In the present study, we separated caveolin-1-enriched Separation of caveolae and noncaveolar lipid microdo- caveolar membranes (caveolae) from the heterogeneous mains noncaveolar lipid microdomains in 3T3-L1 cells by a Caveolae are stable membrane structures that are well modified four-step sucrose density gradient. Cholesterol defined by their invaginated morphology and the pres- and sphingolipids were more enriched in noncaveolar ence of the marker protein, caveolin-1 [1, 2]. The dy- lipid microdomains than in caveolae. The specific asso- namic lipid rafts are composed of unstable nanoclusters ciation of IGF-1 receptor, Thy-1, c-Yes and aquaporin-1 and stabilized coalescences of nanoclusters in the plasma with noncaveolar lipid microdomains suggested that the membrane [10, 11]. These two groups of membrane mi- dynamic lipid rafts might be the functionally important crodomains were floated as a single peak of cholesterol- membrane platform for receptor transmembrane signaling. and sphingolipid-enriched membrane microdomains at the interface of 5-35% sucrose in the conventional three- Results step sucrose density gradient (Figure 2A and 2B). Cho- lesterol, gangliosides (glycosphingolipids) and associated Detergent and non-detergent extraction of membrane proteins (caveolin-1, flotillin-1 and IGF-1 receptor) were lipid microdomains from isolated plasma membranes co-isolated in low-density insoluble membrane fractions 3T3-L1 cells have a high abundance of both caveolae (Figures 1D and 2B). and lipid rafts [5]. Because membrane lipid microdo- In caveolin-1 knockdown 3T3-L1 cells in which cave- mains (caveolae and lipid rafts) are mostly located on olae are absent, the IGF-1 receptor is still associated with the plasma membrane [2, 8], purified 3T3-L1 cell plasma membrane lipid microdomains [5]. In addition, the insu- membranes were used to extract membrane lipid micro- lin receptor is associated with membrane microdomains domains. Plasma membrane purity was examined by enriched with gangliosides, but devoid of caveolin-1 [15]. membrane markers and electron microscopy (Figure 1A- These findings suggest that a ganglioside-enriched lipid 1C). In the purified plasma membrane fraction, which microdomain is separable from caveolae. By inserting was marked by N-cadherin and caveolin-1, there was no an intermediate sucrose density step between the 5% and endoplasmic reticulum (calnexin), mitochondria (cyto- 35% sucrose steps of the conventional 5-35-45% sucrose Cell Research | Vol 19