Transferrin uptake in Trypanosoma cruzi is impaired by interference on cytostome-associated cytoskeleton elements and stability of membrane cholesterol, but not by obstruction of clathrin-dependent endocytosis

Transferrin uptake by Trypanosoma cruzi epimastigotes occurs mainly through the cytostome/cytopharynx. Here, we present evidences for the association of sterol-rich membrane domains with the transferrin endocytic site. Assays using pharmacological treatments to disrupt clathrin-coated pits and hinder caveolae formation showed no association between transferrin uptake and clathrin-dependent endocytosis, but indicated that cholesterol stability in membrane domains is essential for the endocytosis of transferrin. Furthermore, it was observed a connection between the integrity of cytoskeleton elements at the cytopharynx and the function of the cytostome. Our data show that T. cruzi epimastigotes depend on a specialized pathway for transferrin uptake, which is cholesterol-dependent, clathrin-independent, and closely associated with the structural stability of the cytostome/cytopharynx cytoskeleton.     

Trypanosoma cruzi epimastigote endocytic pathway: cargo enters the cytostome and passes through an early endosomal network before storage in reservosomes

It has been known for many years that trypanosomatids require exogenous essential growth factors in order to divide. Two surface domains are involved in starting nutrient endocytosis: the flagellar pocket and the cytostome. Although the flagellar pocket plays a fundamental role in the endocytic process occurring in several trypanosomatids, we have shown the cytostome as the main structure involved in this process in epimastigote forms of T. cruzi. After one minute of endocytosis, cargo is still found at the cytostome entry as well as along the cytopharynx. After two, five and fifteen minutes of endocytosis, cargo was seen inside vesicles and tubules, prior to fusing with reservosomes. Three-dimensional reconstruction of these tubules and vesicles showed they are interconnected, forming an intricate and branched network, distributed from the perinuclear region to the posterior end of the cell. Whole unfixed parasites that had taken up gold-protein conjugates for fifteen minutes were washed and dried on electron microscope grids. Observation with an energy-filtering transmission electron microscope revealed long gold-filled tubules at the posterior end of the cell. Parasites treated with ammonium chloride had their intracellular traffic slowed down, which allowed us to observe many events of vesicle fusion. The acidic nature of this network was evidenced using acridine orange. Based on pH and protein uptake kinetics we propose that the vesicular-tubular network is the early endosome of Trypanosoma cruzi epimastigotes.     

The cytostome of Trypanosoma cruzi epimastigotes is associated with the flagellar complex.

Okuda, K., Esteva, M., Segura, E. L., and Bijovsky, A. T. 1999. The cytostome of Trypanosoma cruzi epimastigotes is associated with the flagellar complex. Experimental Parasitology 92, 223-231. Proliferative forms of Trypanosoma cruzi, amastigotes and epimastigotes, have a cytostome, a specialized structure formed by an invagination of the flagellar pocket's membrane surrounded by microtubules and frequently followed by a row of vesicles. All this assemblage penetrates deeply into the cytoplasm overpassing the nucleus. This structure, together with the flagellar pocket, appears to play an important role in the nutrition of the parasite. We demonstrated that the monoclonal antibody 2C4, made-up against isolated flagellar complex of T. cruzi epimastigotes, recognizes a protein doublet of 76 and 87 kDa in total epimastigotes homogenate. The 76-kDa polypeptide is enriched in the detergent-soluble fraction whereas the 87-kDa polypeptide is highly represented in the insoluble fractions and the purified flagella. Immuno-fluorescence assays show the antigen as a small spot at the flagellar pocket region. Immunogold labeling of ultrathin sections of epimastigote forms reveals gold particles at the opening of flagellar pocket, concentrated in the cytostome region. Immunocytochemistry of epimastigote whole-mount cytoskeletons reveals the labeling on an array of three to four microtubules that appears attached to flagellum, running in the direction of the nucleus. Ultrastructural observations have shown that the posterior region of isolated flagella, corresponding to the level of the flagellar pocket, possesses a microtubular structure compatible with that from the cytostome. The relationship between the cytostome, an endocytic organelle, and the flagellum is here described for the first time.     

Detergent-resistant membrane subfractions containing proteins of plasma membrane, mitochondrial, and internal membrane origins

HEK293 cell detergent-resistant membranes (DRMs) isolated by the standard homogenization protocol employing a Teflon pestle homogenizer yielded a prominent opaque band at approximately 16% sucrose upon density gradient ultracentrifugation. In contrast, cell disruption using a ground glass tissue homogenizer generated three distinct DRM populations migrating at approximately 10%, 14%, and 20% sucrose, named DRM subfractions A, B, and C, respectively. Separation of the DRM subfractions by mechanical disruption suggested that they are physically associated within the cellular environment, but can be dissociated by shear forces generated during vigorous homogenization. All three DRM subfractions possessed cholesterol and ganglioside G_M1, but differed in protein composition. Subfraction A was enriched in flotillin-1 and contained little caveolin-1. In contrast, subfractions B and C were enriched in caveolin-1. Subfraction C contained several mitochondrial membrane proteins, including mitofilin and porins. Only subfraction B appeared to contain significant amounts of plasma membrane-associated proteins, as revealed by cell surface labeling studies. A similar distribution of DRM subfractions, as assessed by separation of flotillin-1 and caveolin-1 immunoreactivities, was observed in CHO cells, in 3T3-L1 adipocytes, and in HEK293 cells lysed in detergent-free carbonate. Teflon pestle homogenization of HEK293 cells in the presence of the actin-disrupting agent latrunculin B generated DRM subfractions A–C. The microtubule-disrupting agent vinblastine did not facilitate DRM subfraction separation, and DRMs prepared from fibroblasts of vimentin-null mice were present as a single major band on sucrose gradients, unless pre-treated with latrunculin B. These results suggest that the DRM subfractions are interconnected by the actin cytoskeleton, and not by microtubes or vimentin intermediate filaments. The subfractions described may prove useful in studying discrete protein populations associated with detergent-resistant membranes, and their potential interactions in cell signaling.     

A cytostome/cytopharynx in green euglenoid flagellates (Euglenales) and its phylogenetic implications

The green, phototrophic euglenoid, Colacium libellae, has a vestigial cytostome and cytopharynx. The membrane forming the simple pocket is decorated with a dense, microfilamentous mesh. The mesh binds the pocket to a band of reinforcing microtubules which is homologous with the bodonid MTR. The opening of the cytostome at the reservoir-canal transition zone implies that the phototrophic euglenoid canal is formed by the invagination of the vestibulum of the phagotrophic euglenoids. Our observations support the hypothesis that the phagotrophic euglenoids arose from a bondonid ancestor and gave rise to the phototrophs by chloroplast acquisition.     

The Form and Function of the Cytostome-Cytopharynx of the Culture Forms of the Elasmobranch Haemoflagellate Trypanosoma raiae Laveran & Mesnil

SYNOPSIS. In the culture forms of the elasmobranch trypanosome Trypanosoma raiae is found a prominent cytopharyngeal complex. This consists of a group of 5 or 6 microtubules associated with a deep invagination of the cell membrane which arises from a cytostome near the opening of the flagellar pocket. This structure is a constant feature of the various epimastigote and trypomastigote forms that this flagellate has in culture. Replication of the cytopharyngeal apparatus is completed before cytokinesis.
Experiments using ferritin as an electron dense tracer show that endocytosis occurs from the blind ending of the cytopharynx both in the exponential and stationary phases of growth in vitro. Ferritin is transported from the cytopharynx by endocytotic vesicles to large, membrane-bound vacuoles in the posterior region of the cell. Ultrastructural location of non-specific acid phosphatase within these digestive vacuoles and also within the Golgi apparatus is reported.
Coated vesicles found in association with the flagellar pocket are another route of uptake of ferritin by T. raiae.     

Large-scale quantitative LC-MS/MS analysis of detergent-resistant membrane proteins from rat renal collecting duct

In the renal collecting duct, vasopressin controls transport of water and solutes via regulation of membrane transporters such as aquaporin-2 (AQP2) and the epithelial urea transporter UT-A. To discover proteins potentially involved in vasopressin action in rat kidney collecting ducts, we enriched membrane "raft" proteins by harvesting detergent-resistant membranes (DRMs) of the inner medullary collecting duct (IMCD) cells. Proteins were identified and quantified with LC-MS/MS. A total of 814 proteins were identified in the DRM fractions. Of these, 186, including several characteristic raft proteins, were enriched in the DRMs. Immunoblotting confirmed DRM enrichment of representative proteins. Immunofluorescence confocal microscopy of rat IMCDs with antibodies to DRM proteins demonstrated heterogeneity of raft subdomains: MAL2 (apical region), RalA (predominant basolateral labeling), caveolin-2 (punctate labeling distributed throughout the cells), and flotillin-1 (discrete labeling of large intracellular structures). The DRM proteome included GPI-anchored, doubly acylated, singly acylated, cholesterol-binding, and integral membrane proteins (IMPs). The IMPs were, on average, much smaller and more hydrophobic than IMPs identified in non-DRM-enriched IMCD. The content of serine 256-phosphorylated AQP2 was greater in DRM than in non-DRM fractions. Vasopressin did not change the DRM-to-non-DRM ratio of most proteins, whether quantified by tandem mass spectrometry (LC-MS/MS, n = 22) or immunoblotting (n = 6). However, Rab7 and annexin-2 showed small increases in the DRM fraction in response to vasopressin. In accord with the long-term goal of creating a systems-level analysis of transport regulation, this study has identified a large number of membrane-associated proteins expressed in the IMCD that have potential roles in vasopressin action.     

Interplay between Toxin Transport and Flotillin Localization

The flotillin proteins are localized in lipid domains at the plasma membrane as well as in intracellular compartments. In the present study, we examined the importance of flotillin-1 and flotillin-2 for the uptake and transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin and we investigated whether toxin binding and uptake were associated with flotillin relocalization. We observed a toxin-induced redistribution of the flotillins, which seemed to be regulated in a p38-dependent manner. Our experiments provide no evidence for a changed endocytic uptake of Stx or ricin in cells silenced for flotillin-1 or -2. However, the Golgi-dependent sulfation of both toxins was significantly reduced in flotillin knockdown cells. Interestingly, when the transport of ricin to the ER was investigated, we obtained an increased mannosylation of ricin in flotillin-1 and flotillin-2 knockdown cells. The toxicity of both toxins was twofold increased in flotillin-depleted cells. Since BFA (Brefeldin A) inhibits the toxicity even in flotillin knockdown cells, the retrograde toxin transport is apparently still Golgi-dependent. Thus, flotillin proteins regulate and facilitate the retrograde transport of Stx and ricin.     

Distinct Lipid Rafts in Subdomains from Human Placental Apical Syncytiotrophoblast Membranes

We report on the characteristics of raft domains in the apical membrane from human placental syncytiotrophoblast (hSTB), an epithelium responsible for maternal-fetal exchange. Previously, we described two isolated fractions of the hSTB apical membrane: a classical microvillous membrane (MVM) and a light microvillous membrane (LMVM). Detergent-resistant microdomains (DRMs) from MVM and LMVM were prepared with Triton X-100 followed by flotation in a sucrose gradient and tested by Western and dot blot with raft markers (placental alkaline phosphatase, lipid ganglioside, annexin 2) and transferrin receptor as a nonraft marker. DRMs from both fractions showed a consistent peak for these markers, except that the DRMs from MVM had no annexin 2 mark. Cholesterol depletion modified the segregation in both groups of DRMs. Our results show two distinguishable lipid raft subsets from MVM and LMVM. Additionally, we found significant differences between MVM and LMVM in cholesterol content and in expression of cytoskeletal proteins. MVM is enriched in ezrin and beta-actin; in contrast, cholesterol and cytokeratin-7 are more abundant in LMVM. These differences may explain the distinct properties of the lipid raft subtypes.     

uPA binding increases UPAR localization to lipid rafts and modifies the receptor microdomain composition

UPAR is a GPI anchored protein, which is found in both lipid rafts and in more fluid regions of the plasma membrane. We have studied the role of the ligand uPA on uPAR localization and on the composition of the lipid membrane microdomains. We have analyzed the glycosphingolipid environment of uPAR in detergent resistant membrane (DRM) fractions prepared by cell lysis with 1% Triton X-100 and fractionated by sucrose gradient centrifugation obtained from HEK293-uPAR cells. The uPAR specific lipid membrane microdomain has been separated from the total DRM fraction by immunoprecipitation with an anti-uPAR specific antibody under conditions that preserve membrane integrity. We have also tested uPA-induced ERK phosphorylation in the presence of methyl-beta-cyclodextrin, which is known to disrupt lipid rafts by sequestering cholesterol from such domains. Our results show that uPAR is partially associated with DRM and this association is increased by ligands, is independent of the catalytic activity of uPA, and is required for intracellular signalling. In the absence of ligands, uPAR experiences a lipid environment very similar to that of total DRM, enriched in sphingomyelin and glycosphingolipids. However, after treatment of cells with uPA or ATF the lipid environment is strongly impoverished of neutral glycosphingolipids.     

uPA binding increases UPAR localization to lipid rafts and modifies the receptor microdomain composition

UPAR is a GPI anchored protein, which is found in both lipid rafts and in more fluid regions of the plasma membrane. We have studied the role of the ligand uPA on uPAR localization and on the composition of the lipid membrane microdomains. We have analyzed the glycosphingolipid environment of uPAR in detergent resistant membrane (DRM) fractions prepared by cell lysis with 1% Triton X-100 and fractionated by sucrose gradient centrifugation obtained from HEK293-uPAR cells. The uPAR specific lipid membrane microdomain has been separated from the total DRM fraction by immunoprecipitation with an anti-uPAR specific antibody under conditions that preserve membrane integrity. We have also tested uPA-induced ERK phosphorylation in the presence of methyl-β-cyclodextrin, which is known to disrupt lipid rafts by sequestering cholesterol from such domains. Our results show that uPAR is partially associated with DRM and this association is increased by ligands, is independent of the catalytic activity of uPA, and is required for intracellular signalling. In the absence of ligands, uPAR experiences a lipid environment very similar to that of total DRM, enriched in sphingomyelin and glycosphingolipids. However, after treatment of cells with uPA or ATF the lipid environment is strongly impoverished of neutral glycosphingolipids.     

PTOV1 enables the nuclear translocation and mitogenic activity of flotillin-1, a major protein of lipid rafts

PTOV1 is a mitogenic protein that shuttles between the nucleus and the cytoplasm in a cell cycle-dependent manner. It consists of two homologous domains arranged in tandem that constitute a new class of protein modules. We show here that PTOV1 interacts with the lipid raft protein flotillin-1, with which it copurifies in detergent-insoluble floating fractions. Flotillin-1 colocalized with PTOV1 not only at the plasma membrane but, unexpectedly, also in the nucleus, as demonstrated by immunocytochemistry and subcellular fractionation of endogenous and exogenous flotillin-1. Flotillin-1 entered the nucleus concomitant with PTOV1, shortly before the initiation of the S phase. Protein levels of PTOV1 and flotillin-1 oscillated during the cell cycle, with a peak in S. Depletion of PTOV1 significantly inhibited nuclear localization of flotillin-1, whereas depletion of flotillin-1 did not affect nuclear localization of PTOV1. Depletion of either protein markedly inhibited cell proliferation under basal conditions. Overexpression of PTOV1 or flotillin-1 strongly induced proliferation, which required their localization to the nucleus, and was dependent on the reciprocal protein. These observations suggest that PTOV1 assists flotillin-1 in its translocation to the nucleus and that both proteins are required for cell proliferation.     

Affinity of PIP-aquaporins to sterol-enriched domains in plasma membrane of the cells of etiolated pea seedlings

The hypothesis that sterol-enriched domains represent sites of preferred localization of PIP-aquaporins was tested in experiments on plasma membranes isolated from cells of etiolated pea (Pisum sativum L.) seedlings. Plasma membranes were isolated from microsomes by the partition in the aqueous two-phase polymer system and separated into vesicle fractions of different buoyant density by flotation in discontinuous OptiPrep gradient. Two types of plasma membrane preparations were used: one was treated with cold 1% Triton X-100 and the other was not. In untreated preparations, three populations of plasma membrane vesicles were obtained, while in the case of treated preparations, fractions of detergent-resistant membranes (DRM) and solubilized membrane proteins were obtained. In all membrane fractions collected after OptiPrep flotation, the amounts of proteins, sterols, and PIP-aquaporins were determined. The highest sterol content was detected in the membrane fraction with buoyant density 1.098 g/cm³ and in the DRM fraction (1.146 g/cm³). These fractions contained much more PIP-aquaporins than the other ones. Phase state of the lipid bilayer was determined by measuring generalized polarization excitation of fluorescence (GPEX) of laurdan incorporated into the membranes of different fractions. It was revealed that the lipid bilayer of the membranes with density of 1.098 g/cm³ had a higher extent of ordering than that of the fractions with density of ∼1.146 g/cm³. The results indicated that uppermost local concentrations of PIP-aquaporins were associated with tightly packed sterol-enriched domains. Moreover, upon solubilization of plasma membrane with Triton X-100, PIP-aquaporins mainly resided in DRM, thus exhibiting a high affinity to sterols.     

Detergent-free domain isolated from Xenopus egg plasma membrane with properties similar to those of detergent-resistant membranes

Microdomains known as "rafts" have been isolated from many cell types as detergent-resistant membranes (DRMs) and are enriched in sphingolipids and cholesterol. However, there has been considerable controversy over whether such domains are found in native membranes or are artificially generated by the purification procedure. This controversy is based at least in part on the fact that raft membranes were first detected following detergent extraction in the cold. We isolated two plasma membrane fractions, without detergent treatment, using a discontinuous sucrose density gradient. One fraction was designated "light" and the other "heavy." These fractions were compared with DRMs, which were isolated in the presence of 1% Triton X-100. We found that Xenopus DRMs are enriched with sphingomyelin and cholesterol and exhibit a phase state similar to the liquid-ordered phase. Comparison of DRM complexes with the light and heavy plasma membrane fractions revealed some physical and biochemical similarities between the light fraction of the plasma membrane and the DRM complexes, based on (1) the phosphatidylcholine/sphingomyelin ratio and (2) the protein composition visualized on a two-dimensional gel. These two fractions are also quite similar in their thermotropic phase behavior, and their high levels of ganglioside GM1. We conclude that the light membrane fraction isolated in a detergent-free environment has many of the characteristics normally associated with DRMs.     

Lipid raft proteome of the human neutrophil azurophil granule

Detergent-resistant membrane domains (DRMs) are present in the membranes of azurophil granules in human neutrophils (Feuk-Lagerstedt et al., J. Leukoc. Biol. 2002, 72, 970). Using a proteomic approach, we have now identified 106 proteins in a DRM preparation from these granule membranes. Among these proteins were the lipid raft structural proteins flotillin-1 and -2, cytoskeletal proteins such as actin, vimentin and tubulin, and membrane fusion promoting proteins like annexins and dysferlin. Our results suggest that the azurophil granule membrane, in similarity to the plasma membrane, is an elaborate structure that takes part in intracellular signaling and functions other than the mere delivery of bactericidal effector molecules to the phagosome.     

Attachment of flagellum to the cell body is important to the kinetics of transferrin uptake by Trypanosoma cruzi

The flagellar pocket and the cytostome are surface domains of Trypanosoma cruzi epimastigote involved in acquisition of nutrients. The cytostome is physically connected to the flagellar complex. To investigate if this association plays a role in endocytosis in T. cruzi, the endocytic activity in wild type and gp72 null mutant (flagellum-cell body attachment region is absent) epimastigotes was compared. Both wild type and mutant cells were incubated with transferrin conjugated with Alexa 543 or gold particles over different time periods and thereafter qualitatively and quantitatively analyzed by flow cytometry and transmission electron microscopy. Flow cytometry analysis showed a reduction in transferrin uptake by null mutant after 30 min of incubation. In addition, at this time period, signals detected by fluorescence microscopy were slightly lower in null mutant cells. At lower incubation times, no differences between wild type and mutant epimastigotes could be observed. Quantitative data obtained by morphometric and flow cytometry analysis suggested that the speed of the endocytic process in the null mutant was similar to wild type cells, although null mutants were not able to bind cargo and therefore internalize as much as wild type epimastigotes. Our observations suggest that the physical association between cytostome and the flagellar complex plays a role in endocytosis efficiency by epimastigotes of T. cruzi.     

Calmodulin and Ca2+/calmodulin-binding proteins are involved in Tetrahymena thermophila phagocytosis

The ciliated protist, Tetrahymena thermophila, possesses one oral apparatus for phagocytosis, one of the most important cell functions, in the anterior cell cortex. The apparatus comprises four membrane structures which consist of ciliated and unciliated basal bodies, a cytostome where food is collected by oral ciliary motility, and a cytopharynx where food vacuoles are formed. The food vacuole is thought to be transported into the cytoplasm by a deep fiber which connects with the oral apparatus. Although a large number of studies have been done on the structure of the oral apparatus, the molecular mechanisms of phagocytosis in Tetrahymena thermophila are not well understood. In this study, using indirect immunofluorescence, we demonstrated that the deep fiber consisted of actin, CaM, and Ca2+/CaM-binding proteins, p85 and EF-1alpha, which are closely involved in cytokinesis. Moreover, we showed that CaM, p85, and EF-1alpha are colocalized in the cytostome and the cytopharynx of the oral apparatus. Next, we examined whether Ca2+/CaM signal regulates Tetrahymena thermophila phagocytosis, using Ca2+/CaM inhibitors chlorpromazine, trifluoperazine, N-(6-aminohexyl)-1-naphthalenesulfonamide, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCI. In Tetrahymena, it is known that Ca2+/CaM signal is closely involved in ciliary motility and cytokinesis. The results showed that one of the inhibitors, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl, inhibited the food vacuole formation rather than the ciliary motility, while the other three inhibitors effectively prevented the ciliary motility. Considering the colocalization of CaM, p85, and EF-1alpha to the cytopharynx, these results suggest that the Ca2+/CaM signal plays a pivotal role in Tetrahymena thermophila food vacuole formation.     

Microtubules, Microfilaments, and Membranes in Phagocytosis: Structure and Function of the Oral Apparatus of the Ciliate Climacostomum virens

Climacostomum virens uses oral membranelles to drive suspended food particles into its buccal cavity. The cavity leads to a buccal tube which extends into the cell by as much as half a cell length. The inner end of this tube is delimited by a haplokinety (two rows of basal bodies). Internal to this zone is the cytostome and cytopharynx where food vacuoles form. The buccal tube is encircled by a ring of fibrous material, the cytostomal cord, in the region of the cytostome immediately below the haplokinety. Ribbons of postciliary microtubules extend from the kinetosomes of the haplokinety, attach to the cytopharyngeal membrane, and pass under the cytostomal cord. They become broader and expand into the cytoplasm. Cytopharyngeal vesicles pass between the microtubular ribbons and fuse with the cytopharyngeal membrane to generate membrane for forming food vacuoles. The cytopharyngeal vesicles contain a material which is secreted into the forming food vacuoles. Ciliates continue to feed after incubation in a medium containing cycloheximide, indicating that they draw on a pre-existing pool of membrane when forming the food vacuole.     

Atomic force microscopy imaging of lipid rafts of human breast cancer cells

Several studies suggest that the plasma membrane is composed of micro-domains of saturated lipids that segregate together to form lipid rafts. Lipid rafts have been operationally defined as cholesterol- and sphingolipid-enriched membrane micro-domains resistant to solubilization by non-ionic detergents at low temperatures. Here we report a biophysical approach aimed at investigating lipid rafts of MDA-MB-231 human breast cancer cells by coupling an atomic force microscopy (AFM) study to biochemical assays namely Western blotting and high performance thin layer chromatography. Lipid rafts were purified by ultracentrifugation on discontinuous sucrose gradient using extraction with Triton X-100. Biochemical analyses proved that the fractions isolated at the 5% and 30% sucrose interface (fractions 5 and 6) have a higher content of cholesterol, sphingomyelin and flotillin-1 with respect to the other purified fractions. Tapping mode AFM imaging of fraction 5 showed membrane patches whose height corresponds to the one awaited for a single lipid bilayer as well as the presence of micro-domains with lateral dimensions in the order of a few hundreds of nanometers. In addition, an AFM study using specific antibodies suggests the presence, in these micro-domains, of a characteristic marker of lipid rafts, the protein flotillin-1.     

Sodium-calcium exchanger and lipid rafts in pig coronary artery smooth muscle

Pig coronary artery smooth muscle expresses, among many other proteins, Na+-Ca2+-exchanger NCX1 and sarcoplasmic reticulum Ca2+ pump SERCA2. NCX1 has been proposed to play a role in refilling the sarcoplasmic reticulum Ca2+ pool suggesting a functional linkage between the two proteins. We hypothesized that this functional linkage may require close apposition of SERCA2 and NCX1 involving regions of plasma membrane like lipid rafts. Lipid rafts are specialized membrane microdomains that appear as platforms to co-localize proteins. To determine the distribution of NCX1, SERCA2 and lipid rafts, we isolated microsomes from the smooth muscle tissue, treated them with non-ionic detergent and obtained fractions of different densities by sucrose density gradient centrifugal flotation. We examined the distribution of NCX1; SERCA2; non-lipid raft plasma membrane marker transferrin receptor protein; lipid raft markers caveolin-1, flotillin-2, prion protein, GM1-gangliosides and cholesterol; and cytoskeletal markers clathrin, actin and myosin. Distribution of markers identified two subsets of lipid rafts that differ in their components. One subset is rich in caveolin-1 and flotillin-2 and the other in GM1-gangliosides, prion protein and cholesterol. NCX1 distribution correlated strongly with SERCA2, caveolin-1 and flotillin-2, less strongly with the other membrane markers and negatively with the cytoskeletal markers. These experiments were repeated with a non-detergent method of treating microsomes with sonication at high pH and similar results were obtained. These observations are consistent with the observed functional linkage between NCX1 and SERCA2 and suggest a role for NCX1 in supplying Ca2+ for refilling the sarcoplasmic reticulum.