erlin-1 and erlin-2 are novel members of the prohibitin ...invaginations of the plasma membrane...

3149Research Article

IntroductionIt has long been appreciated that the plasma membrane (PM)is not a random distribution of lipids and proteins. Rather, itcontains discrete domains responsible for mediating specificcellular functions (Harder and Engelhardt, 2004). Lipid rafts,an example of a class of such domains, are PM microdomainsenriched in glycosphingolipids and cholesterol (Brown andLondon, 1998). Owing to their distinct morphology, the firstlipid raft domains to be identified were caveolae (Palade,1953; Yamada, 1955). Caveolae are 50-100 nm flask-shapedinvaginations of the plasma membrane containing thecholesterol-binding protein caveolin-1 (Holthuis et al., 2001).Biochemically, caveolae are characterized by their insolubilityin non-ionic detergents at low temperatures (such as solutionsof Triton X-100 at 4°C) and their buoyancy in sucrose gradients(Brown and Rose, 1992). It is by these biochemical criteria thatnon-caveolin-containing lipid rafts were isolated from andidentified in various hematopoietic cells (Fra et al., 1994;Robbins et al., 1995).

As a result of their lack of visibility as morphologicalstructures, non-caveolar lipid rafts are notoriously difficult tocharacterize. The most commonly used method for studyingthese structures has been biochemical isolation of the buoyant,insoluble material from cells lysed in non-ionic detergent andsubjected to sucrose gradient centrifugation, a feature that hasearned these structures the biochemically descriptive name

‘detergent-resistant membranes’ or DRMs (Brown and Rose,1992). Thus, protein markers that co-fractionate with thesesphingolipid-rich domains have been instrumental in thecharacterization of the size, composition, and functions ofthese membrane microdomains, through indirect means in bothbiochemical and microscopy studies (Rietveld and Simons,1998; Subczynski and Kusumi, 2003).

To characterize non-caveolar lipid rafts in cells ofhematopoietic origin, our laboratory has undertaken acombined immunologically based, proteomics approach. Tothis end, low-density, Triton X-100-insoluble lipid raftpreparations were isolated from differentiated U937 and HL60cells and inoculated into mice to produce a panel ofmonoclonal antibodies. Herein, we focus on the identificationand characterization of two novel proteins enriched in lipid raftfractions from myelomonocytic cells: KE04p and its highlyrelated family member, chromosome 8 open reading frame 2(C8orf2). Both proteins fall within the growing family ofprohibitin domain-containing (PHB) proteins, which includesthe prohibitins, the stomatins and the flotillins (also known asreggies). Like other PHB family members, both KE04p andC8orf2 are found in the detergent-insoluble, low-densityfractions of cell lysates of many different cell types. Neither ofthese proteins localized to the PM; instead, they were observedto localize to the endoplasmic reticulum (ER). This wassomewhat surprising considering that the ER contains

Our laboratory was interested in characterizing themolecular composition of non-caveolar lipid rafts. Thus, wegenerated monoclonal antibodies to lipid raft proteins ofhuman myelomonocytic cells. Two of these proteins, KE04pand C8orf2, were found to be highly enriched in thedetergent-insoluble, buoyant fraction of sucrose gradientsin a cholesterol-dependent manner. They contain anevolutionarily conserved domain placing them in theprohibitin family of proteins. In contrast to other familymembers, these two proteins localized to the ER.Furthermore, the extreme N-termini of KE04p and C8orf2were found to be sufficient for heterologous targeting ofGFP to the ER in the absence of classical ER retrievalmotifs. We also demonstrate that all prohibitin familymembers rely on sequences in their extreme N-termini for

their distinctive subcellular distributions including themitochondria, plasma membrane and Golgi vesicles.Owing to their subcellular localization and their presencein lipid rafts, we have named KE04p and C8orf2, ER lipidraft protein (erlin)-1 and erlin-2, respectively. Interestingly,the ER contains relatively low levels of cholesterol andsphingolipids compared with other organelles. Thus, ourdata support the existence of lipid-raft-like domains withinthe membranes of the ER.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/119/15/3149/DC1

Key words: Lipid rafts, KE04, C8orf2, Flotillin, Stomatin, SPFH

Summary

Erlin-1 and erlin-2 are novel members of the prohibitinfamily of proteins that define lipid-raft-like domains ofthe ERDuncan T. Browman, Mary E. Resek, Laura D. Zajchowski and Stephen M. Robbins*Southern Alberta Cancer Research Institute, Departments of Oncology and Biochemistry and Molecular Biology, University of Calgary, Calgary,Alberta, T2N 4N1, Canada *Author for correspondence (e-mail: [email protected])

Accepted 10 May 2006Journal of Cell Science 119, 3149-3160 Published by The Company of Biologists 2006doi:10.1242/jcs.03060

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extremely low levels of sphingolipids and cholesterol (Holthuiset al., 2001; Prinz, 2002; van Meer and Lisman, 2002).Collectively, our data provide evidence for the existence oflipid-raft-like domains within the membranes of the ER.Because of their subcellular localization and their enrichmentin lipid rafts we propose the names erlin-1 and -2 (forendoplasmic reticulum lipid raft protein) for KE04p andC8orf2, respectively.

ResultsIdentification of known and novel lipid-raft-residentproteinsWe were interested in determining the molecular compositionof non-caveolar lipid rafts in cells of hematopoeitic origin. Tothis end we used an immunologically based, proteomicsapproach. Briefly, U937 and HL60 cells were differentiatedalong the macrophage-like, and granulocytic lineage, usingtetradecanoyl phorbyl myristate acetate (TPA) anddimethylsulphoxide (DMSO), respectively. Detergent resistantmembrane (DRM) isolates were prepared from these cells andused to immunize mice for the generation of a panel ofmonoclonal antibodies.

Out of 2880 original hybridomas cultured, approximatelyone-third (>900) of the clones survived. After screening bywestern blotting, we identified 32 antibodies that recognizedantigens within DRM fractions of these cells. A number ofhybridoma clones reacted with antigens of multiple molecularweights (data not shown), indicating a possible recognition ofcommon epitopes or modifications commonly found on adiverse range of proteins, such as phosphorylation orubiquitylation. For these antibodies we have not yet identifiedthe specific antigens that they recognize. Most of thehybridoma antibodies, however, recognized antigensrepresented by a discrete band, indicating a single proteinspecies. Of these, we were able to identify the specific proteinsrecognized by 26 of these monoclonals using massspectrometry of both 1D and 2D gel samples as well as cDNA

Journal of Cell Science 119 (15)

expression cloning. Confirmation of the identified antigens wasaccomplished by cloning the candidate proteins into amammalian expression system along with epitope tags andwestern blotting with the hybridoma supernatants. Throughthis approach we were able to identify both known andpredicted protein residents of DRMs (Table 1).

The plasma membrane protein CD14 represented the antigenrecognized by the largest number of hybridoma clones (Table1). This was to be expected based on its abundance inmacrophages and its mode of membrane attachment through aglycophosphatidylinositol (GPI) anchor, a moiety known to besufficient for DRM association in a number of different celltypes (Brown et al., 1989; Dai et al., 2005; Lisanti et al., 1989).Other antigens identified include the plasma membrane proteinEMMPRIN/CD147 and the dually acylated Src family kinasemember, Lyn. Consistent with the cell lines used in this study,both EMMPRIN and Lyn have been reported to be upregulatedupon monocyte differentiation (Major et al., 2002; Yamanashiet al., 1989). DRM association has also been reportedpreviously for both of these proteins (Tang and Hemler, 2004;Young et al., 2003).

Other proteins identified were not restricted to the PM, butare known to populate the membranes of intracellularorganelles, including both isoforms of the mitochondrialprotein mitofilin and the ER protein, BAP31 (Breckenridge etal., 2003; Groenendyk and Michalak, 2005; Simmen et al.,2005). Although mitofilin was only recently reported to residein DRMs (Mielenz et al., 2005), to our knowledge this is thefirst report describing a raft localization for BAP31.

Interestingly, two independent hybridoma clones, 10E6 and7D3, identified the same novel antigen: the predicted proteinproduct of the chromosome 10 open reading frame 69 gene(C10orf69), known as KE04p or stomatin-prohibitin-flotillin-HflC/K (SPFH) domain protein 1 (herein renamed erlin-1).While attempting to identify the antigen for the 10E6 and 7D3antibodies from bands of silver-stained acrylamide gels,another highly related protein was identified by mass

Table 1. Monoclonal antibodies to lipid rafts of human myelomonocytic cellsHybridoma clone Mr (kDa) Identity Validated by Possible functions Reference

ANDNANnwonknU0517H612E4, 24D4 88, 90 Mitofilin cDNA expression cloning,

mass spectrometryMitochondrial cristae

morphology; maintainsmitochondrial structure

John et al., 2005;Mielenz et al., 2005

19F7, 24A8, 11E3,16C4, 18G3, 31F7,27B7, 13E4, 13E9, 7F2,26G5, 11B4, 15D4,22D10

55 CD14 cDNA expression cloning GPI-linked protein; co-receptorfor LPS

Dai et al., 2005

-nyL retfa tolb nretseWnyL65 ,356H7specificimmunoprecipitation

Signal transduction Young et al., 2003

/NIRPMME55-059C11 collagenasestimulatoryfactor/CD147

cDNA expression cloning Regulation of MMPs; tumourprogression and metastasis

Tang and Hemler,2004

ANDNANnwonknU242F62 ,3H610E6, 7D3 40 Erlin-1/KE04p Mass spectrometry and

cDNA expressioncloning

siht ni debircseDnwonknUpaper

ANDNANnwonknU54-534B210E7, 7F10 28 BAP31 cDNA expression cloning Regulation of apoptosis;

intracellular traffickingSimmen et al., 2005

NA, not applicable; ND, not determined.

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3151Erlins define lipid raft microdomains of the ER

spectrometry from U937 DRM preparations, known as theprotein product of chromosome 8 open reading frame 2 gene(C8orf2), or the SPFH domain protein 2 (renamed erlin-2).Both proteins have been identified in DRM fractions by massspectrometry in previous reports (Blonder et al., 2005; Blonderet al., 2004b; Blonder et al., 2004c; Brown and London, 1998;Ledesma et al., 2003; Li and Prinz, 2004; Sprenger et al.,2004). We decided to focus on the molecular characterizationof erlin-1 and erlin-2 since they had not been previouslycharacterized in the literature.

Erlin-1 and erlin-2 are highly related members of theprohibitin family of proteinsThe most striking feature of erlin-1 and -2 is their high degreeof relatedness, sharing 83% sequence identity and 89%similarity at the amino acid level (Fig. 1A) (Ikegawa et al.,1999; Li et al., 2000). They belong to the prohibitin family ofproteins by virtue of a conserved prohibitin-homology domain(PHB) of ~160 amino acids (Fig. 1B). In fact, they are moreclosely related to prohibitin, the protein for which this familyis named, than any of the other family members (Fig. 1C).Common properties shared by PHB family members, such asassociation with DRMs, the tendency to form oligomers, andtheir diverse subcellular distribution, guided our studies oferlin-1 and -2.

Erlin-1 can be distinguished from erlin-2 usingmonoclonal antibodiesSince erlin-1 and erlin-2 are highly related proteins, we wantedto ensure the specificity of the hybridoma clones for erlin-1over erlin-2. To this end, we expressed full-length erlin-1 andhemagglutinin-tagged erlin-2 proteins in NIH 3T3 fibroblasts,which do not endogenously express the antigen(s) for either10E6 or 7D3. Western blotting with either 10E6 or 7D3revealed a single, ~40 kDa band in DRM fractions from boththe native U937 cell line as well as NIH 3T3 cells thatectopically express a full-length erlin-1 cDNA (3T3-erlin-1),(Fig. 2A,B). This observed molecular mass corresponds verywell with the predicted mass of 38.925 kDa for the protein (Li

et al., 2000). No immunoreactivity was observed with vectorcontrol 3T3 cells.

Western blotting with the monoclonal anti-hemagglutinin(HA) antibody (12CA5) revealed high levels of expression oferlin-2HA in retrovirally infected NIH 3T3 cells (3T3-erlin-2HA), (Fig. 2C). In addition to a band at ~43 kDa, highermolecular mass bands were readily observed for 3T3-erlin-2HA extracts on western blots (Fig. 2C, arrow). We inferredthat these bands represented higher-order oligomers of theprotein. The propensity to form oligomers is a common featureshared by PHB family members (Morrow and Parton, 2005).

Since erlin-1 and erlin-2 were highly related, we wanted todetermine the specific epitopes on erlin-1 recognized by the10E6 and 7D3 antibodies. To this end, we generated GSTfusions to various truncation mutants of the erlin-1 protein(Fig. 3A). Western blotting with the 10E6 and 7D3 antibodiesrevealed bands in extracts of bacteria expressing GST fusionscontaining residues 305-330 of erlin-1 (Fig. 3A-C).Appropriate expression of all constructs was confirmed bywestern blotting with anti-GST antibody (Fig. 3D). Specificrecognition of this C-terminal region of erlin-1 by theseantibodies is consistent with the lower degree of sequenceidentity with erlin-2 at this site compared with the rest of theprotein (Fig. 2A). This accounts for the inability of theseantibodies to detect erlin-2.

Erlin-1 and erlin-2 are enriched in the buoyant,detergent-insoluble fractions of sucrose gradientsAlthough both erlin-1 and erlin-2 were identified from theTriton-X-100-insoluble, low-density fraction of U937 andHL60 cells using mass spectrometry, some proteins identifiedfrom DRM fractions by mass spectrometry were not enrichedas determined by western blotting such as the transferrinreceptor and calnexin (Fig. 4B, supplementary material Fig.S1). Therefore we wanted to ensure that these proteins wereindeed enriched in DRMs. First, to confirm that erlin-1 anderlin-2 were membrane proteins, we subjected U937, as wellas NIH 3T3 cells stably expressing either erlin-1 (3T3-erlin-1)or HA-tagged erlin-2 (3T3-erlin-2HA) to hypotonic lysis.

Fig. 1. Comparison of erlin-1, erlin-2 and other PHBfamily members. (A) CLUSTALW alignment oferlin-1 and -2. Non-conserved, similar, conservedand identical (all match) amino acid residues areindicated by shading as described in the legend.(B) Schematic alignment of erlin-1 and -2 with otherPHB family members. PHB domains are representedby grey-filled regions and demarcated by borderingamino acid numbers. Putative transmembranedomains are indicated by light-grey regions outsideof the PHB domains and dark grey regions withinthe PHB domains. The overlap between the twoPHB domains of flotillin-1 is represented by theblack filled region. The palmitoylation sites ofstomatin-1a and flotillin-1 are demarcated byasterisks (*). (C) PHYLIP rooted dendrogram ofPHB family members.

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Equivalent proportions of the membrane (pellet) and cytosolic(supernatant) fractions were analyzed by western blotting.Both erlin-1 and erlin-2 were highly enriched in the membranefractions of these cell lines (Fig. 4A). By contrast, minimalimmunoreactivity was observed in lanes representing thecytosolic fractions of cells. The relative purity of membraneand soluble fractions was determined by western blotting forSrc and pyruvate kinase, respectively. As expected, Src wasenriched in cellular membranes (Kaplan et al., 1990) whereasthe ubiquitous, cytosolic protein, pyruvate kinase, was highlyenriched in cytosolic fractions (Fig. 4A).

Having demonstrated their presence in membranes, wewanted to confirm that these proteins were indeed enriched inDRMs. Schuck et al. (Schuck et al., 2003) show that DRMisolation using the non-ionic detergent Triton X-100 results inthe isolation of the least number of proteins in the insolublefractions compared with other detergents tested. Therefore, webased our criteria for DRM-resident proteins on this stringentmethod: insolubility in Triton X-100 at 4°C and buoyancy insucrose gradients (Brown, 1992; Schuck et al., 2003). Westernblotting of equal proportions (by volume) of low-density,insoluble and soluble fractions using 10E6 revealed that erlin-1 was highly enriched in the DRMs of both the U937 and 3T3-erlin-1 cell lines (Fig. 4B).

In accordance with the results for erlin-1, erlin-2 was alsohighly enriched in DRM fractions as assessed by westernblotting (Fig. 4B). Based on the consistent behaviour of erlin-1 in both the native U937 and the ectopically expressing 3T3-erlin-1 cell line, it was concluded that the DRM enrichmentobserved for erlin-2 in 3T3-erlin-2HA cells represents itsnatural cellular distribution. Western blotting for the ubiquitousDRM marker protein, flotillin-1, and the transferrin receptor(CD71), a non-raft transmembrane protein indicated that ourisolation procedure was specific for DRMs and not genericcellular membranes (Fig. 4B). Similar to other PHB familymembers, we were able to demonstrate the enrichment of botherlin-1 and erlin-2 in DRMs.

Erlin-1 and erlin-2 are refractory to plasma membranecholesterol depletionIn addition to defining DRMs merely by detergent resistance,Schuck et al. (Schuck et al., 2003) recommend proceduresaimed at disrupting these structures, such as cholesterol orsphingomyelin depletion, to confirm the presence of proteinsin such microdomains. To assess the requirement of cholesterolfor the ability of erlin-1 and -2 to associate with DRMs wetreated live cells with the cholesterol sequestration agent,methyl-�-cyclodextrin (M-�-C) or mock-treated cells withPBS alone. Unexpectedly, M-�-C treatment had little effect onthe solubility of either protein or on their buoyancy in sucrosegradients (Fig. 5). Similar to mock-treated cells, both erlin-1and -2 remained predominantly in the insoluble fractions inboth the U937 and the 3T3 cells lines (Fig. 5). Concordantly,there was no increase in the presence of either protein in thesoluble fraction. Consistent with other reports, flotillin-1 wasalso observed to be relatively unaffected by M-�-C treatment(Fig. 5) (Gkantiragas et al., 2001).

We envisioned two possible scenarios for explaining theseresults: (1) Erlin-1 and -2 may occupy detergent-resistant,buoyant complexes that do not rely on cholesterol for theirintegrity or, (2) since M-�-C is only able to extract cholesterolfrom the exoplasmic leaflet of the plasma membrane, erlin-1


Fig. 3. Epitopes for both 10E6 and 7D3 map to a 25 amino acidregion in the C-terminus of erlin-1. (A) Schematic representation ofGST-erlin-1 constructs. Numbers indicate bordering amino acidresidues of the various regions of erlin-1. Black-filled regionsrepresent the region wherein the 10E6 and 7D3 epitopes lie asdeduced by the experiments below. Protein extracts from E. coliexpressing GST fusions with erlin-1 were analyzed by westernblotting with (B) 10E6, (C) 7D3 or (D) anti-GST.

Fig. 2. Specificity of the 10E6 and 7D3 monoclonal antibodies forerlin-1. Lipid raft isolates from U937, 3T3-pBabe, 3T3-erlin-1 and3T3-erlin-2HA cells were subjected to SDS-PAGE in quadruplicateand western blotted using (A) 10E6, (B) 7D3, (C) 12CA5 (anti-HA)or (D) anti-flotillin-1. Arrow in C indicates the undissociated,dimeric form of erlin-2HA. Molecular size markers (kDa) areindicated on the left.

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and -2 may not be at the cell surface but instead populateintracellular detergent-resistant membranes (Neufeld et al.,1996). To test these hypotheses we depleted cholesterol frominternal membranes by subjecting cells to hypotonic lysisbefore treatment of pelleted membranes with M-�-C in orderto disrupt intracellular DRMs.

Following resuspension of M-�-C-treated membranes inTriton X-100 and fractionation of continuous sucrose gradientswe observed a dramatic shift in both buoyancy and solubilityof erlin-1 in both the U937 and 3T3-erlin-1 cell lines from thelow-density insoluble fraction to the high-density solublefraction (Fig. 5A,B). Similar results were observed for thecontrol DRM marker protein flotillin-1, which localizes to thecytoplasmic leaflet of the PM as well as intracellularcompartments including the Golgi and endocytic system(Morrow and Parton, 2005). Once again this is consistent witha report that flotillin-1 was only susceptible to cholesterolsequestration once internal Golgi membranes were exposed toM-�-C (Gkantiragas et al., 2001). Mobility in the sucrosegradient as well as increased solubility in Triton X-100 wasalso observed for erlin-2 following M-�-C-treatment ofexposed intracellular membranes (Fig. 5C). The consistency

between the native U937 and the 3T3-erlin-1 cell line, in termsof response to treatment, suggested that the 3T3-erlin-2HA linewas behaving in a manner indicative of its native state. Basedon these results, we conclude that like flotillin-1, erlin-1 anderlin-2 populate intracellular DRM domains.

Erlin-1 and Erlin-2 are localized to the ERSince we deduced that erlin-1 and erlin-2 were present inintracellular DRMs, we further characterized their specificsubcellular localization using microscopy. Although erlin-1was first identified in hematopoietic cells, western blottingrevealed that it was expressed in a wide array of human celllines (data not shown). Therefore, we used the humanHCT116, MCF-7 and HeLa cell lines for staining ofendogenous erlin-1 with the 10E6 antibody. All cellsdisplayed a perinuclear staining pattern consistent withnuclear envelope localization, a substructure of the ER (Fig.6A) (Estrada de Martin et al., 2005). This staining pattern wasreminiscent of those achieved with the ER marker antibodiesanti-calnexin and anti-calreticulin (compare with Fig. 7).10E6 staining was also observed in places where involutionof cell nuclei was apparent, consistent with nuclear envelopestaining (Fig. 6A, arrowheads). Thus, staining forendogenous erlin-1 revealed a subcellular localizationconsistent with the ER.

Since we did not generate a monoclonal antibody to erlin-2we assessed its subcellular distribution by immunostaining ofNIH 3T3 cells expressing epitope-tagged erlin-2 using the anti-HA antibody, 12CA5. Using this approach we observed areticular cytoplasmic pattern upon staining with 12CA5,reminiscent of ER morphology (Fig. 6B). Similar to the resultsachieved with erlin-1, staining of the nuclear envelope was alsoobserved for erlin-2HA, illustrated by examination of a singlefocal plane through the cell (Fig. 6B, NE).

To extend our studies of the subcellular localization of thesemolecules we generated EGFP fusions with full length erlin-1and -2 and transiently transfected these constructs into HeLacells. Both erlin-1 and erlin-2 demonstrated a reticular patternof fluorescence, as well as nuclear envelope staining, consistentwith ER localization (Fig. 6). A high degree of co-localizationwas observed when full-length erlin-1-EGFP- and erlin-2-EGFP-expressing cells were counterstained with the ERmembrane protein marker antibodies anti-calnexin and anti-calreticulin (Fig. 7).

Other PHB family members do not localize to the ERAs an additional control for non-specific accumulation ofoverexpressed membrane proteins in the ER, we tookadvantage of the reported distinct subcellular localizations ofendogenous PHB family proteins as controls for correctsubcellular localization of the erlins. To this end, EGFP-taggedversions of prohibitin-1, found in the mitochondria, flotillin-1,known to localize to the PM and Golgi as well as endocyticcompartments, and stomatin 1 isoform a, known to localize tothe PM were generated (Gkantiragas et al., 2001; Morrow andParton, 2005). None of the overexpressed, EGFP-tagged, PHBfamily members were observed to localize to the ER asassessed by the lack of correspondence of EGFP fluorescencewith anti-calnexin staining (Fig. 8). EGFP-tagged prohibitin-1displayed mitochondrial localization, whereas both stomatin-1a and flotillin-1 were observed at the plasma membrane as

Fig. 4. Erlin-1 and erlin-2 are enriched in cellular membranes andlipid raft fractions. (A) U937, 3T3-pBabe, 3T3-erlin-1 and 3T3-erlin-2HA cells were subjected to hypotonic lysis. Membrane (m) andcytosolic (c) fractions were collected and equal proportions of eachfraction (by fraction volume) were subjected to SDS-PAGE andwestern blotting for erlin-1 (with 10E6) and erlin-2HA (with12CA5). Purity of the membrane and cytosolic fractions wasassessed by western blotting for Src and pyruvate kinase (PK),respectively. (B) Lipid raft isolates were prepared from U937, 3T3-pBabe, 3T3-erlin-1, and 3T3-erlin-2HA cell lines as described.Soluble fractions were collected from the bottom of the sucrosegradients. Equal proportions of the lipid raft (r) and soluble (s)fractions were subjected to SDS-PAGE and western blotted for erlin-1 (using 10E6) and erlin-2HA (using 12CA5). Fraction purity of theraft fraction was assessed by western blotting for flotillin-1; purity ofthe soluble fraction was assessed by western blotting for CD71-transferrin receptor (TfnR).

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well as at intracellular vesicular structures, probablyrepresenting Golgi and/or endocytic compartments (Fig. 8,arrowheads). Consistent with other reports of endogenousprotein, all ectopically expressed, EGFP-tagged PHB familymembers tested were observed at their previously reportedsubcellular locales (Morrow and Parton, 2005) (Fig. 8).

The extreme N-terminus of PHB proteins is sufficient fortargeting to their distinctive subcellular localizationsSince the prohibitin family of proteins exhibits diversity interms of subcellular localization, we characterized thesequences required for the appropriate targeting of several PHBfamily members. To this end we generated EGFP fusions withthe extreme N-termini (including the putative transmembranedomains) of erlin-1, erlin-2, and the PHB family membersprohibitin-1 and stomatin-1a. Schematic representations of thevarious TM-EGFP fusion constructs are outlined in Fig. 9A.

While the extreme N-terminal domains of erlin-1 and -2were sufficient for targeting of EGFP to the ER, the analogousdomains of prohibitin efficiently targeted EGFP to themitochondria (Fig. 9B,C). The longer N-terminal sequence ofstomatin targeted EGFP to the PM as well as to cytoplasmicvesicular structures probably representing Golgi or endocyticmembranes (Fig. 9D, arrowhead). These experimentsdemonstrate that the extreme N-termini of PHB familymembers are sufficient for their distinctive subcellulartargeting.

DiscussionIn the past few years lipid rafts have garnered considerableattention based on the diverse functional roles that have beenattributed to them, including transcytosis (Simionescu et al.,1983), potocytosis (Anderson, 1992), an alternate route forendocytosis and phagocytosis (Nichols, 2003; Parton andRichards, 2003; Smart et al., 1999), internalization of variouspathogens (Fivaz et al., 1999; Kurzchalia, 2003; Manes et al.,

2003; Parton et al., 1994; Shin et al., 2000), cholesteroltransport (Oram and Yokoyama, 1996; Smart et al., 1996),calcium homeostasis (Isshiki and Anderson, 1999), proteinsorting (Simons and Ikonen, 1997) and signal transduction(Anderson, 1993; Simons and Toomre, 2000; Smart et al.,1999; Zajchowski and Robbins, 2002). There is nowcompelling evidence to suggest that there are distinctpopulations of lipid rafts on different cell types as well aswithin the same cell. These data include distinct populationsof lipid rafts on polarized epithelial cells (Roper et al., 2000;Scheiffele et al., 1998) as well as on polarized migrating T cells(Gomez-Mouton et al., 2001). In addition it is clear thatdifferent GPI-anchored proteins are localized to distinctmembrane microdomains (Madore et al., 1999).

Based on the lack of knowledge of the molecularcomposition of lipid rafts we embarked on an immunologicallybased approach to generate unique tools to investigate themolecular heterogeneity and composition of lipid rafts. Wechose to focus our initial studies on human cell lines ofmyelomonocytic origin because they lack caveolae (Fra et al.,1994) and thus would allow us to focus on the composition ofnon-caveolin containing lipid rafts. Using this approach wewere able to produce antibodies to: (1) known lipid-raft-resident proteins, (2) known proteins not previously reportedto reside in lipid rafts as well as (3) identify and characterizeproteins whose function or existence was previously unknownor unconfirmed (Table 1). In the present study we have focusedour attention on two proteins, erlin-1 and erlin-2 (also knownas KE04p and C8orf2, respectively) because of their highdegree of enrichment within lipid raft fractions and becausethey were two previously uncharacterized proteins.

Our studies complement a growing number of large-scaleproteomics studies aimed at characterizing the molecularcomposition of lipid rafts (Bae et al., 2004; Bini et al., 2003;Blonder et al., 2005; Blonder et al., 2004a; Chakraborty et al.,2005; Foster et al., 2003; Karsan et al., 2005; Kim et al., 2004;


Fig. 5. Erlin-1 and -2 are refractory to cholesterol depletion of intact cells. (A) U937, (B) 3T3-erlin-1, and (C) 3T3-erlin-2HA cell lines weretreated with PBS (PBS-treated cells), 20 mM M-�-C/PBS (M-�-C-treated cells) or lysed in hypotonic medium and pelleted membranes treatedwith 20 mM M-�-C/PBS (M-�-C-treated membranes). Fractionated sucrose gradients (1=top; 6=bottom) were further subfractionated bydilution in MBS and centrifugation to obtain an insoluble subfraction (pellet) and a soluble subfraction (supernatant). The insolublesubfractions were resuspended directly in sample buffer whereas protein from the soluble supernatant subfractions was obtained by TCAprecipitation. Samples representing equivalent proportions of each subfraction (by volume) were subjected to SDS-PAGE and western blottingfor erlin-1, erlin-2HA and flotillin-1 with 10E6, 12CA5 and anti-flotillin-1, respectively. Insoluble subfractions 1 to 6, and soluble fraction 6 aredepicted and were exposed to film simultaneously as part of the same gel.

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Li et al., 2003; Li et al., 2004; Mielenz et al., 2005; Nebl et al.,2002; Tu et al., 2004; Wollscheid et al., 2004). Some of thesestudies have identified erlin-1 and erlin-2 within lipid raftfractions of a number of cell types but very little if anyinformation regarding their biochemical and molecularcharacterization is available (Blonder et al., 2005; Blonder etal., 2004b; Blonder et al., 2004c; Ledesma et al., 2003; Li etal., 2004; Sprenger et al., 2004). Although these large-scaleproteomic studies provide valuable insights into the globalmolecular composition of DRMs, they are not able todistinguish between compositionally distinct rafts within thesame cell, nor are they able to further characterize themolecules that they identify.

By contrast, our approach results in the generation of toolsfor the further characterization of identified molecules as wellas the specific microdomains that they inhabit. Using one of

the antibodies that we generated (10E6) we were able toconfirm the enrichment of erlin-1 in lipid raft fractions of bothendogenously and ectopically expressing cells as well as revealits intracellular localization. In addition, using an epitope-tagged allele of erlin-2 we were able to show that it had similarbiochemical properties to erlin-1 and was highly enriched inlipid raft fractions. Using immunofluorescent staining ofendogenous erlin-1 and expression of EGFP-tagged versionsof both proteins we were able to demonstrate that these twomolecules are localized within the endoplasmic reticulum.Based on their biochemical properties and unique subcellularlocalization we have named these proteins ER lipid raftprotein-1 and -2 (erlin-1 and erlin-2) for KE04p/SPFH domainprotein 1 and C8orf2/SPFH domain protein 2, respectively.

Erlin-1 and erlin-2 represent the most recently discoveredmembers of the growing prohibitin family of proteins. Inaddition to sharing a PHB domain, members of the prohibitinfamily of proteins share many common characteristicsincluding: detergent insolubility, buoyancy in sucrosegradients, association with cellular membranes, and apropensity to form higher-order oligomers (Mairhofer et al.,2002; Neumann-Giesen et al., 2004; Nijtmans et al., 2002;

Fig. 6. Immunostaining for endogenous erlin-1 with 10E6 reveals aperinuclear staining pattern. (A) MCF-7, HCT116 and HeLa cells,as indicated, were fixed, permeabilized and stained for erlin-1 usingthe 10E6 antibody. Arrowheads indicate an involution of the nucleusthat is stained with 10E6. Bar, 10 �m (applicable to allmicrographs). (B) NIH 3T3 cells expressing erlin-2HA were fixed,permeabilized and stained with the 12CA5 (anti-HA) antibody.‘Inset’ shows a magnification of the boxed area in the adjacentmicrograph (‘erlin-2’) to illustrate the reticular pattern. Staining ofthe nuclear envelope (NE) is illustrated in a single focal z-plane ofthe same cell. Bars, 10 �m.

Fig. 7. Erlin-1 and -2 localize to the ER. HeLa cells were transfectedwith full-length erlin-1-GFP or erlin-2-GFP as indicated. Cells werecounterstained using antibodies to the ER marker proteins (A)calnexin or (B) calreticulin (red) as indicated and visualized usingwide-field microscopy. Bars, 10 �m.

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Salzer and Prohaska, 2001; Snyers et al., 1998). However, theydiffer in their subcellular distributions, which include theplasma membrane, phagosomes, lipid droplets, Golgi vesicles,mitochondria, the nucleus and now the ER (Dermine et al.,2000; Garin et al., 2001; Gkantiragas et al., 2001; Green et al.,2004; Morrow et al., 2002; Salzer and Prohaska, 2001; Tatsutaet al., 2005; Umlauf et al., 2004). Proteins with a PHB domainare highly conserved throughout evolution and are foundwithin diverse kingdoms including those of plants, bacteria andanimals (Morrow and Parton, 2005; Tavernarakis et al., 1999).

In this study, we established that the most recentlydiscovered members of the PHB family, erlin-1 and -2, likeother family members, are associated with cellular membranesand are enriched in the detergent-insoluble, buoyant fractionsof sucrose gradients (Mairhofer et al., 2002; Mielenz et al.,2005; Morrow et al., 2002; Salzer and Prohaska, 2001; Snyerset al., 1999). We confirmed their association with lipid rafts,as opposed to other detergent-insoluble, buoyant complexes,based on their sensitivity to the cholesterol sequestering agentM-�-C following exposure of intracellular membranes.Another common feature of PHB family members shared bythese proteins is that erlin-1 and especially erlin-2 appear toform oligomeric complexes that are not fully dissociated byheating in protein sample buffer containing SDS and �-mercaptoethanol. This oligomerization can be greatlyenhanced if the reducing reagent is omitted from the samplebuffer (data not shown). We also discovered another commonproperty of PHB family members: they are targeted to theirdistinctive subcellular locations by their extreme N-termini.Within the extreme N-terminus of erlin-1 and erlin-2 is a

predicted transmembrane domain that is sufficient for thetargeting of EGFP to the ER. Similarly, we determined thatother PHB family members rely on putative transmembranesequences in their N-termini for their distinctive subcellulartargeting. Our data extend the observations made in previousreports that the N-terminal sequences of flotillin-1 and theyeast prohibitins (Phb1 and Phb2) are sufficient for localizationto the PM and mitochondria, respectively (Liu et al., 2005;


Fig. 8. Other PHB family members do not localize to the ER. HeLacells were transfected with pEGFP-N1 containing full-length insertsof prohibitin-1, stomatin-1 isoform a, and flotillin-1 as indicated.Cells were counterstained using antibodies to the ER marker proteincalnexin (red). White arrowheads indicate localization tointracellular, vesicular compartments. Bar, 10 �m.

Fig. 9. The extreme N-terminus of each PHB family member issufficient for its appropriate subcellular targeting. (A) Schematicrepresentations of GFP fusion constructs with the N-terminalfragments of PHB family members. Black-filled regions representthe putative transmembrane domains of the proteins. HeLa cells weretransfected with (B) erlin-1, and erlin-2 N-terminal fusions with GFP(as indicated) and counterstained for the transmembrane ER markerprotein, calnexin (red), (C) prohibitin 1 N-terminal fusion with GFPand counterstained with MitoTracker CMX-Ros (red), or (D)stomatin 1 isoform a N-terminal fusion with GFP and counterstainedwith the lipophilic dye SP-DiI18 (red). Bar, 10 �m.

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Morrow et al., 2002; Tatsuta et al., 2005). Furthermore, ourdata suggest that the transmembrane domains of erlin-1 and -2contain ER-targeting information independent of known ERretrieval signals such as di-lysine or di-arginine motifs.

DRMs have been reported to reside within multipleintracellular membranes including the endosomal system,mitochondria, the Golgi, phagosomes and lysosomes (Bae etal., 2004; Dermine et al., 2001; Parton and Richards, 2003).The members of the prohibitin family of proteins representexcellent examples of protein markers that populate diversesubsets of detergent-resistant membranes within the cell(Morrow and Parton, 2005). Adding to the diversity of the PHBfamily in terms of subcellular localization, we demonstrate thatboth erlin-1 and -2 reside in the ER.

It has long been established that DRMs (characterized bytheir insolubility in non-ionic detergent and their buoyancyon sucrose gradients) are not exclusive to the PM. Earlystudies pioneering the biochemical isolation of lipid raftsdemonstrated that association of proteins with DRMs occursas early as the Golgi in the secretory pathway (Brown andRose, 1992; Fra et al., 1994; Paladino et al., 2004).Interestingly, both the early Golgi and the ER are involved inthe de novo synthesis of sphingolipids and cholesterol (Prinz,2002; van Meer and Lisman, 2002). However, although theGolgi contains a substantial proportion of these components,the ER is known as the organelle with the lowest sphingolipidand cholesterol content within the secretory pathway (Holthuiset al., 2001; Prinz, 2002; van Meer and Lisman, 2002). For thisreason the ER has been viewed as an unlikely place for theexistence of lipid raft domains.

Nonetheless, there are reports that DRMs exist at the ER inmammalian cells. For example, proteins involved in thesynthesis and transfer of the GPI anchors to GPI-anchoredproteins (GPI-APs) have been observed at DRMs withinthe ER (Pielsticker et al., 2005; Sevlever et al., 1999).Additionally, the GPI-linked, prion protein PrP has beendemonstrated to associate with DRMs as early as the ER withinthe secretory pathway (Paladino et al., 2004; Sarnataro et al.,2004). Finally, DRMs were reported to exist within asubstructure of the ER, ER-lipid droplets (Hayashi and Su,2003). Hayashi and Su (Hayashi and Su, 2003) demonstratedthat �-1 receptors of the brain are associated with DRMs ofER-lipid droplets; however, they exhibit lower buoyancy onsucrose gradients than plasma membrane lipid rafts and do notco-stain with calnexin. By contrast, we report that the noveltransmembrane proteins erlin-1 and erlin-2 are enriched inbuoyant DRMs and are residents of the ER, supporting thenotion that lipid raft-like domains are present in the ER. Thiscontention is based on the isolation of lipid rafts using thestringent detergent, Triton X-100, as well as by using methodsaimed at disrupting these cholesterol-dependent structures(Schuck et al., 2003). Collectively, our data, as well as theaforementioned reports provide strong evidence for theexistence of lipid raft-like domains in the ER.

There is certainly increasing data to suggest that there aresubdomains of the ER such as specific regions that arecontinuous with a peroxisomal reticulum from which a majorsource of peroxisomes can originate (Bascom et al., 2003;Hoepfner et al., 2005), as well as ER subdomains that give riseto lipid droplets (Ozeki et al., 2005; Robenek et al., 2004). Inaddition there is now increasing evidence to suggest that there

are regions of the ER that are in direct contact with the plasmamembrane as well as in intimate association with mitochondria(Levine and Rabouille, 2005). Specific regions of the ERinteracting with mitochondria regulate important biologicalfunctions including apoptosis and calcium release(Breckenridge et al., 2003; Chandra et al., 2004). It isinteresting to note that we identified the pro-apoptotic proteinBAP31 in our lipid raft screen, a protein known to reside in theER that mediates the pro-apoptotic calcium signal that passesfrom the ER to the mitochondria after BAP31 is cleaved by acaspase-mediated event (Breckenridge et al., 2003; Chandra etal., 2004).

Despite the large body of literature on the PHB family ofproteins, very little is known about their exact biologicalfunctions. The best-defined members of the PHB family are theprohibitins, which are also the closest relatives of the erlinswithin this family (Fig. 1C). They are reported to function aschaperones for mitochondrial membrane proteins (Nijtmans etal., 2000). Specifically, they protect proteins from degradationby the mitochondrial m-AAA protease (Steglich et al., 1999).This function is conserved in the bacterial kingdom where theprohibitin homologues HflK and HflC protect E. colimembrane proteins from the AAA-protease, FtsH (Kihara andIto, 1998). A number of mammalian membrane proteinsincluding the cystic fibrosis transmembrane conductanceregulator (CFTR), (Jensen et al., 1995), HMG CoA reductase(Moriyama et al., 1998) and stearoyl-CoA desaturase(Heinemann and Ozols, 1998), are degraded by ER-residentproteases as a complementary pathway to the classical ER-associated degradation (ERAD) cytosolic degradation route.Given their high degree of similarity to prohibitin and theirpresence in the ER, it is possible that the erlins regulate thestability of proteins by protecting them from degradation byER-resident proteases.

Based on these reports as well as the structure andsubcellular location of erlin-1 and erlin-2, these newlydiscovered PHB family members in particular, and detergent-resistant ER microdomains in general, may representspecialized regions of the ER for mediating diverse activitiessuch as GPI-anchor biosynthesis and attachment, qualitycontrol, protein degradation, proteolytic signal peptideprocessing, chaperone functions and/or antigen processing andpresentation. More work is required to fully understand theroles that the erlins in particular and ER lipid-raft-like domainsin general are playing in various aspects of cellular function.

Materials and MethodsBioinformaticsMultiple amino acid sequence alignment was accomplished using CLUSTALWanalysis. PHYLIP rooted dendrogram was produced based on the foregoinganalysis. Putative transmembrane segments were predicted using the TMAPalgorithm, all available at SDSC Biology Workbench (www.workbench.sdsc.edu).PHB domains were determined based on the conserved domain database withprotein-protein BLAST (BLASTp) available at NCBI.

Antibodies and chemicalsThe following antibodies were used: anti-Src MAb327 (J. Brugge, Harvard MedicalSchool, Boston, MA); anti-flotillin-1 MAb (Transduction Labs); rabbit anti-calnexin(Stressgen); goat anti-pyruvate kinase (Chemicon); rabbit anti-CD71/transferrinreceptor (Santa Cruz); Fugene 6, 12CA5 (Roche); goat anti-mouse Alexa Fluor 488and 568, goat anti-rabbit Alexa Fluor 568, SP-DiIC18 and Mitotracker CMX-Ros(Molecular Probes); goat anti-mouse HRP conjugate (BD Pharmingen); goatanti-rabbit HRP (Santa Cruz); rabbit anti-goat HRP (Pierce). Puromycin,paraformaldehyde, tetradecanoyl phorbol myristate acetate (TPA), DMSO,

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polybrene and methyl-�-cyclodextrin were all from Sigma. RIBI adjuvant was fromImmunoChem Research and pGEX-2T from Amersham.

Generation of monoclonal antibodiesTriton X-100 detergent-insoluble fractions were purified from 1.5�108 U937 cellsdifferentiated with 20 ng/ml TPA for 48 hours and from 1.5�108 HL-60 cellsdifferentiated for 4 days with 1.3% DMSO as per our normal protocol (Robbins etal., 1995). The isolated DRMs from the two cell types were pooled and suspendedin RIBI adjuvant before injecting subcutaneously into two Balb/C mice with one-sixth of the total DRM fraction. Boosters were given to each mouse on days 21 and35 and the spleens isolated from the mice on day 42. Spleen cells were fused withthe Sp2 myeloma cell line that expresses the murine IL-6 gene according to standardprotocols (Fuller et al., 1998). After fusion, cells were plated into 96-well platesand selected in HAT medium. Supernatants from individual hybridomas werescreened by western blotting of DRMs made from the differentiated U937 cells andHL60 cells as described above. Of the approximately 900 hybridomas that grew, 32different antibodies were selected for further study based on their strongimmunoreactivity with the DRM fractions.

Mass spectrometry (peptide mass fingerprinting/MALDI-TOFMS/MS)Protein gel slices were sent to Dr Liang Li (ACB Proteomics Mass SpectrometryFacility, Department of Chemistry, University of Alberta, Canada) and analyzed ona Bruker REFLEX III time-of-flight mass spectrometer using MALDI positive ionmode. Selected peptides for each sample were fragmented using MALDI MS/MSon a PE Sciex API-QSTAR pulsar to obtain sequence information to identify theproteins.

Plasmid constructsErlin-1 (KE04; Acc. No. AF064093), erlin-2HA (C8orf2HA; Acc. No.NM_007175) and prohibitin (Acc. No. NM_002634) were cloned by RT-PCR fromU937 cDNA. Flotillin-1 (NM_005803) and stomatin transcript variant 1 (Acc. No.NM_004099) were cloned from a human RNA library (Stratagene). Except whereindicated, PCR inserts were first cloned into the pGEM-T-Easy vector (Promega),and subcloned into the indicated mammalian expression vectors. Primer pairs forerlin-1 were XhoI-KE04-F, 5�-CCGCTCGAGAATGAATATGACTCAAG-3� andClaI-KE04p-R, 5�-CCATCGATCAACCTGTGCTCTCTTTG-3�; for erlin-2HAwere XhoI-C8orf2-F, 5�-CCGCTCGAGCCGCCATGGCTCAGTTGGGA-3� andClaI-C8orf2HA-R, 5�-CCATCGATTCAAGCGTAATCTGGAACATCGTATGGG-TATCCTCCATTCTCCTTAGTGGCCGTCTCCAAG-3�. Erlin-1 and erlin-2HAwere cloned into pBabepuro3 using XhoI and ClaI restriction sites. For erlin-1 GSTfusions the primer pairs used were GST-N-terminus: BamHI-KE04-F, 5�-GGATCC-GAGAATGAATATGACTCAAGCCC-3� and EcoRI-KE04-191-R, 5�-GAATTCT-CATGTCTTCTCAGCCTCCATTAACTC-3�; GST-C-terminus: BamHI-KE04-184-F, 5�-GGATCCGAGTTAATGGAGGCTGAGAAGAC-3� and EcoRI-KE04-end-R,5�-GAATTCTCAACCTGTGCTCTCTTTGTTTTG-3�; GST-305-346: BamHI-KE04-305-F, 5�-GGATCCTTCGTGGACTCCTCATGTGCTTTG-3� and EcoRI-KE04-end-R. GST-330-346 was made by hybridizing the following oligonu-cleotides and cloning directly into pGEX-2T: KE04-330-F, 5�-GATCCGCT-CTTGAACCCTCTGGAGAGAACGTCATCCAAAACAAAGAGAGCACAGGTT-GAG-3� and KE04-330-R, 5�-AATTCTCAACCTGTGCTCTCTTTGTTTTGGAT-GACGTTCTCTCCAGAGGGTTCAAGAGCG-3�. GST fusions with erlin-1fragments were generated by cloning into pGEX-2T vector with BamHI and EcoRIrestriction sites. For GFP constructs, the primer pairs are as follows: erlin-1-FL-GFP: XhoI-KE04-F and AgeI-KE04-FL-GFP-R, 5�-ACCGGTCCACCTGT-GCTCTCTTTGTTTTGGATG-3�; erlin-2-FL-GFP: XhoI-C8orf2-F and AgeI-C8orf2-FL-GFP-R, 5�-ACCGGTCCATTCTCCTTAGTGGCCGTCTCCAA-3�;prohibitin-FL-GFP: XhoI-prohibitin-F, 5�-CTCGAGCGGCCGCATGGCTGC-CAAAGTGTTTGAGTC-3� and AgeI-prohibitin-FL-GFP-R, 5�-ACCGGTCC-CTGGGGCAGCTGGAGGAGCA-3�; stomatin-FL-GFP: XhoI-stomatin-F, 5�-CTCGAGGGCAGCATGGCCGAGAAGCG-3� and AgeI-stomatin-FL-GFP-R,5�-ACCGGTCCCTCTTTTATAATCTTTATGCACATCC-3�; flotillin-1-FL-GFP:XhoI-flotillin-F, 5�-CTCGAGTGAACCATGTTTTTCACTTGTGGCC-3� andAgeI-flotillin-FL-GFP-R, 5�-ACCGGTCCGGCTGTTCTCAAAGGCTTGTGA-3�;erlin-1-N29-GFP: XhoI-KE04-F and AgeI-KE04-TM29-R, 5�-ACCGGTCCGCC-CTCCTCAATCTTGTGGATGG-3�; erlin-2-N25-GFP: XhoI-C8orf2-F and AgeI-C8orf2-TM25-R, 5�-ACCGGTCCCTTGTGCACAGCTGAGAAGAGAG-3�;prohibitin-N38-GFP: XhoI-prohibitin-F and AgeI-prohibitin-TM38-R, 5�-ACCG-GTCCGATGACAGCTCTGTGCCC AGCATC-3�; stomatin-N59-GFP: XhoI-stomatin-F and AgeI-stomatin-TM59-R, 5�-ACCGGTCCATCTTTATGCACATC-CATATTGAGATTG-3�. Erlin-1, erlin-2, prohibitin-1, stomatin-1 and flotillin-1 full-length and truncation mutants were cloned into pEGFP-N1 using XhoI and AgeIrestriction sites.

Cell lines and cell cultureU937 and HL60 cells were maintained in RPMI complete medium. HeLa and MCF-7 cells were maintained in DMEM 10% FBS, 1% penicillin-streptomycin (P/S).NIH 3T3 cells were maintained in DMEM, 5% FBS, 1% P/S. Erlin-1 and erlin-

2HA stably expressing 3T3 cell lines were created using previously describedmethods and maintained in the presence of 2 �g/ml puromycin (Davy et al., 1999;Robbins et al., 1995).

Treatments with methyl-�-cyclodextrinWhole cells were incubated in PBS or 20 mM methyl-�-cyclodextrin in PBS for 30minutes at 37°C. For treatment of cellular membranes, membrane isolates wereprepared using previously described methods in hypotonic medium (HM; 10 mMHEPES-KOH, pH 7.5, 10 mM KCl, 1 mM MgCl2, 1 mM PMSF, 1 �g/ml aprotinin,10 �g/ml leupeptin) (Robbins et al., 1995). Briefly, cells were subjected to onecycle of freeze-thaw and incubated in HM for 30 minutes on ice. Next, cells werepassed through a 26.5 gauge needle for 20 strokes and membranes pelleted bycentrifugation at 16,000 g for 10 minutes. Membrane pellets were then incubatedin methyl-�-cyclodextrin using the same conditions as whole cells described above.Following treatment, cells or membrane isolates were pelleted by centrifugation andsubjected to sucrose gradient fractionation outlined below.

DRM preparation and cell fractionationDRM isolates were prepared as described previously (Robbins et al., 1995). Theinsoluble (DRM) pellet was resuspended in 1� protein sample buffer [1� PSB:62.5 mM Tris-HCl, pH 6.8, 10% glycerol (w/v), 5% �-mercaptoethanol (v/v), 2.3%SDS (w/v), 0.01% Bromophenol Blue (w/v)]. The soluble, high-density fraction wasobtained by resuspending the bottom 2 ml of the gradient (40% sucrose) in 10 mlof 1� MBS and subjected to centrifugation for a further hour. The supernatant wasremoved and proteins precipitated with trichloroacetic acid (TCA), (20% w/v final)on ice for 10 minutes. Precipitate was centrifuged at 11,000 rpm in a Beckman JA-17 rotor. Pellet was resuspended in a 1:1 mixture of 1 M Tris-HCl pH 11 and 2�PSB. For gradient fractionation 2 ml fractions were collected from the top of thegradient (1) to the bottom of the gradient (6). These fractions were furthersubfractionated into soluble and insoluble subfractions by dilution in 10 ml of 1�MBS and ultracentrifugation for a further hour. Soluble (supernatant) and insoluble(pellet) subfractions were retained. Soluble fractions were obtained by TCAprecipitation, centrifugation and resuspension in 1:1, 1 M Tris-HCl (pH 11): 2�PSB. Insoluble (raft) pellets were resuspended in 1� PSB.

MicroscopyFor immunofluorescent staining with the 10E6 antibody, serum-free hybridomasupernatant was concentrated by ammonium sulphate precipitation. MCF-7 andHeLa cells were prepared by fixation with 4% paraformaldehyde/PBS for 20minutes at RT. HCT116 cells were fixed with methanol:ethanol at 1:1 for 5 minutesat –20°C. Cells were then permeabilized by incubation in 0.2% Triton X-100–PBSfor 10 minutes on ice. Next, cells were incubated in undiluted, concentrated 10E6for 1 hour at RT, washed with PBS and incubated in goat-anti-mouse-Alexa Fluor488 (Molecular Probes) at 20 �g/ml (1:100 dilution) in PBS for 1 hour at RT.Finally, stained cells were washed with PBS, counterstained with 500 nM DAPI andmounted on slides with PPDA/glycerol. NIH 3T3 cells expressing erlin-2HA wereprepared by fixation in paraformaldehyde and permeabilization as outlined above.Staining was performed with ammonium sulphate-precipitated 12CA5 at a dilutionof 1:500 in PBS, and goat-anti-mouse-Alexa Fluor 568 (20 �g/ml; 1:100 dilution)secondary with PBS washes as described above. For EGFP experiments, HeLa cellswere plated onto acid-washed coverslips in 24-well plates on the day beforetransfection. Cell transfection was carried out using 0.2 �g of plasmid DNA and0.6 �l of Fugene 6 according to manufacturer’s instructions. For mitochondrialstaining 100 nM Mitotracker was added 30 minutes before fixation according to themanufacturer’s instructions. Cells were fixed with 4% paraformaldehyde, andpermeabilized as above, 24-48 hours following transfection and washed with PBS.Cells were incubated in rabbit-anti-calnexin at a dilution of 1:1000 or rabbit-anti-calreticulin at a dilution of 1:200 in PBS for 1 hour at RT. Cells were washed withPBS and incubated in goat-anti-rabbit-Alexa Fluor 568 at 10 �g/ml (a dilution of1:200) in PBS, washed again and mounted on slides as outlined above. Cells werevisualized using a PlanApo 1.4/60� oil immersion objective on an Olympus IX70microscope with mercury arc lamp excitation. Band pass filters for visualizingEGFP fluorescence or Alexa Fluor 488 and Alexa Fluor 568 fluorescence wereexcitation/emission 490±10 nm/528±19 nm, and 555±14 nm/617±37 nm,respectively. Acquisition of z-stacks with 0.2 �m intervals was accomplished usinga cooled Photometrics CH350 camera (–40°C) and the DeltaVision RT restorationimaging system. Images were subjected to full iterative digital deconvolution on aSilicon Graphics Octane 2 workstation running IRIX software and checked forconvergence. Images represent an individual focal plane except in Fig. 6B wherethe micrograph with inset magnification represents a projected stack of deconvolvedz-planes. To increase fluorescence detection, images representing full-lengthprohibitin-GFP and calnexin were binned at 2�2 (Fig. 7A). Images were colouredand merged using Adobe Photoshop 7.0.

We would like to thank members of the Robbins’ laboratory forinput during the course of this work. We would like to thank NancyQuintrell for her help with the initial generation and screening of the


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lipid raft monoclonal antibodies. We would also like to thank Dr LiangLi in the Department of Chemistry at the University of Alberta foridentification of proteins by mass spectrometry. We would also liketo express our gratitude to Dr Pina Colarusso and Betty Pollock of theLive Cell Imaging Core Facility associated with the CanadianInstitutes of Health Research (CIHR) Group in Inflammatory Diseasefor guidance and technical assistance with the microscopy studies.This work was supported by an operating grant from the CIHR toS.M.R. S.M.R. currently holds a Canada Research Chair in CancerBiology and is an Alberta Heritage Foundation for Medical ResearchScientist.

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