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First published online 11 July 2006
doi: 10.1242/jcs.03060

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Erlin-1 and erlin-2 are novel members of the prohibitin family of proteins that define lipid-raft-like domains of the ER

Southern Alberta Cancer Research Institute, Departments of Oncology and Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, T2N 4N1, Canada

View larger version (52K): [in a new window]   Fig. 1. Comparison of erlin-1, erlin-2 and other PHB family members. (A) CLUSTALW alignment of erlin-1 and -2. Non-conserved, similar, conserved and identical (all match) amino acid residues are indicated by shading as described in the legend. (B) Schematic alignment of erlin-1 and -2 with other PHB family members. PHB domains are represented by grey-filled regions and demarcated by bordering amino acid numbers. Putative transmembrane domains are indicated by light-grey regions outside of the PHB domains and dark grey regions within the PHB domains. The overlap between the two PHB domains of flotillin-1 is represented by the black filled region. The palmitoylation sites of stomatin-1a and flotillin-1 are demarcated by asterisks (*). (C) PHYLIP rooted dendrogram of PHB family members.

View larger version (39K): [in a new window]   Fig. 2. Specificity of the 10E6 and 7D3 monoclonal antibodies for erlin-1. Lipid raft isolates from U937, 3T3-pBabe, 3T3-erlin-1 and 3T3-erlin-2HA cells were subjected to SDS-PAGE in quadruplicate and 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) are indicated on the left.

View larger version (39K): [in a new window]   Fig. 3. Epitopes for both 10E6 and 7D3 map to a 25 amino acid region in the C-terminus of erlin-1. (A) Schematic representation of GST-erlin-1 constructs. Numbers indicate bordering amino acid residues of the various regions of erlin-1. Black-filled regions represent the region wherein the 10E6 and 7D3 epitopes lie as deduced by the experiments below. Protein extracts from E. coli expressing GST fusions with erlin-1 were analyzed by western blotting with (B) 10E6, (C) 7D3 or (D) anti-GST.

View larger version (44K): [in a new window]   Fig. 4. Erlin-1 and erlin-2 are enriched in cellular membranes and lipid raft fractions. (A) U937, 3T3-pBabe, 3T3-erlin-1 and 3T3-erlin-2HA cells were subjected to hypotonic lysis. Membrane (m) and cytosolic (c) fractions were collected and equal proportions of each fraction (by fraction volume) were subjected to SDS-PAGE and western blotting for erlin-1 (with 10E6) and erlin-2HA (with 12CA5). Purity of the membrane and cytosolic fractions was assessed 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 sucrose gradients. 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 the raft fraction was assessed by western blotting for flotillin-1; purity of the soluble fraction was assessed by western blotting for CD71-transferrin receptor (TfnR).

View larger version (22K): [in a new window]   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 were treated with PBS (PBS-treated cells), 20 mM M-ß-C/PBS (M-ß-C-treated cells) or lysed in hypotonic medium and pelleted membranes treated with 20 mM M-ß-C/PBS (M-ß-C-treated membranes). Fractionated sucrose gradients (1=top; 6=bottom) were further subfractionated by dilution in MBS and centrifugation to obtain an insoluble subfraction (pellet) and a soluble subfraction (supernatant). The insoluble subfractions were resuspended directly in sample buffer whereas protein from the soluble supernatant subfractions was obtained by TCA precipitation. Samples representing equivalent proportions of each subfraction (by volume) were subjected to SDS-PAGE and western blotting for erlin-1, erlin-2HA and flotillin-1 with 10E6, 12CA5 and anti-flotillin-1, respectively. Insoluble subfractions 1 to 6, and soluble fraction 6 are depicted and were exposed to film simultaneously as part of the same gel.

View larger version (71K): [in a new window]   Fig. 6. Immunostaining for endogenous erlin-1 with 10E6 reveals a perinuclear staining pattern. (A) MCF-7, HCT116 and HeLa cells, as indicated, were fixed, permeabilized and stained for erlin-1 using the 10E6 antibody. Arrowheads indicate an involution of the nucleus that is stained with 10E6. Bar, 10 µm (applicable to all micrographs). (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 adjacent micrograph ('erlin-2') to illustrate the reticular pattern. Staining of the nuclear envelope (NE) is illustrated in a single focal z-plane of the same cell. Bars, 10 µm.

View larger version (84K): [in a new window]   Fig. 7. Erlin-1 and -2 localize to the ER. HeLa cells were transfected with full-length erlin-1-GFP or erlin-2-GFP as indicated. Cells were counterstained using antibodies to the ER marker proteins (A) calnexin or (B) calreticulin (red) as indicated and visualized using wide-field microscopy. Bars, 10 µm.

View larger version (146K): [in a new window]   Fig. 8. Other PHB family members do not localize to the ER. HeLa cells were transfected with pEGFP-N1 containing full-length inserts of prohibitin-1, stomatin-1 isoform a, and flotillin-1 as indicated. Cells were counterstained using antibodies to the ER marker protein calnexin (red). White arrowheads indicate localization to intracellular, vesicular compartments. Bar, 10 µm.

View larger version (51K): [in a new window]   Fig. 9. The extreme N-terminus of each PHB family member is sufficient for its appropriate subcellular targeting. (A) Schematic representations of GFP fusion constructs with the N-terminal fragments of PHB family members. Black-filled regions represent the putative transmembrane domains of the proteins. HeLa cells were transfected with (B) erlin-1, and erlin-2 N-terminal fusions with GFP (as indicated) and counterstained for the transmembrane ER marker protein, calnexin (red), (C) prohibitin 1 N-terminal fusion with GFP and counterstained with MitoTracker CMX-Ros (red), or (D) stomatin 1 isoform a N-terminal fusion with GFP and counterstained with the lipophilic dye SP-DiI18 (red). Bar, 10 µm.

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