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First published online 12 September 2006
doi: 10.1242/jcs.03163

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Characterization of erasin (UBXD2): a new ER protein that promotes ER-associated protein degradation

Jing Liang^1,2,*,, Chaobo Yin^2,*, Howard Doong², Shengyun Fang^1,2, Corrine Peterhoff³, Ralph A. Nixon³ and Mervyn J. Monteiro^1,2,

¹ Graduate Program in Molecular Medicine, and Institute for Neurodegenerative Diseases, University of Maryland Biotechnology Institute, 725 West Lombard Street, Baltimore, MD 21201, USA
² Medical Biotechnology Center, University of Maryland Biotechnology Institute, 725 West Lombard Street, Baltimore, MD 21201, USA
³ Center for Dementia Research, Nathan S. Kline Institute, 140 Old Orangeburg Road, Orangeburg, NY 10962, USA

View larger version (73K): [in a new window]   Fig. 1. Homology of erasin proteins found in different species. (A) Sequence alignment of erasin proteins in selective eukaryotic organisms. Homologous amino acids in four or more sequences, including gaps, are shaded. The UBX domain is underlined. The dashed line is the sequence that covers the region that was necessary and sufficient for ER targeting. (B) Reconstructed phylogenetic tree of the erasin homologs using the AssemblyLIGN software.

View larger version (37K): [in a new window]   Fig. 2. Erasin is widely expressed in different tissues. (A) A schematic drawing of the erasin protein. The inferred erasin ORF consists of 508 amino acids. The protein contains a UBX domain from residues 316 to 395 (striped). The probes used in northern blot assay are indicated. Also shown are the two regions against which rabbit polyclonal anti-erasin antibodies 130 and 141 were raised. (B) Northern blot of ERASIN mRNA expression in multiple human tissues. After stripping, the blot was reprobed with ß-actin. The level of ERASIN mRNA expression relative to that in the lung is shown after normalization for actin loading. (C) Characterization of anti-erasin antibodies. Endogenous erasin protein in HeLa cell lysates detected with anti-erasin antibodies 130 and 141. Both antibodies detected a major 64 kDa band that was not detected by their respective preimmune sera (for the preimmune 141 serum see Fig. 6A lane 1). (D) Peptide competition assay demonstrating specificity of antibody 141. Same procedure as Fig. 2C, except antibody 141 was pre-incubated with 0-1 mg/ml of its cognate peptide for 2 hours before immunoblotting. (E) Protein lysates of HeLa cells mock transfected (Control), or transfected with FL untagged erasin, or a C-Myc-tagged erasin expression constructs immunoblotted with anti-erasin 141 (top panel), anti-Myc (middle panel) and anti-actin (bottom panel) antibodies. (F) Immunoblots showing endogenous erasin protein levels in different human cell lines revealed by anti-erasin antibody. (G) Equal amounts of protein lysates from different mouse tissues immunoblotted for erasin with antibody 141 (upper panel) and subsequently with an anti-actin antibody (lower panel).

View larger version (50K): [in a new window]   Fig. 3. Erasin localizes to the ER. (A) Immunofluorescence microscopy. (a-c) Indirect immunofluorescence microscopy of HeLa cells probed with the preimmune 130 serum (a) or with the anti-erasin 130-immune serum (b and c). Cells in panels a and b were untransfected whereas those in panel c were transfected with a FL untagged erasin cDNA expression plasmid. (d-f) Colocalization of erasin and calnexin. HeLa cells transfected with C-Myc erasin and revealed with monoclonal anti-Myc antibody (d), endogenous calnexin revealed with rabbit polyclonal anti-calnexin antibody (e), and the result of merging the erasin and calnexin images (f). Bar, 5 µm for all panels. (B) Immunoblots showing distribution of proteins in HeLa cell homogenates fractionated on 0-25% iodixanol gradients. The upper three panels show the distribution of endogenous erasin, calnexin, and golgin-97 proteins. The lower two panels show the distribution of the two different GFP-tagged erasin constructs expressed in HeLa cells. (C) Immunoblots showing erasin is only found associated with a membrane fraction and is not present in soluble, ER-luminal or nuclear fractions. The different fractions were prepared as described in the Materials and Methods. Controls showing fractionation of calnexin, an ER-membrane associated protein, BiP and calreticulin, two ER luminal proteins, and CENP-B, a nuclear protein. (D) HeLa cell lysates were separated into pellet (P) and supernatant (S) fractions in 1x PBS, in 1 M NaCl, in 0.1 M Na2CO3 (pH 11) or in 1% Triton X-100. These fractions were analyzed by immunoblotting using anti-erasin, anti-calnexin and anti-GM130 antibodies. GM130 is a known peripheral Golgi protein used here as a control.

View larger version (62K): [in a new window]   Fig. 4. Proteinase K protection assay used to reveal the membrane topology of erasin. Microsome membranes were prepared from HeLa cells and incubated with increasing amounts of proteinase K (0 µg/ml for lane1, 0.25 µg/ml for lane2, 1 µg/ml for lane 3, 10 µg/ml for lane 4, 100 µg/ml for lane 5, these samples were treated with proteinase K for 5 minutes; and 10 µg/ml for lane 6 and lane 7, 10 µg/ml and 100 µg/ml proteinase K were incubated with the samples for 1 hour). (A) Immunoblots of the proteinase-K-treated lysates probed with either anti-calnexin-N, and anti-calnexin-C antibodies or (B) anti-erasin 130 and 141 antibodies. (C) Repeat of a similar experiment using a C-terminus GFP-tagged erasin construct and probed with anti-calnexin-N or anti-GFP antibodies. Schematic drawings on the right side depict the known membrane topology of calnexin and of erasin as we propose here. Our model suggests that erasin is anchored in the ER or NE by a hydrophobic patch located between residues 414-434 (see hydrophobicity profile in B) and that both its N- and C-terminus including its hydrophobic domain face the cytoplasm or nucleoplasm. (D,E) Immunogold microscopy of HeLa cells transfected with FL erasin cDNA and probed with anti-N-terminal (130) (D and E) and anti-C-terminal (141) (F) specific erasin antibodies. The majority of gold particles (D,E, arrows) are located on both the nucleoplasmic and cytoplasmic sides of the NE and ER, respectively, with very few particles, if any, in the lumen (the position of the double membrane is indicted by the arrowheads). Immunoreactivity with the C-terminal antibody was weaker, but it too decorated gold particles on the cytoplasmic side of the NE (arrows in F). Bar, 5 nm.

View larger version (70K): [in a new window]   Fig. 5. GFP-tagging experiments indicate that the erasin sequence between residues 414 to 434 is both necessary and sufficient for ER localization. (A) Schematic diagram of GFP-tagged erasin constructs and summary of their localization obtained by live cell imaging and biochemical fractionation on gradients. The hydrophobic patch (HP) that is responsible for ER localization is indicated. (B) Representative examples of the fluorescent images taken of live cells following transfection with different GFP-tagged erasin constructs. (C) Helical wheel representation of the hydrophobic targeting sequence and comparison with that of caveolin-1. Note the position of the putative helix-disrupting proline residue in both sequences (see text).

View larger version (48K): [in a new window]   Fig. 6. Erasin binds p97/VCP proteins primarily through its UBX domain. (A) Immunoprecipitation assays demonstrating that erasin forms a complex with p97/VCP. Lysates were prepared from HeLa cells that were either left untransfected (lanes 1 and 2), or transfected with a FL untagged erasin cDNA plasmid (lane 3), and used to immunoprecipitate erasin using the preimmune 141 serum (lane 1) or with anti-erasin antibody 141 (lanes 2 and 3). Lane 4 contains 1/10 of the lysate prepared from untransfected cells that was used for the immunoprecipitation shown in lane 1 and 2. After separation by SDS-PAGE, the immunoprecipitates were immunoblotted for erasin, p97/VCP and calnexin proteins (upper three panels, respectively). The bottom two panels show 1/10 the amount of total protein lysates from the same sets of cells immunoblotted for erasin, and p97/VCP. (B) Immunoblot demonstrating recombinant p97/VCP is pulled down by GST-erasin fusion protein but not by GST alone. (C) GST pull-down assays showing that erasin binds p97/VCP primarily through its UBX domain. In vitro translated 35S-radiolabeled proteins generated from different erasin constructs (labeled; arrows indicate the major erasin translation product) were analyzed for binding FL p97/VCP GST-fusion protein by pull-down assays (upper panel, autoradiogram of the pull-down results; middle panel, 1/10 portion of the input sample; lower panel, Coomassie-stained gel of pulled down GST-p97/VCP-fusion protein). (D) Immunoprecipitation assays demonstrating that the UBX domain of erasin is important forming a complex with p97/VCP in cells. Lysates prepared from HeLa cells transfected with either FL Myc-tagged erasin or Myc-tagged erasin with deleted UBX domain (UBX) were used to conduct immunoprecipitations with a mouse anti-Myc antibody. The immunoprecipitates, together with 1/10 portion of the lysate, were immunoblotted with anti-p97/VCP (upper panel) and anti-Myc antibodies (lower panel).

View larger version (32K): [in a new window]   Fig. 7. A reduction of erasin levels inhibits ERAD. (A) HEK293 cell cultures were cotransfected with constant amounts of HA-CD3, together with either ERASIN cDNA expression plasmid, or empty vector plasmid, or with 10 nm SMARTpool ERASIN siRNAs. For the knockdown experiments, the cells were transfected with the siRNAs 20 hours before transfection with CD3. 20 hours after CD3 transfection, cycloheximide was added to all cultures and protein lysates were collected at the time intervals indicated. Equal amounts of protein lysates were then immunoblotted for erasin, HA and actin. The arrow and the asterisk indicate the glycosylated and non-glycosylated forms of CD3, respectively. (B) Densitometric analysis of the bands shown in A and expressed relative to the level present at the 0 time point. The Microsoft Excel program was used to produce a best-fit line for each experimental set. (C) A HEK293 cell line stably expressing GFPu was transfected with the nucleic acids as described in A, but omitting HA-CD3. The turnover of GFPu in the different transfected cells was determined by classical pulse-chase studies of [35S]methionine-labeled and immunoprecipitated GFP proteins over a 7-hour period (upper panel). Immunoblot analysis of the cell lysates used for the immunoprecipitation studies confirmed that erasin levels were altered in the expected manner (middle panel), with actin loading of these lysates confirming equal protein loading of the lysates (bottom panel). (D) Graph showing the exponential decline of pulse-labeled GFPu protein over time. The turnover of GFPu was altered little in the three experimental sets.

View larger version (55K): [in a new window]   Fig. 8. Erasin is found in a complex with other ERAD components. (A) Lysates from HeLa cells transfected with a gp78-expression construct or which were left untransfected were used to conduct immunoprecipitations with anti-erasin 130 antibody or its preimmune serum. The immunoprecipitates were immunoblotted with anti-gp78 (upper panel) and anti-erasin antibodies (second panel). The levels of expression of gp78, erasin and actin in equal amounts of protein lysate from these cultures are shown in the bottom three panels. (B) Immunoprecipitation of proteins from untransfected HeLa cells using anti-Derlin-1 antibody or a control IgG antibody and immunoblotted for erasin (upper panel) or Derlin-1. (C) Immunoblot analyses of equal amounts of proteins from normal HEK293 cells (control) and in cells in which erasin levels were reduced using siRNA, probed for the different proteins as listed.

View larger version (31K): [in a new window]   Fig. 9. Erasin is an ER stress-inducible protein. (A) HeLa cells were treated with 7 µM A23187, 2 mM DTT, 5 µM thapsigargin and 2 µg/ml tunicamycin for 16 hours. Cell lysates were collected and immunoblotted using polyclonal anti-erasin antibody 141, anti-BiP and anti-actin antibodies. (B) Levels of erasin protein (mean ± s.d.) seen after the different treatments relative to that of the untreated control were quantified from three independent experiments.

View larger version (106K): [in a new window]   Fig. 10. Erasin immunocytochemistry in Alzheimer's disease and neuropathologically normal control brain. Paraffin sections (5 µm thickness) of prefrontal cortex immunolabeled with erasin 141 antibody show immunostaining patterns in neurons and within the neuropil from different cases of neuropathologically normal controls (A-C) and moderate to severe AD (SAD) (D-I). Immunoreactivity in control brains is minimal (A-C). Counterstaining with thioflavin-S reveals the location of neurofibrillary tangles in one erasin-positive neuron (arrowhead, L,M) but not in another (arrow, M versus L) and the location of ß-amyloid peptide in a senile plaque (K) surrounded by erasin-positive dystrophic neurites (arrows in J). Bars, 5 µm.

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