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ERp19 and ERp46, New Members of the Thioredoxin Family of Endoplasmic Reticulum Proteins*

  • Barbara Knoblach
    Affiliations
    Canadian Institutes of Health Research Membrane Protein Research Group and the Department of Biochemistry, Edmonton, Alberta, Canada T6G 2H7
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  • Bernd O. Keller
    Affiliations
    Department of Biochemistry Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
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  • Jody Groenendyk
    Footnotes
    Affiliations
    Canadian Institutes of Health Research Membrane Protein Research Group and the Department of Biochemistry, Edmonton, Alberta, Canada T6G 2H7
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  • Sandi Aldred
    Affiliations
    Canadian Institutes of Health Research Membrane Protein Research Group and the Department of Biochemistry, Edmonton, Alberta, Canada T6G 2H7
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  • Jing Zheng
    Affiliations
    Department of Biochemistry Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
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  • Bernard D. Lemire
    Affiliations
    Canadian Institutes of Health Research Membrane Protein Research Group and the Department of Biochemistry, Edmonton, Alberta, Canada T6G 2H7
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  • Liang Li
    Affiliations
    Department of Biochemistry Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
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  • Marek Michalak
    Correspondence
    Canadian Institutes of Health Research Senior Investigator and Alberta Heritage Foundation for Medical Research Medical Scientist. To whom correspondence should be addressed. Tel.: 780-492-2256; Fax: 780-492-0886;
    Affiliations
    Canadian Institutes of Health Research Membrane Protein Research Group and the Department of Biochemistry, Edmonton, Alberta, Canada T6G 2H7
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  • Author Footnotes
    * This work was supported by the Canadian Institutes of Health Research and the Alberta Cancer Board. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ¶ Recipient of a studentship from the Alberta Heritage Foundation for Medical Research and from the Canadian Institutes of Health Research.
Open AccessPublished:August 19, 2003DOI:https://doi.org/10.1074/mcp.M300053-MCP200
      Using a proteomic analysis of the luminal environment of the endoplasmic reticulum (ER), we have identified 141 proteins, of which 6 were previously unknown. Two newly discovered ER luminal proteins, designated ERp19 and ERp46, are related to protein disulphide isomerase. Western and Northern blot analyses revealed that both ERp19 and ERp46 and their respective mRNAs are highly expressed in the liver as compared with other tissues. Both proteins were enriched in purified liver ER vesicles and were localized specifically to the ER in McA-RH7777 hepatocytes. Functional analysis with yeast complementation studies showed that ERp46 but not ERp19 can substitute for protein disulphide isomerase function in vivo.
      The endoplasmic reticulum (ER)
      The abbreviations used are: ER, endoplasmic reticulum; MS, mass spectrometry; MS/MS, tandem mass spectrometry; IEF, isoelectric focusing; PIPES, 1,4-piperazinediethanesulfonic acid; 2D, two-dimensional; PDI, protein disulphide isomerase; MALDI, matrix-assisted laser desorption/ionization.
      1The abbreviations used are: ER, endoplasmic reticulum; MS, mass spectrometry; MS/MS, tandem mass spectrometry; IEF, isoelectric focusing; PIPES, 1,4-piperazinediethanesulfonic acid; 2D, two-dimensional; PDI, protein disulphide isomerase; MALDI, matrix-assisted laser desorption/ionization.
      is a centrally located intracellular organelle involved in protein and lipid synthesis and Ca2+ storage and release (
      • Baumann O.
      • Walz B.
      ). Disruption of ER homeostasis results in organellar disease with detrimental effects at both cellular and systemic levels including metabolic, developmental, and neurodegenerative conditions and protein folding disorders (
      • Mesaeli N.
      • Nakamura K.
      • Zvaritch E.
      • Dickie P.
      • Dziak E.
      • Krause K.-H.
      • Opas M.
      • MacLennan D.H.
      • Michalak M.
      ,
      • Scheuner D.
      • Song B.
      • McEwen E.
      • Liu C.
      • Laybutt R.
      • Gillespie P.
      • Saunders T.
      • Bonner-Weir S.
      • Kaufman R.J.
      ,
      • Mattson M.P.
      • Chan S.L.
      • Camandola S.
      ,
      • Brooks D.A.
      ). This is not surprising, as more than 30% of all proteins are synthesized in the ER before being distributed to other locations in the cell. Many Ca2+-binding chaperones reside in the lumen of the ER and are involved in virtually every aspect of ER function, including the regulation of Ca2+ homeostasis, the folding, oligomerization, and glycosylation of proteins, and the formation and isomerization of disulphide bonds within proteins (
      • Meldolesi J.
      • Pozzan T.
      ,
      • Corbett E.F.
      • Michalak M.
      ). While the complexity of ER function is recognized, the protein composition of the ER and its luminal environment remains to be fully analyzed.
      The analysis of complex protein mixtures has recently been facilitated by the development of two-dimensional (2D) gel electrophoresis with immobilized pH gradient strips, allowing for the separation of hundreds of proteins on a single gel. In addition, mass spectrometry (MS) provides a sensitive analytical tool that allows for the identification of very small amounts of individual proteins. These techniques have been combined successfully to determine the protein compositions of subcellular structures (
      • Rout M.P.
      • Aitchison J.D.
      • Suprapto A.
      • Hjertaas K.
      • Zhao Y.
      • Chait B.T.
      ,
      • Garin J.
      • Diez R.
      • Kieffer S.
      • Dermine J.F.
      • Duclos S.
      • Gagnon E.
      • Sadoul R.
      • Rondeau C.
      • Desjardins M.
      ,
      • Journet A.
      • Chapel A.
      • Kieffer S.
      • Roux F.
      • Garin J.
      ) and specialized cell types (
      • O'Neill E.E.
      • Brock C.J.
      • von Kriegsheim A.F.
      • Pearce A.C.
      • Dwek R.A.
      • Watson S.P.
      • Hebestreit H.F.
      ). This study emphasizes the luminal proteome of mouse liver ER. A 2D protein map of the ER luminal environment, which included over 2,000 spots, was generated. Peptide analyses by matrix-assisted laser desorption/ionization mass spectroscopy and tandem mass spectrometry (MALDI-MS/MS) allowed unambiguous identification of more than 140 different proteins, including several that were not previously known. Two of these “new” luminal ER proteins, ERp19 and ERp46, contain thioredoxin motifs that are typically found in PDI-like proteins. Functional studies revealed that ERp46 but not ERp19 can compensate for the loss of PDI function in yeast. This work clearly illustrates and deepens our understanding of the complexity of the ER lumen and suggests a multiplicity of PDI family members.

      EXPERIMENTAL PROCEDURES

      Fractionation and Characterization of the Liver Membrane—

      ER vesicles, Golgi apparatus, plasma membrane, nuclei, and mitochondria were purified from livers of Balb/C mice (
      • Croze E.M.
      • Morre D.J.
      ) with some modifications to accommodate 2D gel electrophoresis requirements. ER vesicles were centrifuged twice on discontinuous sucrose gradients followed by two washes with 10 mm Tris-HCl, pH 7.5. One-mg aliquots of the isolated ER vesicles were pelleted by centrifugation at 90,000 × g for 60 min and stored at −80 °C until use.
      For electron microscopy analysis, an aliquot of freshly prepared ER vesicles was fixed for 60 min on ice in the dark with 2.5% glutaraldehyde and 1% OsO4 (Polyscience, Warrington, PA) in 100 mm PIPES, pH 7.0. Samples were washed twice with 100 mm PIPES, pH 7.0, embedded in 0.5% low melting point agarose in 100 mm PIPES, pH 7.0, dehydrated, and polymerized in Spurr's resin (Electron Microscopy Sciences, Washington, PA). Ultrathin sections were contrasted with a 2% aqueous solution of uranyl acetate followed by 15 mm lead citrate and inspected in a Hitashi H-7000 electron microscope.
      Western blot analysis was carried out as described by Dammann et al. (
      • Dammann H.
      • Hellstern S.
      • Husain Q.
      • Mutzel R.
      ). Nitrocellulose membranes were probed with rabbit anticalreticulin (1:300), rabbit anticalnexin (1:10,000), rabbit antimannosidase II (1:2,000), monoclonal antiporin (1:1,000) (Calbiochem), and monoclonal anti-Na+/K+-ATPase (1:200) antibodies (Hybridoma Bank, Iowa City, IA). Horseradish peroxidase-conjugated goat anti-rabbit or rabbit anti-mouse secondary antibodies (1:10,000) (Jackson Immuno Research, West Grove, PA) were used. Immunoreactive protein bands were visualized using the ECL system (Amersham Biosciences).

      Two-dimensional Gel Electrophoresis and Image Analysis—

      ER vesicles were suspended in 1 ml of an IEF buffer containing 8 m urea, 2 m thiourea, 4% (w/v) CHAPS, 15 mm dithiothreitol, 1 mm tris[2-carboxyethyl]phosphine (Pierce), 0.7% pharmalytes, and a trace of bromophenol blue followed by a 3-h incubation at room temperature. Insoluble material was removed by centrifugation at 40,000 × g for 45 min at 23 °C. Semipreparative IEF was carried out with 300 μg/18-cm immobilized pH gradient strip. Strips with linear gradients of pH 3–10, 4.0–5.0, 4.5–5.5, 5.0–6.0, 5.5–6.7, and 6.0–11.0 were used. Prior to use, each strip was rehydrated for 12 h at 30 V. The IEF parameters were as follows: 150 V, 1 h; 500 V, 1 h; 1,000 V, 1 h; a gradient to 8,000 V, 30 min; and 8,000 V to the steady state, depending on the pH interval used (48,000 Volt hours (Vhrs) for pH 3–10, 120,000 Vhrs for narrow range strips, and 150,000 Vhrs for pH 6.0–11.0 strips). Strips were equilibrated for 12.5 min in 10 ml of a solution containing 50 mm Tris-HCl, pH 8.8, 6 m urea, 30% (w/v) glycerol, 2% (w/v) SDS, 1% (w/v) dithiothreitol, and a trace of bromophenol blue. Second dimension electrophoresis was carried out simultaneously on 4–8-slab SDS-PAGE containing 11% acrylamide (250 × 200 × 1.5 mm) using a Hoefer DALT tank (Amersham Biosciences). Gels were stained with silver and scanned at a resolution of 600 dots/inch, converted to 8-bit grayscale mode, and imported as TIFF files into ImageMaster 2D Elite software (Amersham Biosciences). The parameters for spot detection were as follows: sensitivity, 9,500; noise factor, 5; operator size, 95; and background factor, 120. The images were edited manually and aligned, and spots were excised in a laminar flow hood. Matched spots from 2–4 gels were pooled prior to tryptic digestion. All equipment and supplies for IEF (IPGphor, immobilized pH gradient strip holders and dry strips, and pharmalytes) were from Amersham Biosciences.

      Peptide Extraction and Mass Spectrometric Analysis—

      Protein spots excised from 2D gels were transferred to 0.6-ml siliconized plastic vials, washed three times with H2O, dehydrated with acetonitrile, and dried under a vacuum. They were incubated in the presence of 5 mm dithiothreitol in 100 mm NH4HCO3 for 30 min at 56 °C, cooled to ambient temperature, and resuspended in 10 mm iodoacetamide in 100 mm NH4HCO3 for 30 min in the dark. Trypsin (10 ng/μl) was added in 100 mm NH4HCO3 followed by an overnight incubation at 30 °C. The resulting peptides were sequentially extracted under agitation with 20%, 50% (twice), and 85% acetonitrile in 0.1% trifluoroacetic acid. The pooled extracts were evaporated to a final volume of 5–10 μl. MALDI-MS spectra were collected on a Bruker Reflex III time-of-flight mass spectrometer (Bruker Daltonics, Bremen/Leipzig, Germany) equipped with a SCOUT 384 multiprobe inlet and a 337-nm nitrogen laser in positive ion mode with delayed extraction using the reflectron option. Spectra were obtained by averaging 100–300 individual laser shots and then processed with the Bruker supporting software. The spectra were internally calibrated with trypsin autolysis peptide peaks and matrix peaks. MALDI-MS/MS spectra were collected on an Applied Biosystems/MDS-SCIEX QSTAR Pulsar tandem quadrupole time-of-flight instrument (Concord, Ontario, Canada) equipped with an orthogonal MALDI source employing a 337-nm nitrogen laser. The instrument was operated in positive ion mode, and collision-induced dissociation of peptides was achieved with argon as collision gas. Spectra were acquired and processed using SCIEX support software. The obtained peptide maps were submitted to the MASCOT peptide mass fingerprint tool (www.matrixscience.com) using several databases (the Swiss Protein Database, the mass spectrometry protein sequence database (MSDB), and the NCBI Protein Database). MALDI-MS/MS analysis was also carried out when necessary. Parent and fragment ion m/z values were submitted to the MASCOT MS/MS ion search tool using the same databases as for peptide fingerprint maps. Proteins were considered confidently identified if the fragmentation spectra of isolated peptides could be matched to the sequence of a peptide from the tryptic peptide map or if the significance threshold (provided and established by the MASCOT software) was exceeded. The original spectra shown in Fig. 3 were processed with the Igor Pro software (Wavemetrics, Lake Oswego, OR). Spectra were normalized using the highest signal and smoothed using a Savitzky-Golay algorithm.
      Figure thumbnail gr3
      Fig. 3MALDI-MS and MS/MS spectra of ERp46.A, MALDI-MS spectrum of the in-gel tryptic digestion of ERp46 (see Fig. 2). Peaks matching ERp46 (TrEMBL entry Q91W90) are labeled 1. Peaks marked with an arrow were later used for MS/MS analysis. T- and K-labeled peaks correspond to trypsin autolysis products or tryptic peptides from human keratins, respectively. Another protein in the same spot was identified as serum paraoxonase/arylesterase 1 (Swiss-Prot accession number P52430). The peak used for MS/MS analysis to confirm this protein is labeled 2. Arb., arbitrary. B, MALDI-MS/MS spectrum of m/z 1,301.69 representing the peptide with sequence DLDSLHSFVLR. This sequence tag is specific for ERp46. The fragmentation pattern showing numerous y, b, and some a ions confirms the proposed sequence. The histidine-specific immonium ion is labeled H, and internal fragment ions are labeled i.

      Isolation of cDNAs Encoding ERp19 and ERp46 and Northern Blot Analysis—

      ERp19 and ERp46 were identified by protein-specific MS/MS peptide analysis: m/z 1124.5, GFGDHIHWR; m/z 1702.9, VRPEIINESGNPSYK (ERp19) and m/z 1079.6, GYPTLLLFR; m/z 1301.7, DLDSLHSFVLR; m/z 1555.7, SFEDTIAQGITFVK (ERp46). A nucleotide sequence encoding ERp19 was isolated by PCR-driven amplification of an embryonic mouse cDNA library (Stratagene, La Jolla, CA). A cDNA encoding ERp46 was obtained from the American Type Culture Collection (Manassas, VA). Nucleotide sequence of cDNAs was verified by nucleotide sequence analysis. Sequences were analyzed by BLAST (
      • Altschul S.F.
      • Madden T.L.
      • Schaffer A.A.
      • Zhang J.
      • Zhang Z.
      • Miller W.
      • Lipman D.J.
      ), and algorithms available through the Expert Protein Analysis System (ExPASy) molecular biology server, TMpred, SignalP, and Motif Scan. Genomic configuration of the ERp19 and ERp46 genes was established using BLAST analysis against the Ensembl mouse genome server (www.ensembl.org/Mus_musculus). Multitissue Northern blots were from Clontech (Palo Alto, CA), and they were hybridized under stringent conditions using Rapid-Hyb buffer. The entire coding regions of ERp19 and ERp46 were used as DNA probes. A human β-actin-specific probe was used to normalize for RNA loading. Probes were labeled using the Prime-It II random primer labeling kit (Stratagene).

      Antibody Production and Confocal Microscopy—

      DNA fragments encoding residues Ser21-Leu170 of ERp19 and Gly76-Leu417 of ERp46 were cloned into the periplasmic expression vector pBAD (Invitrogen). His-tagged proteins were expressed in Escherichia coli and purified on nickel-nitrilotriacetic acid-agarose. Polyclonal anti-ERp19 and anti-ERp46 antibodies were raised in rabbits followed by affinity purification on recombinant ERp19 and ERp46 affinity columns prepared using the UltraLink Biosupport medium system (Pierce). For immunostaining, McA-RH7777 hepatocytes were grown on glass coverslips, fixed (
      • Humbel B.M.
      • Biegelmann E.
      ), and immunostained with anti-ERp19 and anti-ERp46 affinity-purified antibodies and anti-PDI monoclonal antibodies (MA3–018, Affinity Bioreagents, Golden, CO) at a concentration of 1 μg/ml. Cy3-conjugated donkey anti-rabbit IgG and Cy2-conjugated donkey anti-mouse IgG (Jackson Immuno Research) were used at a dilution of 1:200. Confocal microscopy was performed on a Zeiss LSM 510. Western blot analysis of ERp19 and ERp46 was carried out using affinity-purified antibodies at concentrations of 0.3 μg/ml followed by the ECL detection system.

      Yeast Complementation Assay—

      A diploid yeast strain heterozygous for the deletion of the PDI1 gene, PDI1/pdi1Δ::KanMX (BY4743-YCL043C, Saccharomyces genome deletion record 23450: MATa/MATα, his3Δ1/his3Δ1, leu2Δ2/leu2Δ2, lys2Δ0/+, met15Δ0/+, ura3Δ0/ura3Δ0) was obtained from the American Type Culture Collection. cDNAs encoding the open reading frames of ERp19, ERp46, and yeast PDI were cloned into pYES2 (Invitrogen) under control of the GAL1 promoter. The plasmids were introduced into the diploid knockout strain, and transformants were selected for their ability to grow on uracil-deficient media. The genotypes of the haploid progeny were confirmed by Southern blot hybridization of restriction enzyme-digested genomic DNA. The ability of the mammalian PDIs to complement for the deletion of the chromosomal PDI gene was assessed by growth on galactose and glucose media. Yeast strains were cultivated at 30 °C in minimal medium containing 6.8 g/liter N-base without amino acids, 2% glucose or 2% galactose, and auxotrophic requirements. Rich medium contained 1% yeast extract, 2% bactopeptone, and 2% glucose or 2% galactose.

      RESULTS

      ER Fractionation and Proteome Analysis by Two-dimensional Gel Electrophoresis—

      ER vesicles were fractionated according to a previously developed procedure (
      • Croze E.M.
      • Morre D.J.
      ). This membrane preparation has been extensively characterized with respect to ER markers (
      • Milner R.E.
      • Baksh S.
      • Shemanko C.
      • Carpenter M.R.
      • Smillie L.
      • Vance J.E.
      • Opas M.
      • Michalak M.
      ). In this study, the quality of the fractionation was further assessed by ultrastructural and immunological analysis. A morphometric analysis of electron micrographs obtained in a hierarchical sampling procedure from 9 experimental animals was carried out. Volume densities of the profiles were estimated by point counting (
      • Weibel E.R.
      ). 75% of all profiles contained attached ribosomes and therefore corresponded to rough ER, whereas 10% did not and were thus likely derived from smooth ER, vesicles of the secretory pathway, and/or the plasma membrane (Fig. 1A). The remaining 15% of the profiles comprised visible contaminants of mitochondrial and peroxisomal origin. Immunoblotting indicated that the preparation was enriched in calnexin and calreticulin, both of which are known to be ER proteins (Fig. 1B) (
      • Michalak M.
      • Corbett E.F.
      • Mesaeli N.
      • Nakamura K.
      • Opas M.
      ). Mannosidase II was undetectable in the ER fraction, indicative of a low Golgi content (Fig. 1B). Very low levels of porin (a mitochondrial outer membrane protein), of Na+/K+-ATPase, and of nuclear pore protein Gp210 were found in the ER preparation used in this study (Fig. 1B). This is not surprising because the ER is in contact with both mitochondria and the plasma membrane and shows continuity with the nuclear envelope.
      Figure thumbnail gr1
      Fig. 1Electron microscopy (A) and Western blot analysis (B) of mouse liver fractionation. Endoplasmic reticulum, Golgi, mitochondria (Mito), nuclei, and plasma membrane (PM)-containing fractions were isolated from mouse liver as described under “Experimental Procedures.” The bar represents 0.5 μm. Western blots were probed with antibodies against calnexin (CNX), calreticulin (CRT), mannosidase II, porin, Gp210, and Na+/K+-ATPase.
      The proteome of the ER lumen was analyzed as follows. Proteins were extracted from ER vesicles using an IEF buffer, producing a urea-soluble fraction containing predominantly ER peripheral membrane proteins and luminal proteins. These proteins were separated by isoelectric focusing, either using wide or narrow range pH strips, followed by standard SDS-PAGE. The wide range 2D gels produced ∼500 discernible silver-stained protein “spots” with the most abundant proteins located in the acidic portion of the gel (Fig. 2A). This protein pattern was not observed in 2D gels of proteins found in total liver homogenates (results not shown), demonstrating that the membrane fraction used in this study was specifically enriched for ER proteins. The extracted proteins were resolved further using narrow range pH strips, which covered pH units of 4.0–5.0, 4.5–5.5, 5.0–6.0, 5.5–6.7, and 6.0–11.0. The overlap in pH ranges led to data redundancy with multiple identifications of the majority of the proteins. Importantly, these high resolution gels (
      • Wildgruber R.
      • Harder A.
      • Obermaier C.
      • Boguth G.
      • Weiss W.
      • Fey S.J.
      • Larsen P.M.
      • Gorg A.
      ) enabled us to identify new ER proteins present in very small amounts (Fig. 2B).
      Figure thumbnail gr2
      Fig. 2Two-dimensional gel electrophoresis of ER-enriched fractions. Proteins were separated in the first dimension based on their isoelectric point on wide range (pH 3.0–10.0, top) and narrow range pH strips (pH 4.5–5.5) and in the second dimension based on their molecular mass in SDS-PAGE (11% acrylamide). The gels were stained with silver. Identified proteins are annotated; novel proteins of the secretory pathway are depicted in red.

      Identification of Proteins Extracted from the ER—

      Protein spots were systematically excised and subjected to in-gel trypsin digestion to generate peptides for MS analysis. We used the following criteria for identification of specific proteins. Peptide mass fingerprinting was considered sufficient for identification if several peptides specific for a given protein were found and if at least 15% of the amino acid sequence of the protein was covered. Also, the observed molecular weight and pI determined for a protein by 2D gel electrophoresis had to agree with calculated values. MALDI-MS/MS analysis was performed on proteins that could not be identified unambiguously using peptide mass fingerprinting. This approach allowed us to identify the 141 proteins listed in Table I.
      Table IProteins associated with the ER
      Most of the proteins we identified are associated with the ER and are resident in the lumen, peripheral membrane proteins, or intracellular cargo. Numerous ribosomal proteins and tRNA binding attenuation protein (TRAP) δ, a subunit of the signal sequence receptor, were identified as components of the cellular protein synthesis machinery. Not surprisingly, ER chaperones such as calreticulin, BiP, endoplasmin, and PDI were most prominent (Fig. 2A). Several less abundant chaperones and protein-processing enzymes were also identified, including GRP170, α-glucosidase, several isozymes of peptidylprolyl isomerase, and the PDI-like proteins, P5 (CaBP1), ERp57, and ERp29.
      While ER luminal chaperones are important in protein folding and posttranslational modification, they also contribute to Ca2+ buffering and Ca2+ signaling in the ER by providing low affinity Ca2+-binding sites. These include calreticulin, BiP, GRP94, and several PDI-like proteins. We also identified high affinity Ca2+-binding and Ca2+-signaling molecules, including calumenin, an EF-hand Ca2+-binding protein that resides in the ER lumen (
      • Yabe D.
      • Nakamura T.
      • Kanazawa N.
      • Tashiro K.
      • Honjo T.
      ), the cytosolic protein p22, which is implicated in vesicular trafficking, and deaminated (Asn97 → Asp) calmodulin. Furthermore, we found that protein kinase Cβ, the protein kinase C activator 14-3-3ε, and the G protein β-subunit were associated with the ER membrane.
      Our analysis of the ER proteome also revealed that detoxifying enzymes of various families are abundant constituents of the ER. Most prominent were members of the carboxylesterase family, which hydrolyze a variety of structurally unrelated xenobiotics and endogenous substances into their respective acids (
      • Satoh T.
      • Hosokawa M.
      ). High concentrations of cytochrome b5, NADH-cytochrome b5 reductase, and members of the cytochrome P450 family were found. These enzymes, which are membrane-bound and oriented with their active domains exposed to the cytosol, are involved in lipid and drug metabolism. Other enzymes that we identified included dimethylaniline oxidase 5 and epoxide hydrolase (both microsomal and soluble), which increase the water solubility of aliphatic epoxides by converting them into dihydrodiols, and the cell-surface-bound enzymes arylesterase/paraoxonase 1 and 3, which hydrolyze organophosphate substrates and aromatic carboxylic acid esters.
      We identified multiple ER cargo proteins. A series of abundant, acidic, low molecular weight proteins constitute members of a family of major urinary proteins. These pheromone-binding proteins are synthesized in the liver and released in the urine of male rodents where they play a role in chemosensory signaling (
      • Cavaggioni A.
      • Mucignat-Caretta C.
      ). Other secreted proteins that we identified are the thyroid hormone-binding protein transthyretin and the iron-binding protein transferrin. As would be expected, hemoglobins α and β were found in relatively small amounts because their synthesis is down-regulated in the liver of adult animals. More prominent cargo proteins were mouse serum albumin, apolipoprotein A, which mediates the reverse transport of cholesterol from tissues to the liver, and apolipoprotein E, which mediates the binding, internalization, and catabolism of lipoprotein particles. Other proteins involved in lipid metabolism were also identified including nonspecific lipid transfer protein and microsomal triglyceride transfer protein. These proteins mediate the transfer of phospholipids, gangliosides, and cholesterol between membranes and are required for the secretion of plasma lipoproteins. Microsomal estradiol 17β-dehydrogenase and a membrane-associated progesterone receptor component that is involved in hormonal regulation were also identified in the ER proteome.

      Identification of New Proteins in the ER—

      In our MS analysis, we detected several ER proteins that have not been identified previously. In particular, two protein spots yielded peptide masses that could not be matched with any entry in current databases. Peptide sequencing will be required to identify these apparently novel ER proteins. In contrast, MALDI-MS/MS data for 6 other proteins corresponded to entries in the GenBank™ expressed sequence tag database (Table I). In this study we have designated these proteins as ERp19, ERp46, ERp73, p16, p17, and p36. The term “ERp” (endoplasmic reticulum protein) followed by the calculated molecular mass of the protein is used for those proteins that contained a putative N-terminal hydrophobic signal sequence and a C-terminal KDEL ER retrieval sequence. The letter “p” followed by the calculated molecular mass of the protein is used for those proteins that are not luminal ER residents (Table I).
      PSI-BLAST searches (
      • Altschul S.F.
      • Madden T.L.
      • Schaffer A.A.
      • Zhang J.
      • Zhang Z.
      • Miller W.
      • Lipman D.J.
      ) reveal that the small acidic protein p17 (Translated European Molecular Biology Laboratory Nucleotide Sequence Database (TrEMBL) entry Q9CQ92) does not share significant sequence homology with any other known protein. It contains a putative C-terminal transmembrane domain and one TRP repeat, a motif involved in protein-protein interactions. Its existence in the human genome has been predicted by comparative gene identification using the completed Caenorhabditis elegans proteome as a template (
      • Lai C.-H.
      • Chou C.-Y.
      • Ch'ang L.-Y.
      • Liu C.-S.
      • Lin W.-C.
      ). Protein p16 (gi‖21314834) contains an N-terminal transmembrane region that serves as a signal sequence for ER translocation and an EF-hand Ca2+-binding site. It has been identified as a secretory protein in neural stem cells where it plays a role in cell survival.
      H. Toda, K. Tashiro, J. Takahashi, N. Hashimoto, I. Nakano, K. Kobuke, M. Tsuji, and T. Honjo, unpublished data.
      The protein p36 (gi‖20911791) also contains an N-terminal signal peptide but no KDEL C-terminal ER retrieval signal, strongly suggesting that it is a secreted protein. It contains a comparative gene identification domain, which is typically found in large multisubunit protein complexes, and its C-terminal region is enriched in glutamic acid and arginine residues and might play a role in ion binding and/or protein-protein interactions.
      The protein designated ERp73 (gi‖20910231) shares sequence identity with C. elegans aminoacylase-1, which is a member of the M20 peptidase family. The remaining two proteins, ERp19 (TrEMBL entry Q9CQU0) and ERp46 (TrEMBL entry Q91W90), are members of the thioredoxin protein family. The MS spectra used to identify ERp46 are depicted in Fig. 3. Seven tryptic peptides, which matched a theoretical digest of the protein, were found (Fig. 3A). Four of these were subjected to additional MALDI-MS/MS analysis. The b and y ion fragmentation pattern of the DLDSLHSFVLR peptide allows for unambiguous identification of ERp46. The relatively strong abundance of the y10 and the y8 ions, which result from enhanced cleavage C-terminal to aspartic acid residues in a quadrupole time-of-flight mass spectrometer equipped with a MALDI source (
      • Sullivan A.G.
      • Brancia F.L.
      • Tyldesley R.
      • Bateman R.
      • Sidhu K.
      • Hubbard S.J.
      • Oliver S.G.
      • Gaskell S.J.
      ), provides additional evidence for the proposed sequence of the peptide (Fig. 3B).

      Amino Acid Sequence Analysis of ERp19 and ERp46—

      Analysis of the amino acid sequences of ERp19 and ERp46 reveals that both proteins contain N-terminal signal sequences (Met1 → Thr20 and Leu8 → Ala27, respectively) and C-terminal ER retrieval signals (QDEL and KDEL, respectively; Fig. 4). ERp46 harbors a second sequence of hydrophobic amino acids (Met56 → Cys75), which may serve as an additional membrane anchor or translocation signal (Fig. 4). Our sequence analysis also revealed that ERp19 and ERp46 contain one and three thioredoxin motifs, respectively, enabling their classification as novel members of the thioredoxin protein family (Fig. 4). BLAST searches against the mouse genome server (www.ensembl.org/Mus_musculus/) identified a single gene for ERp19 at 106.6 Mb on chromosome 4. The gene sequence predicts a primary transcript of 1,275 nucleotides in length and a protein of 19,048 Da with a pI of 4.92. The BLAST analysis also indicated that the gene for ERp46 is located on chromosome 13.
      Figure thumbnail gr4
      Fig. 4Primary amino acid sequences of murine ERp19 (A) and ERp46 (B). The dotted underlining indicates hydrophobic amino acid segments; the solid underlining represents ER retrieval signals. Thioredoxin motifs are boxed.

      Tissue Distribution and Localization of ERp19 and ERp46—

      The relative amounts and tissue-specific distributions of ERp19 and ERp46 transcripts were studied on multiple tissue Northern blots. A probe specific for ERp19 hybridized with transcripts at 1.5 and 3.7 kb. The highest levels of expression were observed in liver, heart, kidney, and testis (Fig. 5A). A probe specific for ERp46 hybridized with transcripts of 2.6 and 1.8 kb, with the high molecular weight species being more abundant. ERp46 transcript levels were highest in liver and were equally abundant in heart, brain, spleen, lung, kidney, and testis (Fig. 5A). ERp19 and ERp46 transcripts were undetectable in skeletal muscle.
      Figure thumbnail gr5
      Fig. 5Tissue distribution of ERp19 and ERp46 mRNA and protein.A, Northern blot analysis of ERp19 and ERp46 mRNA transcripts on multiple tissue Northern blots. A β-actin probe was used to normalize for RNA loading. B, 100 μg of total protein from various tissues were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with affinity-purified anti-ERp19 and anti-ERp46 antibodies. C, 30 μg of total protein of liver homogenate and ER vesicles were separated on SDS-PAGE, transferred to nitrocellulose, and probed with specific antibodies.
      We raised and affinity-purified antibodies specific for ERp19 and ERp46 to study the relative abundance and intracellular distributions of the two proteins. The apparent molecular masses of the proteins detected by anti-ERp19 and anti-ERp46 antibodies in Western blotting experiments correspond to the apparent molecular masses of the proteins that were determined using the 2D gels (Figs. 2 and 5B). The anti-ERp19 antibody recognized a protein band of ∼20 kDa in various tissues (Fig. 5B). In heart and kidney, an additional protein band of 40 kDa was detected (Fig. 5B). Relative amounts of ERp19 protein corresponded to the amounts of its transcript in all tissues (Fig. 5, A and B). The largest amounts of ERp19 were found in the liver, followed by testis, heart, and kidney (Fig. 5B). Relatively low amounts of ERp19 were observed in the brain, spleen, and lungs (Fig. 5B). ERp19 was immunologically undetectable in skeletal muscle (Fig. 5B). In contrast to ERp19, ERp46 protein and transcript levels did not correlate in all tissues (Fig. 5, A and B), suggesting regulation at a posttranscriptional level. Anti-ERp46 antibodies detected a single protein band with an apparent molecular mass of 51,000 Da in the brain, spleen, lung, liver, kidney, and testis (Fig. 5B). ERp46 was immunologically undetectable in both cardiac and skeletal muscle. Most importantly, both ERp19 and ERp46 proteins were highly enriched in ER fractions as compared with total liver homogenate (Fig. 5C), suggesting that they are ER-associated proteins.
      To establish the precise intracellular localizations of ERp19 and ERp46, we labeled McA-RH7777 hepatocytes either with anti-ERp19 antibodies or with anti-ERp46 antibodies and visualized them by confocal microscopy (Fig. 6). In each case, the hepatocytes were double-labeled using anti-PDI monoclonal antibodies as a control. Fig. 6 shows that the distributions of ERp19 and ERp46 label were limited to the intricate tubular system of the ER including the cisternae of the nuclear envelope. Both proteins were detected in the ER throughout the entire network (Fig. 6, A and D). The ER marker PDI showed a similar distribution (Fig. 6, B and E) as illustrated in the merged images (Fig. 6, C and F).
      Figure thumbnail gr6
      Fig. 6Intracellular localization of ERp19 and ERp46. McA-RH7777 hepatocytes were double-labeled with affinity-purified anti-ERp19 or anti-ERp46 antibodies and anti-PDI monoclonal antibodies and subjected to confocal microscopy. ERp19 and ERp46 fluorescence is shown in red (A and D, respectively), and PDI fluorescence is shown in green (B and E). C and F show merged images of ERp19/PDI and ERp46/PDI, respectively. Bar, 10 μm.

      Functional Analysis of ERp19 and ERp46—

      To determine whether ERp19 and ERp46 function as PDI-like molecules, we performed a complementation study in the yeast Saccharomyces cerevisiae. PDI deficiency is lethal in this organism (
      • LaMantia M.
      • Miura T.
      • Tachikawa H.
      • Kaplan H.A.
      • Lennarz W.J.
      • Mizunaga T.
      ) making it an excellent model in which to determine whether ERp46 or ERp19 can complement for the missing PDI function. cDNAs encoding ERp19, ERp46, and yeast PDI were cloned into a yeast expression vector under control of the GAL1 promoter, a strong galactose-inducible and glucose-repressible promoter. The recombinant plasmids were introduced into a diploid yeast strain heterozygous for the deletion of the PDI1 gene, PDI1/pdi1Δ::KanMX (
      • Winzeler E.A.
      • Shoemaker D.D.
      • Astromoff A.
      • Liang H.
      • Anderson K.
      • Andre B.
      • Bangham R.
      • Benito R.
      • Boeke J.D.
      • Bussey H.
      • Chu A.M.
      • Connelly C.
      • Davis K.
      • Dietrich F.
      • Dow S.W.
      • El Bakkoury M.
      • Foury F.
      • Friend S.H.
      • Gentalen E.
      • Giaever G.
      • Hegemann J.H.
      • Jones T.
      • Laub M.
      • Liao H.
      • Davis R.W.
      ). After sporulation and germination on medium containing galactose or glucose, the genotypes and growth properties of the haploid progeny were analyzed. Geneticin-resistant haploid progeny will not be viable unless PDI function is provided by the introduced plasmids.
      If the diploid parental strain had not been transformed prior to sporulation, no geneticin-resistant colonies would have been found, and the genotypes of the viable haploid offspring would have revealed the presence of the endogenous PDI1 gene (Fig. 7, B and C). As expected, the introduction of a multicopy plasmid carrying the yeast PDI gene rescued the lethality of the PDI1 gene disruption under galactose induction but not under glucose repression (Fig. 7, B and C). We also transformed the yeast strain with recombinant ERp46 and ERp19 cDNAs. Western blot analysis was used to confirm the expression of ERp19 and ERp46 proteins in the diploid transformants (Fig. 7A). Fig. 7 indicates that ERp46 complemented for the PDI1 gene disruption, as did the recombinant yeast PDI. In contrast, ERp19 did not complement for PDI activity as all isolated colonies in this experiment were sensitive to G418 and retained the endogenous PDI1 gene (data not shown). These experiments demonstrate that ERp46, which contains 3 thioredoxin repeats, exhibits a PDI-like activity in yeast. In contrast, ERp19, which contains only 1 thioredoxin repeat, does not.
      Figure thumbnail gr7
      Fig. 7Yeast complementation assay.A, a diploid yeast strain heterozygous for the deletion of the PDI1 gene was transformed with yeast expression vectors encoding ERp19 or ERp46. Fifty μg of protein extract were separated by SDS-PAGE, transferred to nitrocellulose, and probed with affinity-purified anti-ERp19 antibodies and anti-ERp46 antibodies. - and + designate samples from non-transformed or transformed strains, respectively. B, Southern blot analysis of HincII-digested genomic DNA of various yeast strains was carried out using a probe corresponding to the 3′-noncoding region of the PDI1 gene. The endogenous PDI1 allele is represented by a 2.5-kb band, and the gene replacement allele is represented by a 4.2-kb band. C, growth of various yeast strains on YPGal (induction) and YPD (repression) solid media. The strains used in B and C are PDI1 haploid. 1, pdi1Δ haploid complemented with PDI1; 2, pdi1Δ haploid complemented with ERp46.

      DISCUSSION

      In this investigation, we have used a proteomic approach to study the protein composition of the ER and to identify new ER proteins with a special emphasis on luminal proteins. We identified 141 proteins of which 6 have not been described previously. Two newly discovered ER luminal proteins, designated ERp19 and ERp46, are members of the PDI family of proteins. They contain one and three thioredoxin motifs, respectively. Functional analysis of ERp19 and ERp46, using yeast complementation, showed that ERp46 but not ERp19 complemented for PDI function in vivo.
      The organization of proteins in the ER is of great significance because this organelle serves as an “entry point” for proteins that are destined for secretion or localization within compartments of the secretory pathway. Many severe disorders such as cystic fibrosis, α1-antitrypsin deficiency, altered metabolism, and developmental disorders have been attributed to malfunctions of certain ER components that affect protein folding and transport or ER-dependent Ca2+ homeostasis (
      • Mesaeli N.
      • Nakamura K.
      • Zvaritch E.
      • Dickie P.
      • Dziak E.
      • Krause K.-H.
      • Opas M.
      • MacLennan D.H.
      • Michalak M.
      ,
      • Rutishauser J.
      • Spiess M.
      ,
      • MacLennan D.H.
      ,
      • Li J.
      • Puceat M.
      • Perez-Terzic C.
      • Mery A.
      • Nakamura K.
      • Michalak M.
      • Krause K.-H.
      • Jaconi M.E.
      ). Here, we have completed the first systematic identification of proteins associated with the ER in the liver of healthy mice. Fig. 8 shows that the luminal constituents of the ER that were identified in this study can be grouped into three categories: ER-resident chaperones, proteins involved in detoxification and hormone signaling, and secretory proteins that were “caught in transit.” The most abundant membrane-anchored proteins are cytochrome b5 and members of the cytochrome P450 family, enzymes which are involved in drug and lipid metabolism. Various signaling proteins, such as calmodulin, G protein β-subunit, protein kinase Cβ and its regulator 14-3-3ε (
      • McConnell J.E.
      • Armstrong J.F.
      • Hodges P.E.
      • Bard J.B.
      ), and p22, a protein required for vesicular trafficking (
      • Barroso M.R.
      • Bernd K.K.
      • DeWitt N.D.
      • Chang A.
      • Mills K.
      • Sztul E.S.
      ), were found to be associated with the ER membrane. Some members and regulators of the protein translation machinery were also identified, including PAI-RBPI, a recently discovered mRNA-binding protein (
      • Heaton J.H.
      • Dlakic W.M.
      • Dlakic M.
      • Gelehrter T.D.
      ). The appearance of certain peroxisomal and mitochondrial proteins in ER fractions (Table I) reflects the close association of these organelles with the ER (
      • Bascom R.A.
      • Chan H.
      • Rachubinski R.A.
      ,
      • Rizzuto R.
      • Pinton P.
      • Carrington W.
      • Fay F.S.
      • Fogarty K.E.
      • Lifshitz L.M.
      • Tuft R.A.
      • Pozzan T.
      ,
      • Newmeyer D.D.
      • Ferguson-Miller S.
      ).
      Figure thumbnail gr8
      Fig. 8Schematic representation and putative localization of selected proteins identified in the ER proteome. Luminal constituents of the ER are grouped according to their functions. ER-resident chaperones are depicted in green, proteins involved in detoxification and hormonal regulation are shown in blue, and secretory proteins are yellow. Novel proteins are shown in red letters and grouped into their respective categories.
      Molecular chaperones reside in the lumen of the ER and assist in protein folding processes. These proteins have a variety of specific functions. For example, BiP and GRP94 bind to temporarily exposed stretches of hydrophobic amino acids, thus preventing protein aggregation, the lectin-like chaperones calnexin and calreticulin interact with intermediates of glycoproteins, and peptidylprolyl isomerases accelerate protein folding by interconversion of cis and trans proline imidic peptide bonds (
      • Ellgaard L.
      • Helenius A.
      ). Thiol disulphide oxidoreductases constitute another important group of ER chaperones. They catalyze the formation, breakage, and rearrangement of disulphide bonds (
      • Sevier C.S.
      • Kaiser C.A.
      ). The catalytic activity of these proteins depends on a pair of active cysteines arranged in a W-C-X-X-C motif. This motif is also found in the small redox protein thioredoxin. Mammalian members of this family include PDI, P5 (CaBP1), ERp72, ERp57, PDIp, PDIr (
      • Ferrari D.M.
      • Söling H.-D.
      ), and the recently discovered ERp44 (
      • Anelli T.
      • Alessio M.
      • Mezghrani A.
      • Simmen T.
      • Talamo F.
      • Bachi A.
      • Sitia R.
      ), ERp29 (
      • Sargsyan E.
      • Baryshev M.
      • Szekely L.
      • Sharipo A.
      • Mkrtchian S.
      ), and JPDI (
      • Hosoda A.
      • Kimata Y.
      • Tsuru A.
      • Kohno K.
      ). Not surprisingly, tissues that secrete large amounts of disulphide-bonded proteins contain the largest amounts of thiol disulphide oxidoreductases, with PDI typically being the most abundantly expressed family member (
      • Noiva R.
      ).
      Our systematic analysis of the ER luminal proteome has enabled the discovery of two new members of the PDI family of proteins, which have been designated ERp19 and ERp46. Although PDI apparently “handles” the majority of incompletely folded protein substrates, other family members appear to interact with specific protein partners or alternatively, to regulate the redox status of the more prominent members of the family by transferring oxidizing equivalents, as has been demonstrated for ERO1p in yeast (
      • Frand A.R.
      • Kaiser C.A.
      ,
      • Frand A.R.
      • Kaiser C.A.
      ). Interestingly, a previous study has shown that ERp57, which contains two thioredoxin motifs, does not complement for deficiency of endogenous PDI in yeast (
      • Gunther R.
      • Srinivasan M.
      • Haugejorden S.
      • Green M.
      • Ehbrecht I.-M.
      • Kuntzel H.
      ). This study suggests that ERp57 possesses a specific substrate-binding site, and it is now known that ERp57 binds to calreticulin and calnexin and participates in folding of N-glycosylated proteins (
      • Oliver J.D.
      • van der Wal F.J.
      • Bulleid N.J.
      • High S.
      ,
      • Zapun A.
      • Darby N.J.
      • Tessier D.C.
      • Michalak M.
      • Bergeron J.J.M.
      • Thomas D.Y.
      ,
      • Oliver J.D.
      • Roderick H.L.
      • Llewellyn D.H.
      • High S.
      ). In contrast, we found that ERp46, which contains three thioredoxin motifs, is able to complement for PDI function in yeast. This suggests that ERp46 may exhibit a more universal protein disulphide isomerase activity than ERp57. Importantly, our experiments also indicate that levels of ERp46 may be subject to posttranscriptional regulation in mice, which is suggestive of a regulatory rather than general function of this protein.
      ERp19 contains only one active site with the amino acid sequence W-C-G-A-C. Although this conforms to the thioredoxin consensus sequence (W-C-X-X-C), the motifs actually found in thioredoxin (W-C-G-P-C) and PDI (W-C-G-H-C) differ slightly. Site-specific mutagenesis studies have indicated that mutations of the internal proline and histidine residues in the active sites of thioredoxin and PDI can alter their activities (
      • Hawkins H.C.
      • Blackburn E.C.
      • Freedman R.B.
      ,
      • Lu X.
      • Gilbert H.F.
      • Harper J.W.
      ). It is therefore possible that the alanine in the active site of ERp19 contributes to the specific function of this protein. Furthermore, the single thioredoxin motif of ERp19 is probably not sufficient for full PDI-like activity, as both thioredoxin motifs in PDI are required for its isomerase function. The inactivation of one thioredoxin motif in PDI reduces its activity by about 50%, whereas disruption of both motifs leaves it dysfunctional (
      • Vuori K.
      • Pihlajaniemi T.
      • Myllyla R.
      • Kivirikko K.I.
      ). Given these observations, it is unlikely that ERp19 acts as a “typical” isomerase, and its function remains to be determined. Interestingly, PDI-like molecules that contain a single thioredoxin active site but which are capable of rearranging disulphide bonds and displaying transglutaminase activity have recently been discovered in the lower eukaryote Giardia lamblia (
      • Knodler L.A.
      • Noiva R.
      • Mehta K.
      • McCaffery J.M.
      • Aley S.B.
      • Svard S.G.
      • Nystul T.G.
      • Reiner D.S.
      • Silberman J.D.
      • Gillin F.D.
      ). These findings may necessitate some revision of existing models of PDI function in the future.
      In summary, our proteomic analysis of the ER has resulted in the discovery of two new PDI-like proteins, making this family of proteins the largest class of the chaperones that reside in the ER. It is currently unknown why there should be such an abundance of PDI-like proteins. Disulphide bonds do play a significant part in the determination of protein structure and function. They can also enhance protein stability, protect proteins from damage, and increase their half-lives. The formation of disulphide bonds is reversible and might be a key component in the regulation of protein stability. Given these many roles for disulphide bonds, there is quite likely a requirement for diverse PDI-like proteins with diverse substrate specificities to assure correct disulphide bond formation and maintenance in the ER lumen and the extracellular space.

      Acknowledgments

      We thank M. Dabrowska for superb technical assistance and L. Guo for helpful discussions. We thank R. Wozniak (University of Alberta) and T. Hobman (University of Alberta) for anti-Gp210 and antimannosidase antibodies, respectively.

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