Mutant Fibrinogen Cleared from the Endoplasmic Reticulum via Endoplasmic Reticulum-Associated Protein Degradation and Autophagy
2006; Elsevier BV; Volume: 168; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2006.051097
ISSN1525-2191
AutoresKristina B. Kruse, Amy Dear, Erin Kaltenbrun, Brandan E. Crum, Peter M. George, Stephen O. Brennan, Ardythe A. McCracken,
Tópico(s)Autophagy in Disease and Therapy
ResumoThe endoplasmic reticulum (ER) quality control processes recognize and remove aberrant proteins from the secretory pathway. Several variants of the plasma protein fibrinogen are recognized as aberrant and degraded by ER-associated protein degradation (ERAD), thus leading to hypofibrinogenemia. A subset of patients with hypofibrinogenemia exhibit hepatic ER accumulation of the variant fibrinogens and develop liver cirrhosis. One such variant named Aguadilla has a substitution of Arg375 to Trp in the γ-chain. To understand the cellular mechanisms behind clearance of the aberrant Aguadilla γ-chain, we expressed the mutant γD domain in yeast and found that it was cleared from the ER via ERAD. In addition, we discovered that when ERAD was saturated, aggregated Aguadilla γD accumulated within the ER while a soluble form of the polypeptide transited the secretory pathway to the trans-Golgi network where it was targeted to the vacuole for degradation. Examination of Aguadilla γD in an autophagy-deficient yeast strain showed stabilization of the aggregated ER form, indicating that these aggregates are normally cleared from the ER via the autophagic pathway. These findings have clinical relevance in the understanding of and treatment for ER storage diseases. The endoplasmic reticulum (ER) quality control processes recognize and remove aberrant proteins from the secretory pathway. Several variants of the plasma protein fibrinogen are recognized as aberrant and degraded by ER-associated protein degradation (ERAD), thus leading to hypofibrinogenemia. A subset of patients with hypofibrinogenemia exhibit hepatic ER accumulation of the variant fibrinogens and develop liver cirrhosis. One such variant named Aguadilla has a substitution of Arg375 to Trp in the γ-chain. To understand the cellular mechanisms behind clearance of the aberrant Aguadilla γ-chain, we expressed the mutant γD domain in yeast and found that it was cleared from the ER via ERAD. In addition, we discovered that when ERAD was saturated, aggregated Aguadilla γD accumulated within the ER while a soluble form of the polypeptide transited the secretory pathway to the trans-Golgi network where it was targeted to the vacuole for degradation. Examination of Aguadilla γD in an autophagy-deficient yeast strain showed stabilization of the aggregated ER form, indicating that these aggregates are normally cleared from the ER via the autophagic pathway. These findings have clinical relevance in the understanding of and treatment for ER storage diseases. Fibrinogen, a large plasma protein synthesized in hepatocytes, plays a critical role in blood coagulation. Mature circulating fibrinogen is a symmetric dimeric molecule in which each half consists of three polypeptide chains: Aα, Bβ, and γ. The two halves of the molecule associate into a trinodal D-E-D structure with the N termini of all six chains in the central E domain, the C termini of Bβ and γ forming the two globular D domains, and the C termini of the Aα-chains forming a less compact structure associated with the E domain.1Mosesson MW Fibrinogen and fibrin structure and function.J Thromb Haemost. 2005; 3: 1894-1904Crossref PubMed Scopus (1325) Google Scholar, 2Weisel JW Fibrinogen and fibrin.Adv Protein Chem. 2005; 70: 247-299Crossref PubMed Scopus (678) Google Scholar Assembly of all six chains into the fibrinogen molecule occurs within the hepatocyte endoplasmic reticulum.3Roy S Yu S Banerjee D Overton O Mukhopadhyay G Oddoux C Grieninger G Redman CM Assembly and secretion of fibrinogen: degradation of individual chains.J Biol Chem. 1992; 267: 23151-23158Abstract Full Text PDF PubMed Google Scholar, 4Huang S Cao Z Chung DW Davie EW The role of betagamma and alphagamma complexes in the assembly of human fibrinogen.J Biol Chem. 1996; 271: 27942-27947Crossref PubMed Scopus (56) Google Scholar, 5Redman CM Xia H Fibrinogen biosynthesis: assembly, intracellular degradation, and association with lipid synthesis and secretion.Ann NY Acad Sci. 2001; 936: 480-495Crossref PubMed Scopus (116) Google Scholar Individual unassembled fibrinogen chains are retained within the endoplasmic reticulum (ER) and ultimately degraded in a proteasome-dependent manner, classifying them as substrates of ER-associated protein degradation (ERAD).5Redman CM Xia H Fibrinogen biosynthesis: assembly, intracellular degradation, and association with lipid synthesis and secretion.Ann NY Acad Sci. 2001; 936: 480-495Crossref PubMed Scopus (116) Google Scholar, 6Xia H Redman CM The degradation of nascent fibrinogen chains is mediated by the ubiquitin proteasome pathway.Biochem Biophys Res Commun. 1999; 261: 590-597Crossref PubMed Scopus (19) Google Scholar The identification of the genetic defects associated with low levels of circulating fibrinogen (hypofibrinogenemia) has shown that mutations within any of the three chains can lead to misfolding of that chain, recognition of the aberrant polypeptide by ER quality control, and subsequent degradation.7Brennan SO Fellowes AP George PM Molecular mechanisms of hypo- and afibrinogenemia.Ann NY Acad Sci. 2001; 936: 91-100Crossref PubMed Scopus (40) Google Scholar However, studies of individuals carrying various mutant forms of fibrinogen chains have shown that a subset of those individuals with hypofibrinogenemia also exhibit hepatic ER accumulation of fibrinogen that appears to lead to liver disease.8Callea F Brisigotti M Fabbretti G Bonino F Desmet VJ Hepatic endoplasmic reticulum storage diseases.Liver. 1992; 12: 357-362Crossref PubMed Scopus (89) Google Scholar, 9Brennan SO Wyatt J Medicina D Callea F George PM Fibrinogen brescia: hepatic endoplasmic reticulum storage and hypofibrinogenemia because of a gamma284 Gly→Arg mutation.Am J Pathol. 2000; 157: 189-196Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 10Brennan SO Maghzal G Shneider BL Gordon R Magid MS George PM Novel fibrinogen gamma375 Arg→Trp mutation (fibrinogen aguadilla) causes hepatic endoplasmic reticulum storage and hypofibrinogenemia.Hepatology. 2002; 36: 652-658Crossref PubMed Scopus (97) Google Scholar For example, we previously described one family harboring a novel γ-chain mutation, Arg375Trp, termed fibrinogen Aguadilla, in which all carriers present with hypofibrinogenemia, but only a subset have liver inclusions.10Brennan SO Maghzal G Shneider BL Gordon R Magid MS George PM Novel fibrinogen gamma375 Arg→Trp mutation (fibrinogen aguadilla) causes hepatic endoplasmic reticulum storage and hypofibrinogenemia.Hepatology. 2002; 36: 652-658Crossref PubMed Scopus (97) Google Scholar Biopsies from diseased livers showed aggregates of fibrinogen within the ER lumen of hepatocytes.10Brennan SO Maghzal G Shneider BL Gordon R Magid MS George PM Novel fibrinogen gamma375 Arg→Trp mutation (fibrinogen aguadilla) causes hepatic endoplasmic reticulum storage and hypofibrinogenemia.Hepatology. 2002; 36: 652-658Crossref PubMed Scopus (97) Google Scholar This disease pathology, ie, both the loss-of-function hypofibrinogenemia and the gain-of-function liver disease, is similar to α-1-antitrypsin deficiency, another ER storage disease. Individuals who are homozygous for the Z variant of the serum protein α-1-antitrypsin (α-1-protease inhibitor, A1Pi) exhibit low levels of A1Pi and the resulting loss-of-function disease pathology of early onset emphysema.11Crystal RG Alpha 1-antitrypsin deficiency, emphysema, and liver disease: genetic basis and strategies for therapy.J Clin Invest. 1990; 85: 1343-1352Crossref PubMed Scopus (428) Google Scholar In addition, 12 to 15% of A1PiZ homozygous juveniles also display the gain-of-function disease pathology of liver cirrhosis,12Sveger T The natural history of liver disease in alpha 1-antitrypsin deficient children.Acta Paediatr Scand. 1988; 77: 847-851Crossref PubMed Scopus (214) Google Scholar, 13Wu Y Whitman I Molmenti E Moore K Hippenmeyer P Perlmutter DH A lag in intracellular degradation of mutant alpha-1-antitrypsin correlates with the liver disease phentotype in homozygous PiZZ individuals.Proc Natl Acad Sci USA. 1994; 91: 9014-9018Crossref PubMed Scopus (236) Google Scholar, 14Lomas DA Mahadeva R α1-Antitrypsin polymerization and the serpinopathies: pathobiology and the prospects for therapy.J Clin Investig. 2002; 110: 1585-1590Crossref PubMed Scopus (243) Google Scholar, 15Perlmutter DH Alpha1-antitrypsin deficiency: liver disease associated with retention of a mutant secretory glycoprotein in the endoplasmic reticulum.in: Bross P Gregersen N Protein Misfolding and Disease, Principles and Protocols. Humana Press, Totowa, NJ2003: 39-56Crossref Google Scholar and studies of adults indicate a risk of cirrhosis in 30 to 50% of homozygous individuals.16Eriksson S A 30-year perspective on alpha 1-antitrypsin deficiency.Chest. 1996; 110: 237S-242SCrossref PubMed Scopus (89) Google Scholar It has been previously demonstrated, in both mammalian and yeast cell systems, that the intracellular fate of A1PiZ is ERAD.17Teckman JH Burrows J Hidvegi T Schmidt B Hale PD Perlmutter DH The proteasome participates in degradation of mutant alpha 1-antitrypsin Z in the endoplasmic reticulum of hepatoma-derived hepatocytes.J Biol Chem. 2001; 276: 44865-44872Crossref PubMed Scopus (115) Google Scholar, 18Werner ED Brodsky JL McCracken AA Proteasome-dependent endoplasmic reticulum-associated protein degradation: an unconventional route to a familiar fate.Proc Natl Acad Sci USA. 1999; 93: 13797-13801Crossref Scopus (397) Google Scholar However, when this pathway is saturated such that it is insufficient to rid the ER of misfolded A1PiZ, aggregates accumulate within the ER.19Kruse KB Brodsky JL McCracken AA Characterization of an ERAD gene as VPS30/ATG6 reveals two alternative and functionally distinct protein quality control pathways: one for soluble A1PiZ and another for aggregates of A1PiZ.Mol Biol Cell. 2005; 17: 203-212Crossref PubMed Scopus (167) Google Scholar Interestingly, the clearance of these A1PiZ aggregates from the ER is dependent on the autophagic pathway to the vacuole.19Kruse KB Brodsky JL McCracken AA Characterization of an ERAD gene as VPS30/ATG6 reveals two alternative and functionally distinct protein quality control pathways: one for soluble A1PiZ and another for aggregates of A1PiZ.Mol Biol Cell. 2005; 17: 203-212Crossref PubMed Scopus (167) Google Scholar These findings correlate with the observation that there is an increased number of autophagosomes in hepatocytes of A1PiZ homozygous individuals with liver disease.20Teckman JH Perlmutter DH Retention of mutant alpha(1)-antitrypsin Z in endoplasmic reticulum is associated with an autophagic response.Am J Physiol Gastrointest Liver Physiol. 2000; 279: G961-G974PubMed Google Scholar Together, these data suggest that liver disease associated with α-1-antitrypsin deficiency may be due to different inherited efficiencies in ER quality control or possibly to a second mutation within the ERAD or autophagy pathways. Because of the similarity between the pathology of α-1-antitrypsin deficiency and hypofibrinogenemia-associated liver disease, we reasoned that the fibrinogen Aguadilla Argγ375Trp variant would have an intracellular fate similar to that of A1PiZ. Thus, to investigate the mechanism involved in the fibrinogen Aguadilla-related ER storage disease, we decided to express the fibrinogen γD domain with the Arg375Trp mutation in our yeast model system. The rationale for the use of the γD domain comes from previous studies showing that when expressed in Pichia pastoris, the normal γD domain is correctly folded into a stable configuration with both its calcium binding site and polymerization pocket intact.21Yee VC Pratt KP Cote HC Trong IL Chung DW Davie EW Stenkamp RE Teller DC Crystal structure of a 30 kDa C-terminal fragment from the gamma chain of human fibrinogen.Structure. 1997; 15: 125-138Abstract Full Text Full Text PDF Scopus (226) Google Scholar Should the Aguadilla mutation affect folding of the fibrinogen γD domain, we anticipate that the quality control mechanisms that contribute to clearance of the aberrant polypeptide may be identified in our yeast system. Knowledge of these quality control mechanisms could begin to answer questions regarding the molecular mechanisms driving the pathology of hypofibrinogenemia and liver disease. Electrocompetent Escherichia coli strain HB101 (F−, mcrB, mrr, hsdS20(rB−, mB−), recA13, supE44, ara14, galK2, lacY1, proA2, rpsL20(Smr), xyl5, λ−, leu, and mtl) was purchased from Invitrogen Corp. (Carlsbad, CA). The P. pastoris strain used in this study was GS115/his4 (Invitrogen). The Saccharomyces cerevisiae strains used were wild type22Winzeler EA Shoemaker DD Astromoff A Liang H Anderson K Andre B Bangham R Benito R Boeke JD Bussey H Chu AM Connelly C Davis K Dietrich F Dow SW El Bakkoury M Foury F Friend SH Gentalen E Giaever G Hegemann JH Jones T Laub M Liao H Liebundguth N Lockhart DJ Lucau-Danila A Lussier M M'Rabet N Menard P Mittmann M Pai C Rebischung C Revuelta JL Riles L Roberts CJ Ross-MacDonald P Scherens B Snyder M Sookhai-Mahadeo S Storms RK Veronneau S Voet M Volckaert G Ward TR Wysocki R Yen GS Yu K Zimmermann K Philippsen P Johnston M Davis RW Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.Science. 1999; 285: 901-906Crossref PubMed Scopus (3258) Google Scholar: BY4742/MATα, his3Δ1, leu2Δ0, lysΔ0, and ura3Δ0; BY4742: pep4Δ22/MATα, his3Δ1, leu2Δ0, lysΔ0, ura3Δ0, and YPL154c::KANMX (Invitrogen); BY4742: atg14Δ19/MATα, his3Δ1, leu2Δ0, lysΔ0, ura3Δ0, and YBR128c::KANMX4; the proteasome mutant23Heinemeyer W Gruhler A Mohrle V Mahe Y Wolf DH PRE2, highly homologous to the human major histocompatibility complex-linked RING10 gene, codes for a yeast proteasome subunit necessary for chrymotryptic activity and degradation of ubiquitinated proteins.J Biol Chem. 1993; 268: 5115-5120Abstract Full Text PDF PubMed Google Scholar: pre1-1, pre2-2/MATα, leu2-3, 112, his3-11, 15, ura3(Δ5), GAL+, can(s), pre1-1, and pre2-2; and the wild-type isogenic parent23Heinemeyer W Gruhler A Mohrle V Mahe Y Wolf DH PRE2, highly homologous to the human major histocompatibility complex-linked RING10 gene, codes for a yeast proteasome subunit necessary for chrymotryptic activity and degradation of ubiquitinated proteins.J Biol Chem. 1993; 268: 5115-5120Abstract Full Text PDF PubMed Google Scholar: WCG4/MATα, leu2-3, 112, his3-11, 15, ura3(Δ5), and GAL+ (kindly provided by Dieter Wolf, University of Stuttgart, Germany). P. pastoris yeast cells were cultured in synthetic media containing 1.34% yeast nitrogen base without amino acids and ammonium sulfate, 2% peptone, 1% yeast extract, 100 mmol/L potassium phosphate, pH 6.0, 400 ng/ml biotin, and 1% glycerol as a carbon source or with 1% methanol to induce expression of γD fibrinogen. S. cerevisiae yeast cells were cultured in synthetic media containing 0.67% yeast nitrogen base with amino acids, 0.5% casamino acids, and 2% dextrose as a carbon source or with 2% galactose to induce expression of γD fibrinogen. Antibodies used included rabbit anti-human fibrinogen (DakoCytomation California Inc., Carpinteria, CA) and horseradish peroxidase-conjugated goat anti-rabbit (U.S. Biochemical Co., Cleveland, OH), monoclonal mouse anti-yeast phosphoglycerate kinase (Invitrogen), rabbit anti-Kar2p24Brodsky JL Schekman R A Sec63p-BiP complex from yeast is required for protein translocation in a reconstituted proteoliposome.J Cell Biol. 1993; 123: 1355-1363Crossref PubMed Scopus (232) Google Scholar (generously provided by Jeffrey Brodsky, University of Pittsburgh, Pittsburgh, PA), and sheep anti-mouse IgG horseradish peroxidase-conjugated antibody (GE Health Care Bio-Sciences, Little Chalfont, UK). The integrative expression vector, pPIC9 (Invitrogen), carrying the coding regions spanning amino acid residues 143 to 411 of the human fibrinogen γ-gene25Lishko VK Yakubenko VP Hertzberg KM Grieninger G Ugarova TP The alternatively spliced alpha(E)C domain of human fibrinogen-420 is a novel ligand for leukocyte integrins alpha(M)beta(2) and alpha(X)beta(2).Blood. 2001; 98: 2448-2455Crossref PubMed Scopus (43) Google Scholar (hereafter referred to as the fibrinogen γD domain) was kindly supplied by Dr. Tatiana Ugarova (Cleveland Clinic Foundation, Cleveland, OH). The fragment encoding the fibrinogen γD domain was cloned in frame with the yeast α-factor secretion signal and propeptide and driven by an alcohol oxidase 1 methanol-inducible promoter. The fibrinogen Aguadilla mutation, Argγ375Trp,10Brennan SO Maghzal G Shneider BL Gordon R Magid MS George PM Novel fibrinogen gamma375 Arg→Trp mutation (fibrinogen aguadilla) causes hepatic endoplasmic reticulum storage and hypofibrinogenemia.Hepatology. 2002; 36: 652-658Crossref PubMed Scopus (97) Google Scholar was inserted using mutagenic primers (available on request) with a QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA), and the mutation was verified using automated sequencing. Using polymerase chain reaction and primers that introduced unique restriction sites, the α-factor pre-pro and γ-sequences were subcloned into an S. cerevisiae expression vector with a regulatable promoter26Mumberg D Müller R Funk M Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression.Nucleic Acids Res. 1994; 22: 5767-5768Crossref PubMed Scopus (810) Google Scholar: either the inducible GAL1 promoter p426Gal1 (2μ, Ampr, and URA3; American Type Culture Collection, Manassas, VA) or the repressible MET25 promoter p426MET25 (2μ, Ampr, and URA3; American Type Culture Collection). Correct cloning was verified by restriction digest and automated sequencing. The Cell-Porator E. coli Pulser (Invitrogen) was used to electroporate competent E. coli. Plasmids were isolated from bacterial transformants using the Quantum Prep Plasmid Miniprep kit (Bio-Rad, Hercules, CA). P. pastoris transformations were performed using the spheroplasting protocol within the Pichia Expression kit M instruction manual (Invitrogen), and transformants were selected in medium lacking histidine. Southern blot was used to choose transformants with only a single copy27Clare JJ Rayment FB Ballantine SP Sreekrishna K Romanos MA High-level expression of tetanus toxin fragment-C in Pichia-pastoris strains containing multiple tandem integrations of the gene.Bio-Technology. 1991; 9: 455-460Crossref PubMed Scopus (374) Google Scholar of the integrative pPIC9 expression vector. S. cerevisiae transformations were performed using a standard lithium acetate procedure,28Adams A Gottschling DE Kaiser CA Stearns T Techniques and protocols: high-efficiency transformation of yeast.in: Dickerson MM Methods in Yeast Genetics. Cold Spring Harbor Press, New York1997: 99-102Google Scholar and transformants were isolated after growth in selective medium containing 2% dextrose. The colony-blot immunoassays for both S. cerevisiae and P. pastoris were a modification of a previously described procedure.19Kruse KB Brodsky JL McCracken AA Characterization of an ERAD gene as VPS30/ATG6 reveals two alternative and functionally distinct protein quality control pathways: one for soluble A1PiZ and another for aggregates of A1PiZ.Mol Biol Cell. 2005; 17: 203-212Crossref PubMed Scopus (167) Google Scholar In brief, exponentially growing yeast were resuspended to a final optical density (OD; at 600 nm) of 0.001OD/μl to control for cell number. A 3-μl aliquot of these yeast were spotted onto a nitrocellulose disk overlaid on medium containing 1% methanol to induce expression in P. pastoris of the γD off the AOX1 promoter, medium containing 2% galactose to induce expression in S. cerevisiae of the γD off the GAL1 promoter, or synthetic medium lacking methionine to induce expression in S. cerevisiae of the γD off the MET25 promoter. After incubation at 30°C for 18 hours, cells were lysed with solution 1 (0.2 mol/L NaOH, 0.1% sodium dodecyl sulfate (SDS), and 0.05% 2-mercaptoethanol) for 1 hour at room temperature, and blots were then assayed as previously described.29McCracken AA Karpichev IV Ernaga JE Werner ED Dillin AG Courchesne WE Yeast mutants deficient in ER-associated degradation of the Z variant of alpha-1-protease inhibitor.Genetics. 1996; 144: 1355-1362Crossref PubMed Google Scholar Immunoreactive proteins were detected by developing the blots with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and Super RX X-ray film (Fuji, Tokyo, Japan). The density of immunoreactive protein at each colony spot was quantified using Molecular Analyst (Bio-Rad). For pulse-chase protein radiolabeling to assay for γD degradation in P. pastoris, cultures were grown at 30°C in medium containing 1% methanol to induce expression for 24 hours before analysis. Using a protocol based on those previously described,30McCracken AA Kruse KB Selective protein degradation in the yeast exocytic pathway.Mol Biol Cell. 1993; 4: 729-736Crossref PubMed Scopus (35) Google Scholar, 31Brodsky JL Lawrence JG Caplan AJ Mutations in the cytosolic DnaJ homologue, YDJ1, delay and compromise the efficient translation of heterologous proteins in yeast.Biochemistry. 1998; 37: 18045-18055Crossref PubMed Scopus (27) Google Scholar cells were resuspended at 4 OD/ml in medium lacking histidine, cysteine, and methionine. They were incubated at 30°C for 45 minutes, then pulsed with 25 μCi/OD EasyTag Express Protein Labeling Mix (PerkinElmer Inc., Boston, MA) for 10 minutes, and chased with 1 mg/ml cold cysteine and methionine. Aliquots were removed at the desired time points. Cell lysates were prepared and immunoprecipitated with rabbit polyclonal antibody to human fibrinogen. The purified proteins were separated by 10% reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by autoradiography, and signal intensities were quantified using Molecular Analyst (Bio-Rad). The cycloheximide chase analyses of S. cerevisiae were a modification of a previously described procedure.19Kruse KB Brodsky JL McCracken AA Characterization of an ERAD gene as VPS30/ATG6 reveals two alternative and functionally distinct protein quality control pathways: one for soluble A1PiZ and another for aggregates of A1PiZ.Mol Biol Cell. 2005; 17: 203-212Crossref PubMed Scopus (167) Google Scholar In brief, overnight cultures were incubated in medium containing 2% galactose for 4 to 8 hours at 30°C. To control for cell number, cultures were then resuspended to a final OD600 of 2 OD/ml in medium containing 2% dextrose plus 200 μg/ml cycloheximide. They were incubated at 30°C with shaking, 500-μl aliquots were collected, and the cells were harvested by centrifugation at the indicated time points. At each time point, the medium was reserved and represented the secreted material. The cell pellets were resuspended in 50 μl of lysis buffer (160 mmol/L Tris, pH 6.8, 4% SDS. 0.2% bromophenol blue, 200 mmol/L dithiothreitol [DTT], and 20% glycerol) and incubated at 95°C for 2 minutes, and 0.5-mm acid-washed glass beads were added. The cells were disrupted via four sequential 60-second bursts on a Vortex mixer at the highest setting followed by cooling on ice for 60 seconds. Finally, an additional 50-μl aliquot of lysis buffer and the reserved media were added, resulting in 0.0017 OD/μl final concentration. Then, 20-μl aliquots of lysate/media were combined with 10 μl of sample buffer (0.125 mol/L Tris, pH 6.8, 4% SDS, 0.004% bromophenol blue, 10% 2-mercaptoethanol, and 20% glycerol), heated to 95°C for 10 minutes, and resolved by 10% reducing SDS-PAGE. The γD domain was detected by immunoblot analysis, and results were quantified as described above. P. pastoris cultures were grown in medium containing 1% methanol for 72 hours at 30°C. Cell pellets were lysed by vortexing with acid-washed glass beads as described above but in 50 mmol/L Tris-HCl, pH 7.4, and 1 mmol/L EDTA, washed twice with 1% Triton X-100 and then three times with water. The remaining pellet was solubilized in 8 mol/L urea, 100 mmol/L Tris-HCl, pH 8.0, and 15 mmol/L DTT and reduced for 16 hours at room temperature. The high and low molecular weight γD species were separated by reverse phase high performance liquid chromatography (HPLC) on a 5-μm Jupiter C4 column (Phenomenex, Torrence, CA) with a 0.05% trifluoroacetic acid/acetonitrile gradient similar to that used for reduced human fibrinogen chains.32Brennan SO Electrospray ionisation analysis of human fibrinogen.Thromb Haemost. 1997; 78: 1055-1058PubMed Google Scholar Peaks were collected and assayed at the Protein Microchemistry Facility at the University of Otago, New Zealand for N-terminal protein sequencing using established methods.33Hubbard MJ Mchugh NJ Carne DL Isolation of ERp29, a novel endoplasmic reticulum protein, from rat enamel cells: evidence for a unique role in secretory-protein synthesis.Eur J Biochem. 2000; 267: 1945-1956Crossref PubMed Scopus (60) Google Scholar A 20-μl sample of each peak crest was also analyzed by electrospray ionization mass spectrometry (ESI MS) on a VG Platform II quadrupole analyzer (Micromass, Manchester, UK) as previously described.32Brennan SO Electrospray ionisation analysis of human fibrinogen.Thromb Haemost. 1997; 78: 1055-1058PubMed Google Scholar For peptide mapping, HPLC peaks were dried, redissolved in 50 μl of 50 mmol/L ammonium carbonate and digested with 2 μg of trypsin for 16 hours at 37°C. After being dried under vacuum with P2O5 and redissolved in 50 μl of 50% acetonitrile and 0.1% formic acid, 20 μl of each digest was analyzed by ESI MS. S. cerevisiae cultures were grown in medium containing 2% galactose for 40 hours at 30°C to induce expression of γD. Yeast were pelleted, and the medium was reserved and concentrated fourfold for use as the extracellular, secreted fraction. Spheroplasts were formed by incubating cells with 10 mg/ml lyticase in 1.2 mol/L sorbitol and 0.1 mol/L K2HPO4, pH 7.2, and spheroplasts and the periplasm fraction were separated by centrifugation through a cushion of 0.8 mol/L sucrose, 1.5% Ficoll, and 20 mmol/L HEPES, pH 7.4, at 4000 × g for 10 minutes at 4°C. After the spheroplasts were washed with 1.2 mol/L sorbitol and 0.1 mol/L K2HPO4, pH 7.2, they were resuspended in ice-cold Spheroplast lysis buffer (0.1 mol/L sorbitol, 50 mmol/L KOAc, 2 mmol/L EDTA, 20 mmol/L HEPES, pH 7.4, 1 mmol/L DTT, and 1 mmol/L phenylmethylsulfonyl fluoride) and lysed by 10 strokes with a dounce homogenizer on ice. Resulting cell lysates were layered over a cushion of 1 mol/L sucrose, 50 mmol/L potassium acetate, 20 mmol/L HEPES, pH 7.4, and 1 mmol/L DTT and subjected to 6500 × g centrifugation for 10 minutes at 4°C. The top fraction, above the interface, was collected and subjected to further centrifugation at 22,000 × g for 10 minutes at 4°C. The resulting pellet was the microsome fraction, and the supernatant/cytosol was further cleared of small vesicles by centrifugation at 100,000 × g for 1 hour at 4°C. Protease protection assays were performed on the microsome fraction by exposing washed microsomes either to 0.4 mg/ml trypsin in buffer 88 (20 mmol/L HEPES, pH 6.8, 150 mmol/L KOAc, 250 mmol/L sorbitol, and 5 mmol/L MgOAc) or to 0.4 mg/ml trypsin and 2% Triton X-100 in buffer 88 for 30 minutes on ice. Specific proteins were then visualized by immunoblot analysis. Microsomes were isolated as described above from S. cerevisiae producing γD during 40 hours of induction. The microsomes were lysed in buffer 88 that had been supplemented with 0.5% Triton X-100 and complete protease inhibitor cocktail (Sigma, St. Louis, MO) by incubation on ice for 2 hours, with brief vortex mixing every 30 minutes. The total protein content of the lysates was determined using Coomassie Plus Protein Assay (Pierce). An aliquot of 1 mg of total protein from each lysate was loaded onto a 5 to 60% sucrose gradient34Kabani M Kelley SS Morrow MW Montgomery DL Sivendran R Rose MD Gierasch LM Brodsky JL Dependence of endoplasmic reticulum-associated degradation on the peptide binding domain and concentration of BiP.Mol Biol Cell. 2003; 14: 3437-3448Crossref PubMed Scopus (88) Google Scholar, 35Schmidt BZ Perlmutter DH Grp78, Grp94 and Grp170 interact with alpha-1-antitrypsin mutants that are retained in the endoplasmic reticulum.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G444-G455Crossref PubMed Scopus (90) Google Scholar and centrifuged in a Beckman (Beckman Coulter, Inc., Fullerton, CA) SW50 at 145,000 × g for 20 hours at 4°C. Fractions of 250 μl were collected, and proteins were concentrated by precipitation in 10% trichloroacetic acid. Specific proteins were visualized by immunoblot analysis. Microsomes were prepared as described above and lysed on ice for 30 minutes in denaturing buffer (50 mmol/L sodium acetate, pH 5.2, 0.5% 2-mercaptoethanol, and 0.5% SDS). Samples were heated to 95°C for 3 minutes and then centrifuged 14,000 × g for 5 minutes at 4°C to remove membranes. An equal volume of reaction buffer (50 mmol/L sodium acetate, pH 5.2, 0.5 mmol/L phenylmethylsulfonyl fluoride, and 0.5% NP-40) was added to the supernatant, and the sample was split in two. One sample received 0.05 units of endoglycosidase H (Roche Diagnostics, Indianapolis, IN), both samples were incubated at 37°C for 1 hour, and proteins were visualized by immunoblot analysis. The ∼30-kd C-terminal domain of the γ-chain of human fibrinogen (Val143 through Val411) cloned in-frame behind the yeast α-factor pre-pro sequence was used for these studies because this γD domain had previously been shown to correctly fold and function.21Yee VC Pratt KP Cote HC Trong IL Chung DW Davie EW Stenkamp RE Teller DC Crystal structure of a 30 kDa C-terminal fragment from the gamma chain of human fibrinogen.Structure. 1997; 15: 125-138Abstract Full Text Full Text PDF Scopus (226) Google Scholar,
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