Degradation of Mutated Bovine Pancreatic Trypsin Inhibitor in the Yeast Vacuole Suggests Post-endoplasmic Reticulum Protein Quality Control

• DOI: 10.1074/jbc.m309673200
• Publication Date: 2004-04-02T22:18:24Z
• Authors: Christina M. Coug...; Jennifer L. Walker; Jared C. Cochran; K. Dane Wittrup; Jeffrey L. Brodsky
• Keywords: Secretory protein; Endoplasmic-reticulum-associated protein degradation; Proteolysis

The rate-limiting step in protein secretion is folding, which occurs the endoplasmic reticulum (ER) lumen, and almost all secreted proteins contain disulfide bonds that form ER stabilize native state. Secreted unable to fold may aggregate or they be subject ER-associated degradation. To examine fate of aberrant forms a well characterized, disulfide-bonded protein, we expressed bovine pancreatic trypsin inhibitor yeast. Bovine single domain, 58-amino acid polypeptide containing three bonds, yeast cells secrete wild type protein. In contrast, Y35L mutant, folds rapidly but unstable, remains soluble not secreted. Surprisingly, proteolysis unaffected mutations genes encoding factors required for degradation stable if artificially retained ER. Rather, diverted from Golgi vacuole degraded. Because only mutant quantitatively proteolyzed these data suggest post-ER quality control check-point diverts unstable Missense small in-frame deletions insertions might affect function compromise ability into its conformation. On occasion, non-native exhibit gain-of-function conformations are cytotoxic. fact, defects folding leading toxic have been implicated pathogenesis several diseases, including antitrypsin deficiency, Alzheimer's disease, prion-related diseases (1Ma J. Woollmann R. Lindquist S. Science. 2002; 298: 1781-1785Crossref PubMed Scopus (430) Google Scholar, 2Ma 1785-1788Crossref (268) 3Kopito R.R. Ron D. Nat. Cell Biol. 2000; 11: E207-E209Crossref (321) 4McCracken A.A. Brodsky J.L. BioEssays. 2003; 25: 868-877Crossref (194) 5Coughlan C.M. Bross P. Gregersen N. Methods Molecular Biology, Protein Misfolding Disease: Principles Protocols. 232. Humana Press Inc., Totowa, NJ2003: 77-90Google Scholar). However, misfolded do necessarily harm cell as instead For example, 1The abbreviations used are: ER, reticulum; ERAD, reticulum-associated degradation; UPR, unfolded response; BPTI, inhibitor; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MES, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline; BSA, serum albumin. -associated (ERAD) mechanism detected retrotranslocated cytoplasm, degraded by proteasome (4McCracken 6Hampton R.Y. Curr. Opin. 14: 476-482Crossref (401) 7Fewell S.W. Travers K.J. Weissman J.S. Annu. Rev. Genet. 2001; 35: 149-191Crossref (264) 8Tsai B. Ye Y. Rapoport T.A. Mol. Cell. 3: 246-255Crossref (551) 9Ellgaard L. Helenius A. 13: 431-437Crossref (333) lumenal chaperones, such BiP calnexin facilitate ERAD soluble, polypeptides (10Skowronek M.H. Hendershot L.M. Haas I.G. Proc. Natl. Acad. Sci. U. 1998; 95: 1574-1578Crossref (84) 11Hendershot Wei Gaut Melnick Aviel Argon 1996; 93: 5269-5274Crossref (131) 12McCracken 132: 291-298Crossref (346) 13Helenius Aebi M. 291: 2364-2369Crossref (1986) 14Plemper R.K. Bohmler Bordallo Sommer T. Wolf D.H. Nature. 1997; 388: 891-895Crossref (472) 15Brodsky Werner E.D. Dubas M.E. Goeckeler Kruse K.B. McCracken Chem. 1999; 274: 3453-3460Abstract Full Text PDF (232) 16Kabani Kelley S.S. Morrow M.W. Montgomery D.L. Sivendran Rose M.D. Gierasch 3437-3448Crossref (88) It thought recently characterized lectins (17Molinari Calanca V. Galli C. Lucca Paganetti 299: 1397-1400Crossref (389) 18Oda Hosokawa Wada I. Nagata K. 1394-1397Crossref (391) 19Sifers R.N. 1330-1331Crossref (32) Scholar), select then target them retrotranslocation. accumulation can also induce response (UPR), an intracellular signaling pathway leads expansion induces expression chaperones other biogenesis transport (20Ng D.T. Spear Walter 150: 77-88Crossref (275) 21Patil 349-355Crossref (677) Consequently, like reduces stress when concentration aberrant, rises. lumen efficiencies bond formation linked (22Bardwell J.C. Microbiol. 1994; 199-205Crossref (198) 23Braakman EMBO 1992; 1717-1722Crossref (331) 24Dunn Luz J.M. Natalia Gamble J.A. Freedman R.B. Tuite M.F. Biochem. Soc. Trans. 1995; 23: 78SCrossref (9) Loss affects unpredictable ways, ranging aggregation retention, degradation, dramatic acceleration (25Gillece Lennarz W.J. de la Cruz F.J. Romisch 147: 1443-1456Crossref (154) 26Hammond 7: 523-529Crossref (587) 27Kowalski Parekh Wittrup K.D. Biochemistry. 37: 1264-1273Crossref (67) 28Kowalski Mso 273: 19453-19458Abstract (99) 29Taniyama Yamamoto Nakao Kikuchi Ikehara Biophys. Res. Commun. 1988; 152: 962-967Crossref (54) 30Omura F. Otsu Eur. 205: 551-559Crossref (12) 31Suganuma Matzuk M.M. Boime 1989; 264: 19302-19307Abstract 32Bedows E. Huth J.R. Suganuma Bartels C.F. Ruddon R.W. 1993; 268: 11655-11662Abstract As result, one cannot yet predict how mutation will alter begin address this question, (BPTI) BPTI structure contains formed C14–C38, C5–C55, C30–C51, distinct intermediates N* (C5–C55, C14–C38), N′ (C30–C51, NSH,SH C5–C55). Two most highly populated N′, conversion represents during vitro (33Weissman Kim P.S. Struct. 2: 1123-1130Crossref (80) 34Creighton T.E. Goldenberg D.P. 1984; 179: 497-526Crossref (261) Tyr at position 35 buried immobile (35Wlodawer Nachman Gilliland G.L. Gallagher W. Woodward 1987; 198: 469-480Crossref 36Wagner G. Bruhwiler Wuthrich 196: 227-231Crossref (56) Although final conformation radically altered, some normally contacts become solvent-exposed relative (37Naderi H.M. Thomason J.F. Borgias B.A. Anderson James T.L. Kuntz I.D. Nall B.T. Dill K.A. Conformations Forces Folding. American Association Advancement Science, Washington, C.1991: 86-114Google 38van Mierlo C.P. Darby N.J. Neuhaus Creighton 1991; 222: 353-371Crossref 39Hurle M.R. Eads C.D. Pearlman D.A. Seibel Kosen P.A. Kollman 1: 91-106Crossref (30) 40Zhang J.X. 32: 14075-14081Crossref (39) however, rate overall enhanced (40Zhang C5A intermediate states form, quite because C5–C51 hydrophobic core Not surprisingly, significant conformational deviations between noted (38van fates secretory were examined previously, although neither was secreted, accumulated (27Kowalski Based on (41Hagihara 99: 6619-6624Crossref 42Kjeldsen Ludvigsen Diers Balschmidt Sorensen A.R. Kaarsholm N.C. 277: 18245-18248Abstract (61) Scholar) thermodynamic stability appears determine competence. study, set out investigate whether derivatives handled and/or aggregated. We found escaped vacuole. These "second line" pathway. Yeast Methods—The strains study listed Table Plasmids constitutive type, Y35L, described previously (43Parekh Forrester Expression Purif. 6: 537-545Crossref (72) DNA inserts corresponding promoter elements YE112-GPD-BPTI subcloned PRS426 (44Christianson T.W. Sikorski R.S. Dante Shero J.H. Hieter Gene (Amst.). 110: 119-122Crossref (1434) When resulting plasmids introduced cells, glyceraldehyde-3-phosphate dehydrogenase promoter. N terminus synthetic prepro sequence derived prepro-α factor lacks oligosaccharyl consensus sites ("G" BPTI). proregion cleaved Kex2p protease, added glutamate-alanine C-terminal existing cleavage site improves processing. Similar levels evident bona fide fused glycosylation (data shown). individual vectors transformed desired using lithium acetate (45Ito H. Fukuda Murata Kimura Bacteriol. 1983; 153: 163-168Crossref transformants selected growth complete medium lacking uracil glucose 2%. Cells expressing grown mid-log phase glycerol stocks made stored -70 °C. All experiments performed freshly broken-out onto solid selective medium.Table IStrains studyStrainGenotypeSourceRSY 801Mata, ura 3-52 leu2-3,112 ade 2-101Ref. ScholarMS 1111Mata leu2-3, 112 2-101 kar2-1Ref. ScholarW303-16Mata ade2-1, can1-100, ura3-1, leu2-3,112, trp1-1, his3-11,15Ref. 47Parlati Dominguez Bergeron J.J. Thomas D.Y. 270: 244-253Abstract (144) ScholarYFP73Mata his3-11,15 cnel::LEU2Ref. ScholarWCG4Mata ura3 (D5)Ref. 48Heinemeyer Gruhler Mohrle Mahe 5115-5120Abstract ScholarWCG4/11-21Mata pre1-1 pre2-1Ref. ScholarRSY620Mata ade2-1 trp1-1 ura3-1 pep4::TRP1R. SchekmanRSY367Mata his3-11,15R. SchekmanBJ5146Mata can1-100 ade2 kex2::HIS3-5E. JonesFY834Mata his3 leu2 lys2 trp1C. BarloweCBY557Mata trp1 sec12-4C. BarloweBJ5558Mata ura3-52 his1 leu2-D1E. JonesBJ5557Mata leu2-D1 pep3::LEU2E. JonesBJ4785Mata pep1-35E. JonesBJ4763Mata trp1E. Jones Open table new tab Cycloheximide Chase Assay Monitor Maturation Degradation—Yeast liquid 26 °C collected resuspended OD (optical density 600 nm) 4/ml same medium. incubated 37 10 min before cycloheximide 200 μg/ml. At indicated times, 1.6 values harvested. supernatant (∼500 μl) centrifugation step, pool concentrated Centricon 3 microconcentrators (Amicon) volume 15 μl assayed below. pellet, represented material, 100 Stop buffer (20 mm Tris-HCl, pH 8, 50 ammonium acetate, 500 iodoacetamide, 20 sodium azide, 0.45 cycloheximide, 5 EDTA) Extract (10 25 EDTA, 10% trichloroacetic acid, 1 phenylmethylsulfonyl fluoride, μg/ml pepstatin A, leupeptin) added. A total 1.5 g zirconia/silica beads (0.5 diameter, Biospec Products, Bartlesville, OK) 20% disrupted with 60-s burst mini-Bead Beater (Biospec followed cooling ice 60 s. 500-μl washes Wash 2 acid) combined original lysate. Each wash included s bursts. This mixture centrifuged (16,000 × 4 °C), aspirated, pellet 40 Resuspension (100 Tris, 11, dithiothreitol, 3% SDS). samples heated 72 160 Dilution (60 7.4, 190 NaCl, 6 1.25% Triton X-100) μlofa0.5 m stock iodoacetamide solution mixed vigorously, Omnisorb (Calbiochem) added, 30 min. 16,000 supernatant, clarified, cellular lysate, gel samples. NuPage 4× LDS sample (Invitrogen) extracellular material (see above), dilution 1× supplemented 0.06 dithiothreitol Proteins resolved 4–12% bis-Tris gradient gels SDS-MES running (Invitrogen). transferred PROTRAN nitrocellulose membranes (pore size = 0.2 μm; Bioscience) room temperature TBS-T 7.5, 0.5 0.05% Tween 20) 5% nonfat dry milk azide mm. filters polyclonal anti-BPTI antiserum (8371T) diluted 1:1,000 TBS-T, milk, 16 h Filters washed times secondary antibody (horseradish peroxidase-conjugated donkey anti-rabbit IgG; Amersham Biosciences) 1:5,000 h. treated Super Signal West Pico chemiluminescence reagent (Pierce Chemical), exposed x-ray film, where quantified films linear range Image Gauge software (Fuji Film). Sucrose Density Gradient Analysis—A ml exponentially growing (OD 0.5–0.8) pelleted, washed, STED10 (10% (w/v) sucrose, Tris-Cl, 7.6, protease inhibitors (1 leupeptin, fluoride) split two microcentrifuge tubes, subsequently placed ice. Zirconia/silica meniscus lysed (as above) An additional 400 tube 0.3 extract layered 30–70% sucrose gradient, layering 2.2 STED decreasing amounts (from bottom): STED70 (70% buffer), STED60, STED50, STED40, STED30. gradients 18 100,000 Beckman SW41 rotor. Fractions 800 top (Fraction 1) bottom 13) remaining STED30 aggregated 14). extraction each fractions resultant precipitated resuspension (3% SDS, dithiothreitol). Samples prepared separated above. Indirect Immunofluoresence—The distribution visualized protocol supplied laboratory Snyder. 2J. Vogel Snyder, Yale University, personal communication. °C, 1/10th 37% formaldehyde gentle shaking. harvested, twice Solution (1.2 sorbitol, KPO4, 7.0) μl/ml mg/ml Z100T zymolase (made double-distilled water (ddH2O)). 30–40 Polylysine slides incubation; brief, polylysine (Sigma) ddH2O 75-mm Teflon-coated immunofluorescence slide (Polysciences Inc.), temperature, air dried had incubating, harvested 1–2 allowed adhere Unbound aspirated wells rinsed gently 0.1% BSA Nonidet P-40, BSA. primary (BPTI, 1:50; Vma2p, 1:25, BSA) overnight time PBS Alexa antibodies (goat anti-rabbit, 488; goat anti-mouse, 594; Probes), 1:500 Finally, once 4,6-Diamidino-2-phenylindole dihydrochloride mounted mount glycerol/PBS 2% n-propyl gallate). sealed coverslip clear nail polish, dry, immunofluoresence either assessed immediately -20 dark months. fluorescence Olympus BX60 microscope fitted Hamamamatsu digital camera images analyzed QED Imaging (Pittsburgh, PA). Concanavalin Immunoprecipitation—A 300 concanavalin (500 1% X-100, NaN3) (added 1:1 buffer) chase above). those bound buffer, bead:sample ratio. min, Western blotting above protocol. Degradation Is Independent BiP, Calnexin, Proteasome Function—Previous studies intracellularly (28Kowalski via pathway, BPTI-expressing kar2-1 allele gene (KAR2 (46Rose Misra J.P. 57: 1211-1221Abstract (527) Scholar)), strain (cne1Δ (47Parlati pre1-1pre2-1 ∼95% activity abrogated (48Heinemeyer others substrates defective ER-derived microsomes cytosol mutants (12McCracken 49Werner 13797-13801Crossref (390) 50Zhang Nijbroek Sullivan M.L. Watkins S.C. Michaelis 12: 1303-1314Crossref (227) Isogenic parallel. control, URA-marked 2-μ vector, insert. under "Experimental Procedures" Bis-Tris polyacrylamide (4–12% acrylamide) resolve ER-resident, immature proregion, mature results Kex2 protease-mediated removal Golgi. proregion-containing species immunoblot analysis extracts antiserum. signal absent immunoblotted vector probed shown Fig. 1, time-dependent decrease amount ("pro") ER-resident proteins, regardless examined. level decreased remained constant, suggesting "chased" species, case (Ref. Scholar; see below), variable population stable. differentiate possibilities, radiolabeled [35S]methionine/cysteine, fluid/media isolated Procedures"), immunoprecipitation radiography Goo