Article Figures & Data
Figures
- FIG. 1.
During folding, Tg forms intermolecular disulfide-bonded complexes. (A) PC-Cl3 cells were labeled as outlined in Materials and Methods and chased for the indicated times. Cell lysates and culture media were immunoprecipitated with anti-Tg antibodies. Immunoprecipitates were resolved by SDS-PAGE under nonreducing and reducing conditions. The positions of Tg secreted to the medium (M), 330-kDa Tg, Golgi Tg (G), and intermolecular disulfide-linked adducts (A, B, and C) are indicated. The gels were intentionally overexposed in order to optimally visualize the presence of bands A, B, and C under nonreducing conditions and their absence under reducing conditions. For densitometry, shorter exposures were used. (B) Kinetics of band A, B, and C disappearance as well as Tg coimmunoprecipitation with CRT and CNX. The means of three separate experiments have been plotted. max, maximum. (C) Kinetics of recovery for each of the various forms of Tg. The term “330 kDa” refers to Tg recovered intracellularly at the 330-kDa position as indicated in panel A. The mean values from three separate experiments have been plotted.
- FIG. 2.
Intermolecular disulfide-linked Tg adducts are endo H sensitive and cosediment with monomeric Tg. (A) PC-Cl3 cells were labeled as outlined in Materials and Methods and chased for the indicated times. Cell lysates were immunoprecipitated with anti-Tg antibodies and digested or mock digested with endo H. (B) PC-Cl3 cells were labeled as outlined in Materials and Methods and chased for 90 min. Cell lysates were subjected to sucrose gradient centrifugation, and selected fractions from the dimer (DIM) and monomer (MON) peaks were immunoprecipitated with anti-Tg antibodies and mock-digested or digested with endo H. For both panels, immunoprecipitates were resolved by SDS-PAGE under nonreducing (lanes 1 to 4) and reducing (lanes 5 to 8) conditions. The positions of Golgi Tg (G) and intermolecular disulfide-bonded Tg complexes (A, B, and C) are indicated.
- FIG. 3.
The A, B, and C species represent intermediates of the productive folding pathway of Tg. (Top) PC-Cl3 cells were labeled as outlined in Materials and Methods and chased for up to 4 h in the absence or presence (during labeling and chase) of 10 μM MG132 or 100 μM kifunensine (KIF). Cell lysates (lanes 1 to 9) and culture media (lanes 10 to 18) were immunoprecipitated with anti-Tg antibodies. Immunoprecipitates were resolved by nonreducing SDS-PAGE. The intermolecular disulfide-bonded Tg complexes (A, B, and C) are clearly visible at time zero but subsequently disappear. (Bottom) Time course of the disappearance of the species A, B, and C in the absence or presence of 10 μM MG132. Each value shown is the mean from three experiments.
- FIG. 4.
High-molecular-mass Tg complexes include noncovalently associated chaperones as well as adducts involving ERp57-Tg and PDI-Tg mixed disulfides. (A) PC-Cl3 cells were metabolically labeled continuously for 48 h. Immunoprecipitated Tg was analyzed by nonreducing SDS-PAGE. The radioactive sample was exposed to film and realigned with the original sample, and the disulfide-linked Tg adducts A, B, and C were excised, eluted, and reanalyzed by SDS-PAGE under reducing conditions. Associated bands liberated upon reduction of Tg adducts are highlighted with arrowheads; the positions of molecular mass markers (in kiladaltons) are shown at right. (B) CRT and CNX associate noncovalently with bands A, B, and C. PC-Cl3 cells were labeled as outlined in Materials and Methods and chased for 15 min. Cell lysates were immunoprecipitated (IPPT) under nondenaturing or denaturing conditions with nonimmune serum (NI) or anti-Tg, anti-CRT, or anti-CNX antibody (Ab) and resolved by nonreducing SDS-PAGE. The positions of the 330-kDa Tg, intermolecular disulfide-linked Tg adducts (A, B, and C), CRT, and CNX are indicated. (C) Immunoprecipitation of ERp57 and PDI recovers Tg adducts representing mainly the B species. PC-Cl3 cells were labeled and chased as described for panel B. Cell lysates were immunoprecipitated under nondenaturing and denaturing conditions with nonimmune serum or anti-Tg, anti-ERp57, anti-EsR (a negative control), or anti-PDI antibody and resolved by nonreducing SDS-PAGE. The positions of the 330-kDa Tg, intermolecular disulfide-linked Tg adducts (A, B, and C), ERp57, and PDI are indicated. (D) Direct proof that ERp57 and PDI are engaged in mixed disulfides with Tg. PC-Cl3 cells were labeled and chased as described for panel B. Cell lysates were first immunoprecipitated with anti-Tg antibodies and then boiled in SDS and finally reimmunoprecipitated with nonimmune serum or anti-Tg, anti-ERp57, anti-EsR (a negative control), or anti-PDI antibody and resolved by nonreducing and reducing SDS-PAGE. The positions of the 330-kDa Tg and intermolecular disulfide-linked Tg adducts (A, B, and C) are indicated. Retardation of a low-molecular-mass protein (near bottom of the gel) is indicative of reduction of proteins in the gel.
- FIG. 5.
ERp57 operates early in the folding pathway of Tg and in a CRT/CNX-dependent manner, while PDI action is more extended in time and is not dependent on CRT/CNX. PC-Cl3 cells were pretreated or not with CST and then labeled as outlined in Materials and Methods and chased for 10 and 60 min in the presence or absence of CST. Cell lysates were immunoprecipitated under nondenaturing conditions with nonimmune serum (NI) or anti-Tg, anti-ERp57, anti-EsR (a negative control), or anti-PDI antibody (Ab) and resolved by nonreducing SDS-PAGE. The positions of the 330-kDa Tg, intermolecular disulfide-bonded complexes (A, B, and C), ERp57, and PDI are indicated.
- FIG. 6.
Translation of truncated Tg bearing only the first N-glycosylation site, and association of the truncated protein with ERp57 and PDI. A cDNA encoding a truncated rat Tg lacking a stop codon was prepared as outlined in Materials and Methods. Using a coupled in vitro transcription/translation system, the truncated protein was synthesized in the presence of 35S-amino acids and pancreatic microsomes. (A) The translation period was 25 min and was concluded without the use of puromycin. The labeled products were analyzed by SDS-PAGE after mock-digestion or digestion with endo H. (B) The in vitro translation either omitted (control [Con]) or included (+Cst) 1 mM CST, and all translations were terminated by incubation with 2 mM puromycin to release nascent chains. The translation mix was divided into four equal portions that were either precipitated with 10% trichloroacetic acid (TCA) or immunoprecipitated with one of the antibodies shown. Molecular mass markers (in kilodaltons) are shown at sides of gels.
- FIG. 7.
CST inhibits but does not block Tg secretion and adduct maturation. PC-Cl3 cells were preincubated with 1 mM CST for 1 h and then labeled and chased in the presence of CST. Cell lysates and culture media were immunoprecipitated with anti-Tg antibodies. (Top) Immunoprecipitates were resolved by SDS-PAGE. The positions of Tg secreted to the medium (M), 330-kDa Tg, Golgi Tg (G), and intermolecular disulfide-bonded Tg complexes (A, B, and C) are indicated. The gels were intentionally overexposed in order to improve the visualization of bands A, B, and C under nonreducing conditions and to document their absence under reducing conditions. (Bottom) The disappearance of bands A, B, and C (triangles, X's, and filled squares, all overlapping) and 330-kDa Tg (circles) and the appearance of Tg in the medium (open squares) have been quantitated from the means of three experiments under control and CST pretreatment conditions, as shown.
- FIG. 8.
CST inhibits efficient folding of Tg. (A) CST effect on oligosaccharide processing. PC-Cl3 cells were pretreated with CST, labeled, chased, and analyzed by sucrose gradient centrifugation exactly as described for Fig. 2B. Tg secreted to the media was also collected. All samples were immunoprecipitated for Tg and either mock-digested (−) or digested (+) with endo H. The samples were finally resolved by SDS-PAGE under reducing conditions. mon, monomer; dim, dimer. (B) CST inhibits oxidative folding. PC-Cl3 cells, pretreated (+) or not (−) with CST, were labeled as outlined in Materials and Methods and chased for 90 min. Cell lysates and culture media were immunoprecipitated with anti-Tg antibodies and mock digested (−) or digested (+) with PNGase F. Immunoprecipitates were analyzed by nonreducing and reducing SDS-PAGE as indicated and were run for an extra period of time for improved resolution. (C) CST inhibits dimerization. PC-Cl3 cells, pretreated or not with CST, were labeled as outlined in Materials and Methods and chased for 120 min. Equal fractions of cell lysates and medium were mixed to obtain total Tg for analysis by sucrose gradient centrifugation. The gradient fractions were immunoprecipitated with anti-Tg antibodies. The bottom panel provides arbitrary densitometry units for quantitation of the relative abundance of dimers versus monomers shown above. Squares, CST pretreatment; diamonds, control. (D) CST activates ERAD. PC-Cl3 cells pretreated for 1 h with no drugs (control), 1 mM CST, 100 μM kifunensine (kif), or 1 mM CST plus 100 μM kifunensine (cst + kif), were pulse-labeled and chased for the times indicated in the presence of these drugs. Newly synthesized Tg was recovered from cell lysates and culture media by immunoprecipitation and reducing SDS-PAGE, followed by quantitative densitometry of autoradiographs. Total Tg recovery (the sum of intracellular and secreted Tg) was quantified at each chase time, normalized to that recovered at the 15-min chase time.
- FIG. 9.
CST compromises quality control, causing increased secretion of Tg monomers. (A) CST causes secretion of Tg monomers. PC-Cl3 cells, pretreated or not with CST, were labeled as outlined in Materials and Methods and chased for 120 min. Chase media were analyzed by sucrose sedimentation velocity centrifugation, and the gradients were collected beginning from the bottom of the tube. Each gradient fraction was immunoprecipitated with anti-Tg antibodies. The Tg dimer peak is to the left (lower fraction numbers), and the monomer peak is to the right (higher fraction numbers). (B) Tg monomers secreted following CST pretreatment are at least partially misfolded. PC-Cl3 cells, pretreated (+) or not (−) with CST, were labeled as outlined in Materials and Methods and chased for 90 min. Dimers and monomers from cell lysates and culture media were separated by sucrose velocity gradient centrifugation. Selected fractions from dimer and monomer peaks were immunoprecipitated with anti-Tg antibodies and mock digested (−) or digested (+) with PNGase F. Immunoprecipitates were analyzed by nonreducing SDS-PAGE run for an extra period of time for improved resolution. Brackets highlight a subtle distinction in the mobilities of partially oxidized Tg monomers and better-oxidized intracellular Tg dimers. (C) CST causes a dramatic increase of Tg binding to BiP and GRP94. PC-Cl3 cells, pretreated or not with CST, were labeled as outlined in Materials and Methods and chased for various times. Intact cells were cross-linked with DSP as outlined in Materials and Methods and lysed. Cell lysates were subjected to immunoprecipitation with anti-BiP and anti-GRP94 antibodies. Immunoprecipitates were resolved by reducing SDS-PAGE. The positions of Tg, BiP, and GRP94 are indicated. (D) Delayed maturation of disulfide-linked Tg adducts in cells treated with thapsigargin. PC-Cl3 cells were labeled as outlined in Materials and Methods and chased for the times indicated. Newly synthesized Tg was recovered by immunoprecipitation and SDS-PAGE under nonreducing conditions. The positions of the 330-kDa Tg and intermolecular disulfide-bonded complexes (A, B, and C) are indicated. The gels were intentionally overexposed for optimal detection of disulfide-linked Tg adducts.
- FIG. 10.
CST treatment causes the upregulation of ER chaperone levels indicative of UPR activation. FRTL-5 cells growing in complete media were treated with 1 mM CST for the number of days indicated. Cells were lysed, and 25 μg of each lysate was analyzed by SDS-PAGE and Western blotting with each of the antibodies shown. The positions of selected molecular mass markers are indicated (in kilodaltons). CRT migrated as an ∼58-kDa band. This experiment was repeated four times, revealing an approximately threefold increase in GRP94, BiP, and CRT after both 1 and 2 days of treatment.
- FIG. 11.
Prevention of escape from the CRT/CNX cycle is also deleterious for labeled Tg progression through the secretory pathway. PC-Cl3 cells were not pretreated but labeled as outlined in Materials and Methods and then mock incubated (−) or incubated (+) in the presence of CST only during the chase periods shown. Cell lysates and culture media were immunoprecipitated with anti-CNX, anti-BiP, and anti-Tg antibodies (Ab) and resolved by nonreducing SDS-PAGE. Note that CST treatment postpulse caused intracellular Tg retention in association with CNX.
