FIGURE 1.

PDI-KD enhances proinsulin export from ER. A, at 72 h after transfection with either scrambled or ERO1β (panels at left) or PDI siRNA duplexes (panels at right) (40 nm), INS1 cells were pulse-labeled for 15 min with [³⁵S]Cys/Met and lysed in SDS buffer with 10 mm NEM. Lysates were immunoprecipitated in duplicate with anti-insulin and analyzed by Tris-Tricine-urea-SDS-PAGE under nonreduced or reduced (100 mm DTT) conditions. These data were replicated in three experiments. Recovery of the native proinsulin disulfide isomer (N) divided by total proinsulin recovery under reduced conditions (P) was quantified in these experiments with mean ± S.D. shown below; the asterisks signify p < 0.05. B, INS1 cells treated with siRNA duplexes (100 nm) designed for knockdown of the luciferase mRNA (a negative control, C1), GFP mRNA (negative control, C2), or PDI-KD were lysed at 72 h after siRNA transfection. Upper eight set of bands: Western blotting for the antigens indicated, normalized to protein (10 or 20 μg, depending on antigen), with γ-tubulin serving as a loading control. BiP and GRP94 were identified with anti-KDEL antibodies. From four such experiments, no significant differences were observed in PDI-KD cells except for the PDI protein itself. Bottom row: XBP1 mRNA was measured by RT-PCR using primers that amplify both unspliced (u) and spliced (s) forms (19). As a positive control for ER stress, cells were treated with thapsigargin (downward arrowhead in first lane: 1 μm for 3 h). From three such experiments, no significant differences were observed in PDI-KD cells. C, at 72 h after transfection with either scrambled (Con) or PDI siRNA (KD) duplexes (100 nm), INS1 cells were pulse-labeled for 20 min with [³⁵S]Cys/Met and either lysed immediately or chased for 1 h, and both media (M) and cell lysates (C) were collected. Samples were immunoprecipitated with anti-insulin and analyzed by reducing Tris-Tricine-urea-SDS-PAGE. Note that two-thirds of the radioactivity of insulin is lost upon reducing SDS-PAGE because the A-chain is not recovered. Upper panel: Western blotting of PDI in the cells used in this experiment. D, at 48 h after transfection of INS1 cells with either scrambled (light bars) or PDI siRNA duplexes (dark bars), the media were changed, and fresh growth media (11 mm glucose) were applied and collected after an additional 24 h. At that time, the cells were lysed, and total insulin (i.e. insulin plus proinsulin) in both cell lysates and media was measured by RIA. The data are the mean of three experiments with six independent replicates; control values were set to 100%. When comparing control versus PDI-KD, either cells or media, the asterisks signify p < 0.05. E, Min6 cells were transiently transfected to express empty vector (Control), FLAG-PDI-AGHA,AGHA′ (PDI-DEAD), FLAG-WT-PDI, or FLAG-PDI-CGHA (PDI-TRAP). At 72 h after transfection, the Min6 cells were pulse-labeled for 20 min with [³⁵S]Cys/Met and lysed in SDS-containing lysis buffer with 15 mm NEM. The lysates were immunoprecipitated with either anti-FLAG or anti-insulin as indicated. The anti-FLAG immunoprecipitates (IP) were analyzed on the same SDS gel with phosphorimaging (i.e. identical exposure) under nonreducing or reducing conditions as indicated; the anti-insulin immunoprecipitates come from an independent gel analysis of the same samples. The positions of FLAG-PDI, PDI-proinsulin adduct, and newly synthesized proinsulin (under reducing conditions) are indicated.