The Role of TRAPγ/SSR3 in Preproinsulin Translocation Into the Endoplasmic Reticulum

Abstract

In the endoplasmic reticulum (ER), the translocation-associated protein complex (TRAP), also called signal sequence receptor (SSR), includes four integral membrane proteins TRAPα/SSR1, TRAPβ/SSR2, and TRAPδ/SSR4 with the bulk of their extramembranous portions primarily in the ER lumen, whereas the extramembranous portion of TRAPγ/SSR3 is primarily cytosolic. Individually diminished expression of either TRAPα/SSR1, TRAPβ/SSR2, or TRAPδ/SSR4 mRNA is known in each case to lower TRAPα/SSR1 protein levels, leading to impaired proinsulin biosynthesis, whereas forced expression of TRAPα/SSR1 at least partially suppresses the proinsulin biosynthetic defect. Here, we report that diminished TRAPγ/SSR3 expression in pancreatic β-cells leaves TRAPα/SSR1 levels unaffected while nevertheless inhibiting cotranslational and posttranslational translocation of preproinsulin into the ER. Crucially, acute exposure to high glucose leads to a rapid upregulation of both TRAPγ/SSR3 and proinsulin protein without change in the respective mRNA levels, as observed in cultured rodent β-cell lines and confirmed in human islets. Strikingly, pancreatic β-cells with suppressed TRAPγ/SSR3 expression are blocked in glucose-dependent upregulation of proinsulin (or insulin) biosynthesis. Most remarkably, overexpression of TRAPγ/SSR3 in control β-cells raises proinsulin levels, even without boosting extracellular glucose. The data suggest the possibility that TRAPγ/SSR3 may fulfill a rate-limiting function in preproinsulin translocation across the ER membrane for proinsulin biosynthesis.

Figure 1

The TRAPγ/SSR3 subunit of the TRAP/SSR complex contributes to the efficiency of recombinant proinsulin biosynthesis. A: Schematic of the TRAP/SSR complex highlights the predominant cytosolic exposure of the TRAPγ/SSR3 subunit, including a hypothetical contact with a signal peptide N-terminus during polypeptide translocation (red line). B: Control (Ctrl) (wild-type [WT] 293T cells) and SSR3-KO 293T cells were transfected with untagged human preproinsulin plasmids. At 48 h posttransfection, cell lysates were analyzed by reducing SDS-PAGE gel and immunoblotting with antiproinsulin and anti-SSR1–4 antibodies as indicated. C: Quantitation (mean ± SD) of preproinsulin, proinsulin, and TRAP/SSR subunit protein levels from four independent experiments like that shown in B (normalized to tubulin). *P < 0.05 compared with Ctrl. D: Ctrl and SSR3-KO 293T cells were transfected with human preproinsulin plasmids. At 48 h posttransfection, the cells were pulse labeled with ³⁵S-Met/Cys for the times indicated; this is a representative pulse labeling from three identical experiments. Cell lysates (normalized to trichloroacetic acid–precipitable counts) were subjected to immunoprecipitation with anti-insulin and analyzed by reducing SDS-PAGE and phosphorimaging; both the absolute and relative amounts of recovered preproinsulin and proinsulin are shown in the graph.

Figure 2

Effect of TRAPγ/SSR3 deficiency on endogenous proinsulin biosynthesis in β-cells. A: INS1E cells were transfected with scrambled oligo or TRAPγ/SSR3-KD (SSR3i). At 72 h posttransfection, cells were lysed and analyzed by reducing SDS-PAGE and immunoblotted with antiproinsulin and anti-SSR3 antibodies. B and C: Quantitation (mean ± SD) of TRAPγ/SSR3 protein and proinsulin protein from three independent experiments (normalized to tubulin). D: At 72 h posttransfection, control (Ctrl) and TRAPγ/SSR3-KD (SSR3i) INS1E cells were pulse labeled with ³⁵S-Met/Cys for the indicated times. Cell lysates (normalized to trichloroacetic acid–precipitable counts) were immunoprecipitated with anti-insulin and analyzed by reducing SDS-PAGE and phosphorimaging. E: Quantitation (mean ± SD) of newly synthesized proinsulin from five independent experiments like that shown in D, with the proinsulin band from Ctrl INS1E cells at 10 min labeling set to 1. *P < 0.05 compared with Ctrl.

Figure 3

TRAPγ/SSR3 deficiency results in a decrease in the steady-state level of stored, processed insulin in β-cells. INS832/13 cells were transfected with TRAPγ/SSR3-targeted siRNA. A: Immunofluorescence with anti-TRAPγ/SSR3 (red) and anti-insulin (green) in INS832/13 cells at 72 h posttransfection. Nuclei were counterstained with DAPI (blue). B: Cells transfected as in A were treated with or without MG132 (10 μmol/L) for 4 h. Cells were lysed and analyzed by reducing SDS-PAGE and immunoblotting with either anti-human-(pre)proinsulin or a battery of antibodies, as indicated. C: Quantitation (mean ± SD, n = 8 independent experiments) of each of the TRAP/SSR subunits in control (Ctrl) cells and TRAPγ/SSR3-KD (SSR3i). D: Quantitation of human proinsulin levels from five independent experiments like that shown in B. E: Quantitation of human preproinsulin levels from five independent experiments like that shown in B. F: Human preproinsulin-to-proinsulin ratios in MG132-treated INS832/13 cells were calculated from the experiments shown in D and E (n = 5). G: Quantitation (mean ± SD) of insulin protein levels from three independent experiments like that shown in B. *P < 0.05 compared with Ctrl. h, human; PI, proinsulin; PPI, preproinsulin.

Figure 4

The ability of β-cells to acutely increase proinsulin (and insulin) level in response to high-glucose stimulation depends on TRAPγ/SSR3. A: INS1E cells were transfected with scrambled oligo or TRAPγ/SSR3-KD (SSR3i). At 72 h posttransfection, cells were preincubated in RPMI medium containing 2.5 mmol/L glucose for 1.5 h followed by a 4-h incubation at either 2.5, 11.1, or 25 mmol/L glucose, respectively. Cells were lysed and analyzed by SDS-PAGE under reducing conditions (left) or nonreducing conditions (right) for immunoblotting with the antibodies indicated. The 4-h media were collected and similarly analyzed by SDS-PAGE and immunoblotting with anti-insulin (bottom left). B: Quantitation (mean ± SD) of proinsulin, insulin, and each TRAP/SSR subunit from five independent experiments. *P < 0.05. Ctrl, control.

Figure 5

TRAPγ/SSR3 re-expression rescues proinsulin protein levels in TRAPγ/SSR3-KO β-cells. TRAPγ/SSR3-KO INS832/13 cells were transfected with empty vector (−) or plasmids encoding Flag-tagged TRAPγ/SSR3 cDNA (+). Wild-type (WT) INS832/13 cells were also transfected with empty vector as control. A: At 48 h posttransfection, cells were lysed and analyzed by reducing SDS-PAGE and immunoblotting for anti-rodent proinsulin and the additionally indicated antibodies. B: Quantitation (mean ± SD) of proinsulin and each TRAP/SSR subunit from four independent experiments like that shown in A; expression levels in WT control cells were set to 1.0. C: TRAPγ/SSR3-KO INS832/13 cells transfected as in A were examined by immunofluorescence with anticalnexin (red), anti-Flag (Flag-SSR3, purple), and anti-rodent proinsulin (green); a merged anti-Flag/antiproinsulin image is shown. D: Quantitation (mean ± SD) of rodent proinsulin–positive TRAPγ/SSR3-KO cells that either re-express or do not re-express TRAPγ/SSR3 protein from three independent experiments like that shown in C. E: Quantitation (mean ± SD) of insulin-positive TRAPγ/SSR3-KO cells that either re-express or do not re-express TRAPγ/SSR3 protein from four independent experiments. *P < 0.05.

Figure 6

Overexpression of TRAPγ/SSR3 in wild-type INS832/13 cells increases the steady-state level of proinsulin. A: Wild-type INS832/13 cells were transfected with plasmids encoding empty vector (−) or Flag-tagged TRAPγ/SSR3 cDNA (+). At 48 h posttransfection, cells were lysed and analyzed in duplicate by reducing SDS-PAGE and immunoblotting with the indicated antibodies. B: Quantitation (mean ± SD) of rodent proinsulin (CCI-17 antibody) and human proinsulin (human C-A junction antibody) and each TRAP/SSR subunit as indicated (n = 4 independent experiments). C: Wild-type INS832/13 cells were transfected with plasmids encoding empty vector or each individual Flag-tagged TRAP/SSR subunit and processed as in A. The red arrows highlight expression of the Flag-tagged constructs. D: Quantitation (mean ± SD) showing increased expression of each of the TRAP/SSR subunits (top) and the impact of that overexpression on rodent proinsulin (bottom left) and human proinsulin (bottom right) levels from five independent experiments. E: Wild-type 293T cells were transfected with human wild-type preproinsulin plasmid plus plasmid encoding empty vector or each individual Flag-tagged TRAP/SSR subunit and processed as in A. F: Quantitation (mean ± SD) of individually overexpressed TRAP/SSR subunits (left) and recombinant human proinsulin (right) from four independent experiments. G: Human islets were preincubated for 90 min in RPMI medium containing 2.5 mmol/L glucose and 0.1% BSA without serum. Aliquots of 130 human islet equivalents were sedimented at 1,000g, and the preincubation medium was removed, followed by a 2-h incubation in 500 μL of fresh medium bearing either 2.5 or 25 mmol/L glucose in the absence or presence of CHX (100 μg/mL). Human islets were then lysed in RIPA buffer; lysates normalized to total protein were analyzed by SDS-PAGE and immunoblotting for the indicated proteins (tubulin was used as a loading control to confirm proper normalization of samples). *P < 0.05. EV, empty vector; PI, proinsulin.

Figure 7

Effects of TRAPγ/SSR3 on the posttranslational translocation of mutant preproinsulin-R6C. Control (Ctrl) 293T cells and SSR3-KO 293T cells were transfected to express untagged human preproinsulin-R6C. A: At 48 h posttransfection, cells were treated with or without MG132 (10 μmol/L) for 2 h. The cells were lysed and analyzed by reducing SDS-PAGE and immunoblotting with the indicated antibodies. B: After transfection as in A, cells were pulse labeled with ³⁵S-Met/Cys for 10 min followed by a 0-, 10-, or 30-min chase in the presence of CHX (10 μg/mL) and MG132 (10 μmol/L). Cell lysates (normalized to trichloroacetic acid–precipitable counts) were subjected to immunoprecipitation with anti-insulin and analyzed by reducing SDS-PAGE and phosphorimaging; both the absolute and the relative amounts of recovered preproinsulin and proinsulin are shown below each lane of the gel.