Deficient endoplasmic reticulum translocon-associated protein complex limits the biosynthesis of proinsulin and insulin

Abstract

The conserved endoplasmic reticulum (ER) membrane protein TRAPα (translocon-associated protein, also known as signal sequence receptor 1, SSR1) has been reported to play a critical but unclear role in insulin biosynthesis. TRAPα/SSR1 is one component of a four-protein complex including TRAPβ/SSR2, TRAPγ/SSR3, and TRAPδ/SSR4. The TRAP complex topologically has a small exposure on the cytosolic side of the ER via its TRAPγ/SSR3 subunit, whereas TRAPβ/SSR2 and TRAPδ/SSR4 function along with TRAPα/SSR1 largely on the luminal side of the ER membrane. Here, we have examined pancreatic β-cells with deficient expression of either TRAPβ/SSR2 or TRAPδ/SSR4, which does not perturb mRNA expression levels of other TRAP subunits, or insulin mRNA. However, deficient protein expression of TRAPβ/SSR2 and, to a lesser degree, TRAPδ/SSR4, diminishes the protein levels of other TRAP subunits, concomitant with deficient steady-state levels of proinsulin and insulin. Deficient TRAPβ/SSR2 or TRAPδ/SSR4 is not associated with any apparent defect of exocytotic mechanism but rather by a decreased abundance of the proinsulin and insulin that accompanies glucose-stimulated secretion. Amino acid pulse labeling directly establishes that much of the steady-state deficiency of intracellular proinsulin can be accounted for by diminished proinsulin biosynthesis, observed in a pulse-labeling as short as 5 minutes. The proinsulin and insulin levels in TRAPβ/SSR2 or TRAPδ/SSR4 null mutant β-cells are notably recovered upon re-expression of the missing TRAP subunit, accompanying a rebound of proinsulin biosynthesis. Remarkably, overexpression of TRAPα/SSR1 can also suppress defects in β-cells with diminished expression of TRAPβ/SSR2, strongly suggesting that TRAPβ/SSR2 is needed to support TRAPα/SSR1 function.

Keywords: diabetes; insulin secretion; pancreatic beta cell; preproinsulin; translocation.

FIGURE 1

SiRNA-mediated suppression of TRAPβ/SSR2 or TRAPδ/SSR4 in INS832/13 cells. A, Structure and subunit composition of the TRAP/SSR complex as predicted by bioinformatic analysis. B, Immunoblotting with the listed antibodies of INS832/13 cell lysates transfected with the indicated siRNAs. Proinsulin was measured with rodent-specific anti-proinsulin. Tubulin is a loading control. C, Quantitation (mean ± SD) of TRAP/SSR subunit protein levels from experiments like that in panel B (n = 4). *P < .05, **P < .01 comparing to WT. D, Quantitation (mean ± SD) of proinsulin and insulin protein levels from experiments like that in panel B (n = 4). *P < .05, **P < .01 compared to WT. E, Scrambled oligo (NC) or TRAPβ/SSR2 or TRAPδ/SSR4 siRNA-mediated gene suppression cells were pulse-labeled with ³⁵S-Met/Cys for 10 minutes (left panel). Cell lysates (normalized to TCA-precipitable counts) were subjected to immunoprecipitation with anti-insulin, followed by SDS-PAGE and phosphorimaging. Quantitation of proinsulin (n = 3) is shown at right. *P < .05, **P < .01

FIGURE 2

Deletion of TRAPβ/SSR2 or TRAPδ/SSR4 in INS832/13 cells. A, TRAPβ/SSR2 or TRAPδ/SSR4 null cells and corresponding control cells (CON) were immunoblotted with the antibodies indicated. Tubulin is a loading control. B, Quantitation of TRAP/SSR subunits from experiments like that in panel A (n = 4). C, Quantitation of proinsulin and insulin from experiments like that in panel A (n = 4). *P < .05, **P < .01 compared to Control. D, TRAPβ/SSR2 or TRAPδ/SSR4 null cells were pulse-labeled with ³⁵S-Met/Cys for 30 minutes (left panel) before immunoprecipitation with anti-insulin, followed by SDS-PAGE and phosphorimaging. Quantitation of newly-synthesized proinsulin (PI, mean ± SD) is shown *P < .05 compared to Control (n = 4)

FIGURE 3

Effect of TRAPβ/SSR2 or TRAPδ/SSR4 deficiency on insulin biogenesis. A, TRAPβ/SSR2 or TRAPδ/SSR4 deficient cells and control cells were pulse-labeled with ³⁵S-Met/Cys for 5 minutes. The cells were lysed and immunoprecipitated with anti-insulin and analyzed by 4%−12% NuPage gel and phosphorimaging. B, TRAPβ/SSR2 or TRAPδ/SSR4 deficient cells were pulse-labeled for 30 minutes followed by 0 or 2 hours chase in complete culture medium. Lysates of cells (C) and 2 hours chase medium (M) were immunoprecipitated with anti-insulin and resolved by 4%−12% NuPage and phosphorimaging. The newly-synthesized insulin / proinsulin ratio after 2 hours chase from experiments like those in panel B or D was quantitated (mean ± SD; n = 4 for Figure 3B and n = 3 for Figure 3D) and is shown in panels C and E, respectively. *P < .05, **P < .01 compared to Control

FIGURE 4

Glucose-stimulated insulin secretion in TRAPβ/SSR2 or TRAPδ/SSR4 deficient cells. The cells (including control cells – CON) were incubated with prewarmed KRBH buffer containing 2.5 mM glucose for 2 hours followed by KRBH buffer containing 25 mM glucose for an additional 2 hours, as shown on the X-axis. Insulin released to the media was measured by ELISA (mean ± SD; n = 5)

FIGURE 5

TRAPβ/SSR2 re-expression rescues proinsulin protein levels in TRAPβ/SSR2 null cells. A, TRAPβ/SSR2 null cells were transfected with empty vector (EV) or plasmid encoding Flag-tagged TRAPβ/SSR2. At 48 hours post transfection, these cells or INS832/13 wild type cells (Con) were lysed and immunoblotted with the indicated antibodies. Tubulin is a loading control. Quantitation of proinsulin and insulin from experiments like that in panel A was shown as panel B and C, respectively (n = 4). *P < .05, **P < .01compared to Control. D, Immunofluorescence of proinsulin (green) and Flag (red) in TRAPβ/SSR2 null cells transfected with Flag-tagged TRAPβ/SSR2 plasmid, containing both transfected and untransfected cells. Nuclei were counter-stained with DAPI (blue). For clarity in the merged image, white arrows point to the nuclei of some of the untransfected cells

FIGURE 6

TRAPδ/SSR4 re-expression rescues proinsulin protein levels in TRAPδ/SSR4 deficient cells. A, TRAPδ/SSR4 null cells were transfected with empty vector (EV) or plasmid encoding Flag-tagged TRAPδ/SSR4. At 48 hours post transfection, these cells or INS832/13 wild type cells (Con) were lysed and immunoblotted with the indicated antibodies. Tubulin is a loading control. The proinsulin and insulin level presented in panel A was quantified as panel B and C (n = 3). *P < .05, **P < .01compared to Control. D, Immunofluorescence of proinsulin (green) and Flag (red) in TRAPδ/SSR4 null cells transfected with Flag-tagged TRAPδ/SSR4 plasmid, containing both transfected and untransfected cells. Nuclei were counter-stained with DAPI (blue)

FIGURE 7

Re-expression of missing TRAP/SSR subunits increases newly-synthesized proinsulin in TRAPδ/SSR4 or TRAPβ/SSR2 deficient β-cells. A, TRAPδ/SSR4 or TRAPβ/SSR2 deficient β-cells were transfected with plasmids encoding Myc-tagged or Flag-tagged TRAPδ/SSR4 or TRAPβ/SSR2-KO, respectively. Both tags worked similarly. At 48 hours post transfection, the cells were pulse-labeled with ³⁵S-Met/Cys for 30 minutes, followed by immunoprecipitation with anti-insulin, and analysis by 4%−12% NuPage gel and phosphorimaging. B, Quantitation of newly-synthesized proinsulin (mean ± SD) from experiments like those in panel A is shown. *P < .05, **P < .01compared to Control (n = 3)

FIGURE 8

Overexpression of TRAPα/SSR1 partially rescues proinsulin and insulin levels in TRAPβ/SSR2 deficient β-cells. A, Two independent TRAPβ/SSR2-deficient clones were transfected with plasmid encoding Myc-tagged TRAPα/SSR1. At 48 hours post-transfection, cell lysates were subjected to immunoblotting under reducing conditions with the antibodies shown, or non-reducing conditions (for insulin). Tubulin is a loading control. Quantitation of proinsulin and insulin bands (mean ± SD) from experiments like those in panel A is shown in B and C, respectively (n = 4). *P < .05, **P < .01