Research ArticleCELL BIOLOGY

Requirement for translocon-associated protein (TRAP) α in insulin biogenesis

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Science Advances  04 Dec 2019:
Vol. 5, no. 12, eaax0292
DOI: 10.1126/sciadv.aax0292
  • Fig. 1 trap-1 loss-of-function enhances DAF-2/InsR signaling.

    (A) Schematic of the trap-1 genomic region and the dpDf665 deletion allele identified in a genetic screen. (B) trap-1 null alleles dp669, dp671, and dp672 (fig. S1) phenocopy dauer suppression caused by dpDf665 deletion. (C) The trap-1(dp672) null mutation suppresses the dauer-constitutive phenotype of a daf-2/InsR loss-of-function mutant, and a TRAP-1::mCherry fusion protein is functional. (D to F) The trap-1(dp672) null mutation inhibits the expression of the DAF-16/FoxO target genes (D) sod-3, (E) mtl-1, and (F) dod-3.

  • Fig. 2 Spatiotemporal expression of a functional TRAP-1::mCherry fusion protein.

    TRAP-1::mCherry is expressed widely in (A) embryos, (B) larvae, and (C) adult animals. Differential interference contrast [top in (A) and (B); left in (C)] and fluorescence [bottom in (A) and (B); right in (C)] images of representative animals are shown. In adults (C), TRAP-1::mCherry is expressed in the pharynx (hashtag), intestine (asterisks), hypodermis (arrows), and vulva (arrowheads). (D) TRAP-1::mCherry colocalizes with the ER signal peptidase GFP-SP12. Two anterior intestinal cells are shown.

  • Fig. 3 TRAPα promotes preproinsulin ER translocation, insulin biogenesis, and insulin secretion in INS 832/13 cells.

    (A) Immunostaining of INS 832/13 cells with anti-TRAPα antibodies reveals colocalization with ER proteins recognized by anti-KDEL antibodies (top) but not with the Golgi protein GM130 (bottom). Nuclei are stained with DAPI (4′,6-diamidino-2-phenylindole). (B) SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and anti-proinsulin immunoblotting of lysates from INS 832/13 wild-type (WT) or TRAPα KO cells. Cells were untreated or treated with the proteasome inhibitor MG132 as indicated. pPI, preproinsulin; PI, proinsulin. (C) SDS-PAGE and anti-proinsulin immunoblotting of lysates from INS 832/13 wild-type or TRAPα KO cells after pretreatment with phosphate-buffered saline (PBS), digitonin (DIG), or Triton X-100 (TRX) and subsequent exposure to Proteinase K (PK). (D) SDS-PAGE and immunoblotting of lysates (left) and anti-TRAPα immunoprecipitates (right) from 293 T cells transfected with either empty vector (EV) or a complementary DNA (cDNA) encoding the A24D MIDY preproinsulin mutant. The arrow denotes preproinsulin present in the anti-TRAPα immunoprecipitate. (E) SDS-PAGE and anti-insulin immunoblotting of lysates from INS 832/13 wild-type and TRAPα KO cells. (F) SDS-PAGE of anti-insulin immunoprecipitates of cell lysates (C) or conditioned media (M) from INS 832/13 wild-type or TRAPα KO cells after pulse-labeling with 35S-Met/Cys and chase for the indicated times. Ins-B, insulin. Quantification of total proinsulin and insulin (C + M) is shown in (G) and (H), respectively. (I) Glucose-stimulated insulin secretion assay on INS 832/13 wild-type, TRAPα KO, and TRAPα KO cells transfected with a TRAPα cDNA. (J) SDS-PAGE of anti–α1-antitrypsin (AAT) immunoprecipitates of cell lysates (C) or conditioned media (M) from INS 832/13 wild-type or TRAPα KO cells after pulse labeling and chase as described for (F). INS 832/13 wild-type or TRAPα KO cells were transfected with a plasmid encoding AAT 48 hours before pulse-labeling. (K) Quantification of total AAT (C + M) at the indicated time points.

  • Fig. 4 TRAPα promotes insulin biosynthesis and reduces ER stress.

    (A) Immunostaining of INS832/13 wild-type or TRAPα KO cells with anti-TRAPα and (A) anti-insulin or (B) anti-proinsulin antibodies. Nuclei are stained with DAPI. (C) Immunostaining of TRAPα KO cells with anti-Myc and anti-insulin antibodies after transfection with a plasmid encoding a Myc-tagged TRAPα cDNA. Nuclei are stained with DAPI. (D) Quantification of insulin-positive cells in INS 832/13 wild-type, TRAPα KO, and TRAPα KO cells expressing exogenous Myc-tagged TRAPα. (E) Expression of the ER stress reporter hsp-4::GFP in second-stage (L2) and fourth-stage (L4) larvae and young adult wild-type (left) and trap-1(dp672) null mutant (right) animals. At each stage, images of wild-type and trap-1 mutant animals were captured with equivalent exposure times. Scale bars, 100 μm. (F) Higher-magnification images of representative adult wild-type (left) and trap-1 mutant (right) hsp-4::GFP transgenic animals. Images were captured with equivalent exposure times. Scale bars, 20 μm. (G) Detection of unspliced and spliced forms of xbp-1 mRNA in INS 832/13 cells after TRAPα knockdown (left) or KO (right). Tunicamycin-treated cells (TM) were used as a positive control. The band corresponding to spliced xbp-1 mRNA is denoted by the arrowheads. M, molecular weight marker; scr, scrambled siRNA control. (H) SDS-PAGE and anti–phospho-Ser51-eIF2 immunoblotting of lysates from INS 832/13 wild-type cells that were transfected with either scrambled (Sc) or TRAPα siRNA as indicated.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/12/eaax0292/DC1

    Fig. S1. trap-1 mutant DNA sequences and predicted effects on TRAP-1 protein.

    Fig. S2. trap-1 and Y71F9AL.1 mutant phenotypes.

    Fig. S3. Alignment of C. elegans TRAP-1 and human TRAPα amino acid sequences.

    Fig. S4. Dauer-defective phenotypes of a trap-1 null mutation.

    Fig. S5. Effect of reduced TRAPα activity on preproinsulin, proinsulin, and insulin levels in INS 832/13 cells.

    Fig. S6. Reexpression of TRAPα in TRAPα KO cells rescues insulin production in a dose-dependent manner.

    Table S1. Description of eight daf-2/InsR alleles assayed in fig. S5A.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. trap-1 mutant DNA sequences and predicted effects on TRAP-1 protein.
    • Fig. S2. trap-1 and Y71F9AL.1 mutant phenotypes.
    • Fig. S3. Alignment of C. elegans TRAP-1 and human TRAPα amino acid sequences.
    • Fig. S4. Dauer-defective phenotypes of a trap-1 null mutation.
    • Fig. S5. Effect of reduced TRAPα activity on preproinsulin, proinsulin, and insulin levels in INS 832/13 cells.
    • Fig. S6. Reexpression of TRAPα in TRAPα KO cells rescues insulin production in a dose-dependent manner.
    • Table S1. Description of eight daf-2/InsR alleles assayed in fig. S5A.

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