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. 2012 Nov;14(11):1223-30.
doi: 10.1038/ncb2593. Epub 2012 Oct 28.

PARP16 is a tail-anchored endoplasmic reticulum protein required for the PERK- and IRE1α-mediated unfolded protein response

Miri Jwa  1 , Paul Chang
Affiliations

PARP16 is a tail-anchored endoplasmic reticulum protein required for the PERK- and IRE1α-mediated unfolded protein response

Miri Jwa et al. Nat Cell Biol. 2012 Nov.

Erratum in

  • Nat Cell Biol. 2013 Jan;15(1):123

Abstract

Poly(ADP-ribose) polymerases (PARPs; also known as ADP-ribosyl transferase D proteins) modify acceptor proteins with ADP-ribose modifications of varying length (reviewed in refs , , ). PARPs regulate key stress response pathways, including DNA damage repair and the cytoplasmic stress response. Here, we show that PARPs also regulate the unfolded protein response (UPR) of the endoplasmic reticulum (ER). Human PARP16 (also known as ARTD15) is a tail-anchored ER transmembrane protein required for activation of the functionally related ER stress sensors PERK and IRE1α during the UPR. The third identified ER stress sensor, ATF6, is not regulated by PARP16. As is the case for other PARPs that function during stress, the enzymatic activity of PARP16 is upregulated during ER stress when it ADP-ribosylates itself, PERK and IRE1α. ADP-ribosylation by PARP16 is sufficient for activating PERK and IRE1α in the absence of ER stress, and is required for PERK and IRE1α activation during the UPR. Modification of PERK and IRE1α by PARP16 increases their kinase activities and the endonuclease activity of IRE1α. Interestingly, the carboxy-terminal luminal tail of PARP16 is required for PARP16 function during ER stress, suggesting that it transduces stress signals to the cytoplasmic PARP catalytic domain.

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Figures

Figure 1
Figure 1. PARP16 is a tail-anchored ER transmembrane protein
HeLa cells used in all figures. a, Cells stained for PARP16 (green) and organelle markers (red). b, PARP16 domain structure. TM: transmembrane domain; PARP: PARP catalytic domain. c, Membrane extraction assay. Immunoblots of input, cytosol, supernatant and pellet of 1 M NaCl or 1% Triton X-100 (TX-100) treated membrane fractions. Molecular weight (MW) (kD) at right of blot. d, Protease protection assay using Untreated, Digitonin- or Proteinase K-treated cells expressing GFP-PARP16 or GFP. e, Left: PARP16 mutants. Right; Untreated or digitonin-treated cells expressing GFP fusions of indicated proteins. f, PARP16 topology within the ER. Dark blue bands mark conserved histidine and tyrosine residues within the PARP catalytic domain. Bars, 10 μm.
Figure 2
Figure 2. (ADP-ribosyl)ation activity of PARP16 is required for the ER stress response
MW at right of blots. a, 32P-NAD + incorporation by recombinant GST-PARP16 and GST-PARP16H152Q Y182A. Asterisk= MW of GST-PARP16. b, EMAA using GFP-PARP16 or GFP-PARP16H152Q Y182A containing microsomes. Asterisk= high MW incorporation of 32P-NAD. c, Cells expressing GFP-PARP16, GFP-PARP16H152Q Y182A, or GFP-PARP16Cb5 for 16h or 28 h, or untransfected cells treated with Brefeldin A (BFA), stained for PARP16 (green) and Calnexin (red). d, Trypan blue staining of control or PARP16 knock-downs at indicated time points after Tunicamycin, Thapsigargin or Brefeldin A treatment. 16.3 and 16.4 are different siRNAs against PARP16 (n=4 for siRNA 16.3, and 2 for siRNA 16.4). For Tunicamycin, Thapsigargin, or Brefeldin A-treated PARP16 knock down cells, 0.001 < p < 0.05. Immunoblots of knock-down shown at right. Bar, 10 μm.
Figure 3
Figure 3. PERK and IRE1α are (ADP-ribosyl)ated in a PARP16-dependent manner during the UPR
MW (kD) at right of blots. (UT)= untreated; (BFA)= Brefeldin A treated; (TG)= Thapsagargin treated; (TUN)= Tunicamycin treated. a, Autoradiogram of EMAA showing ADP-ribose incorporation. Immunoblots of GFP-PARP16 precipitates are shown at right. Asterisk= high MW NAD+ incorporation. n = 5; 0.01 < p of fold increase < 0.05 for all stressors. b, ER microsome based co-immunoprecipitation assays of GFP-fusion proteins. Shown are immunoblots of precipitated GFP fusions. c–d, EMAA using control or PARP16 knock-downs. Shown are autoradiogram and immunoblots of GFP-PERK (c) or GFP-IRE1α immunoprecipitates (d). For both (c) and (d), n=4; 0.005 < p of fold increase < 0.05 for all stressors. PARP16 immunoblots of control and PARP16 knock-down lysates are shown. e, EMAA for SEC61β, ATF6 and PARP16. Shown are autoradiogram and immunoblot of the immunoprecipitated GFP fusions. n=2.
Figure 4
Figure 4. Enzymatic activity of PARP16 is required for activation of PERK- and IRE1α–mediated UPR
MW (kD or bp) at right or left of blots or gels. (UT)= untreated; (BFA)= Brefeldin A; (TG)= Thapsagargin; (TUN)= Tunicamycin; PERKp= phospho-PERK; eIF2αp= phospho-eIF2 α. Asterisk= hybrid amplicons. a, Left, Immunoblots of cells overexpressing GFP or GFP fusions to PARP16 or PARP16H152Q/Y182A. Right, Immunoblots of cells transfected with control or PARP16 siRNA. b, XBP-1 mRNA splicing assay from control, GFP-PARP16 or GFP-PARP16H152Q Y182A expressing cells, or cells transfected with control or PARP16 siRNA. Unspliced (U) and spliced (S) XBP-1 cDNA were amplified via RT-PCR then cut with Pst1 restriction enzyme. Only unspliced XBP-1 is cut by PstI. c, Left, Immunoblots of cell lysates. Right, RT-qPCR analysis of UPR-dependent transcription in control or PARP16 knock-downs treated with Tunicamycin. d and e, ER microsome based (ADP-ribosyl)ation and kinase assays. Microsomes containing GFP-PERK (d) or GFP-IRE1α (e) were (ADP-ribosyl)ated via addition of 0.5 μg GST-PARP16 or GST-PARP16H152Q Y182A in the presence of 32P-NAD+. Duplicate NAD+ incorporation reactions performed under identical conditions using unlabeled NAD+, then kinase activity assayed via γ32P-ATP incorporation. For d and e, n=4. f, ER microsome based (ADP-ribosyl)ation and IRE1α endonuclease assays. (ADP-ribosyl)ation of GFP-IRE1α was performed in a similar manner to (d) using unlabeled NAD+. In vitro transcribed 32P labeled XBP-1 transcript was incubated with the (ADP-ribosyl)ated GFP-IRE1α immunoprecipitates and assayed for splicing as indicated via presence of 5′ and 3′ exons. n=4.
Figure 5
Figure 5. The C-tail of PARP16 is required for activation of PERK- and IRE1α–mediated UPR
a, ER microsome based co-immunoprecipitation assays. GFP-PERK or GFP-IRE1α purified from ER microsomes from cells treated with control (Ctrl) or PARP16 (P-16) siRNA, then analysed for BiP binding via immunoblot. PARP16 blots from each assay are shown. b, Immunoblots of cell expressing GFP, or GFP fusions to PARP16 or PARP16Cb5. c, XBP-1 mRNA splicing assay, similar to (b) but performed on PARP16 or PARP16Cb5 overexpressing cells.
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References

    1. Hottiger MO, Hassa PO, Lüscher B, Schüler H, Koch-Nolte F. Toward a unified nomenclature for mammalian ADP-ribodyltransferases. Trends in Biochem Sci. 2010;35:208–219. - PubMed
    1. Schreiber V, Dantzer F, Ame JC, de Murcia G. Poly(ADP-ribose): novel functions for an old molecule. Nat Rev Mol Cell Biol. 2006;7:517–528. - PubMed
    1. Hassa PO, Hottiger MO. The diverse biological roles of mammalian PARPS, a small but powerful family of poly-ADP-ribose polymerases. Front Biosci. 2008;13:3046–3082. - PubMed
    1. Malanga M, Althaus FR. The role of poly(ADP-ribose) in the DNA damage signaling network. Biochem Cell Biol. 2005;83:354–364. - PubMed
    1. Ménissier-de Murcia J, Molinete M, Gradwohl G, Simonin F, de Murcia G. Zinc-binding domain of poly(ADP-ribose)polymerase participates in the recognition of single strand breaks on DNA. J Mol Biol. 1989;210:229–233. - PubMed

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