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. 2015 Jul;17(7):917-29.
doi: 10.1038/ncb3177. Epub 2015 Jun 15.

Amino-terminal Arginylation Targets Endoplasmic Reticulum Chaperone BiP for Autophagy Through p62 Binding

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Amino-terminal Arginylation Targets Endoplasmic Reticulum Chaperone BiP for Autophagy Through p62 Binding

Hyunjoo Cha-Molstad et al. Nat Cell Biol. .
Free PMC article

Abstract

We show that ATE1-encoded Arg-transfer RNA transferase (R-transferase) of the N-end rule pathway mediates N-terminal arginylation of multiple endoplasmic reticulum (ER)-residing chaperones, leading to their cytosolic relocalization and turnover. N-terminal arginylation of BiP (also known as GRP78), protein disulphide isomerase and calreticulin is co-induced with autophagy during innate immune responses to cytosolic foreign DNA or proteasomal inhibition, associated with increased ubiquitylation. Arginylated BiP (R-BiP) is induced by and associated with cytosolic misfolded proteins destined for p62 (also known as sequestosome 1, SQSTM1) bodies. R-BiP binds the autophagic adaptor p62 through the interaction of its N-terminal arginine with the p62 ZZ domain. This allosterically induces self-oligomerization and aggregation of p62 and increases p62 interaction with LC3, leading to p62 targeting to autophagosomes and selective lysosomal co-degradation of R-BiP and p62 together with associated cargoes. In this autophagic mechanism, Nt-arginine functions as a delivery determinant, a degron and an activating ligand. Bioinformatics analysis predicts that many ER residents use arginylation to regulate non-ER processes.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Bioinformatic analysis of the ER N-end rule pathway, and the generation of antibodies to the arginylated form of the ER chaperone BiP. (a) A summary of this study that describes a dual role for the Nt-Arg residue as a degron for both the UPS and autophagy. The Nt-Arg residue generated through the N-end rule pathway is known to act as a degron for selective proteolysis by the UPS. In the Ub-dependent N-end rule pathway, specific recognition components, called N-recognins, recognize and bind the primary degron Nt-Arg and mediate ubiquitination for targeting to the proteasome. The results of this study show that N-terminal arginylation of ER-residing chaperons generates a cis-acting delivery determinant and degron for lysosomal degradation along with associated cargoes. In this autophagic proteolysis, Nt-Arg of BiP and other ER proteins is recognized by a recognin, the autophagic adaptor p62. The binding of Nt-Arg to p62 activates p62, leading to the delivery of p62, R-BiP, and its cargoes to autophagosomes. In definition, Nt-Arg also acts as a trans-acting degron for its cargoes. (b) A sequence alignment of the N-terminal regions of human BiP and its sequelogs. The red box indicates the N-terminal residues (P1′ sites) of mature BiP proteins from various species. Shown above are secondary structures (solid line, αhelices; arrows, βstrands). (c) Generation of anti-R-BiP DH1 antibody. Shown is a peptide binding/competition assay. An 11-mer R-BiP peptide (R-BiP-peptide) corresponding to the N-terminal region of R-BiP was immobilized on a 96-well plate, followed by incubation with serially diluted anti-R-BiP antibody and, subsequently, anti-goat secondary antibody conjugated with horseradish peroxide. The amounts of R-BiP antibody that bound to immobilized R-BiP peptide were determined based on O.D. value at 450 nm of secondary antibody. As a control, BiP-peptide, a 10-mer peptide corresponding to the N-terminal region of unarginylated BiP, was used. (d) A dot blotting analysis of R-BiP antibody using the peptides corresponding to the N-terminal region of unarginylated (left) or arginylated (right) BiP.
Figure 2
Figure 2
ATE1-dependent N-terminal arginylation of multiple ER-residing proteins is induced by cytosolic foreign dsDNA. (a) BiP is N-terminally arginylated at Nt-Glu19. HeLa cells were transfected with a plasmid expressing X-BiP-flag wherein the flag epitope was inserted between BiP protein body and the C-terminal KDEL sequence. (b) Schematic diagrams of full length BiP in comparison with Ub-X-BiP-myc/his in which Ub is C-terminally conjugated with N-terminal X residue (X= Glu, Arg-Glu, Val) of BiP-myc/his. In the Ub fusion technique, Ub-X-BiP-myc/his is cotranslationally cleaved by Ub hydrolases at the Ub-BiP junction, producing Ub and X-BiP-myc/his. (c) ATE11A7A promotes N-terminal arginylation of Glu19-BiP-myc/his in HEK293 cells cotranslationally generated from Ub-X-BiP-myc/his (X= Arg-Glu19, Glu19, or Val19). (d) N-terminal arginylation of X-BiP-myc/his (X= Arg-Glu19 or Glu19) was measured in ATE1−/− MEFs transiently expressing an ATE1 isoform (ATE11B7A, ATE11B7B, or ATE11A7A) which contains either of alternatively spliced exons (1A, 1B, 7A, and 7B). (e) N-terminal arginylation of endogenous BiP was measured in HeLa cells expressing an ATE1 isoform. (f) ATE1-knockdown inhibits the N-terminal arginylation of BiP in HeLa cells expressing ATE11A7A. (g) N-terminal arginylation of endogenous BiP is not induced by thapsigargin (200 nM) but by overexpressing ATE11A7A in HeLa cells. (h) Cell fractionation assay shows that R-BiP localizes to the cytosol. To, total extracts; Cyt, cytosolic fraction; Mic, microsomal fraction. (i) Lumenal BiP is short-lived, and endogenous R-BiP generated by ATE11A7A exhibits a longer half-life. HeLa cells expressing ATE11A7A were treated with 10 μg/ml cycloheximide, followed by time-course immunoblotting. (j–k) N-terminal arginylation of BiP and CRT is induced by the transfection of dsDNAs. Cells were incubated with various types of nucleic acids in the presence or absence of Lipofectamine LTX. Salmo. sp. DNA, Salmon sperm DNA. (l) DNA-induced R-BiP is cytosolic and down-regulated by ATE1-knockdown. HeLa cells were co-transfected with ATE1 siRNA #204 and a plasmid expressing NHK-GFP, followed by fractionation of cytosolic proteins. (m) N-terminal arginylation of BiP and CRT is induced by poly(dA:dT) dsDNA but not by 5′ppp-dsRNA. (n–q) N-terminal arginylation of ER proteins is triggered by poly(dA:dT) (n), coinduced with autophagy in poly(dA:dT)-treated HeLa cells (o), facilitated by overexpressing ATE11A7A (p), and induced by the transfection of various dsDNAs (q). mNHK-GFP, GFP-fused mouse α1-antitrypsin null Hong-Kong.
Figure 3
Figure 3
R-BiP is targeted to the autophagosome via p62 bodies. Scale bars, 10 μm. (a) Colocalization of cytoplasmic R-BiP puncta with p62 puncta in poly(dA:dT)-treated HeLa cells. (b) Colocalization of R-BiP puncta with LC3 puncta in HeLa cells stably expressing RFP-GFP-LC3 as determined by immunostaining of R-BiP in comparison with RFP signal (red) which represents LC3-positive autophagic vacuoles. (c) Three color colocalization analysis between R-BiP (blue), p62 (red), and LC3 (green) in poly(dA:dT)-treated HeLa cells. (d) HeLa cells expressing RFP-GFP-LC3 were subjected to three color colocalization analysis between R-BiP (blue), acid-resistant RFP (red), and acid-sensitive GFP (green). Most R-BiP puncta show a strong colocalization with LC3 puncta which are positive for both RFP and GFP, indicating the delivery of R-BiP to autophagosomes. (e) Immunohistochemistry of total BiP and LC3 on sections of mouse embryonic hearts at embryonic day 13.5, which reveals BiP puncta that colocalize with LC3 puncta. (f) RNA interference assay of ATE1, BiP, and p62 in poly(dA:dT)-treated HeLa cells, followed by colocalization analysis between R-BiP and p62. Note that knockdown of any of ATE1, BiP, and p62 disrupts the targeting of both R-BiP and p62 to autophagic vacuoles. (g) RNA interference assay of ATE1 and BiP in poly(dA:dT)-treated HeLa cells expressing RFP-GFP-LC3, followed by colocalization analysis between R-BiP and LC3. (h) RNA interference assay of LC3 in poly(dA:dT)-treated HeLa cells, followed by colocalization analysis between R-BiP, p62, and LC3. LC3-knockdown apparently did not affect significantly the delivery of R-BiP to p62 puncta.
Figure 4
Figure 4
The Nt-Arg residue of R-Bip is a delivery determinant to the autophagosome. (a) A schematic diagram showing that the Ub fusion protein Ub-X-BiP-GFP is cotranslationally cleaved into Ub and X-BiP-GFP by Ub hydrolases. Also shown is how Ub-X-BiP19–124-GFP (Ub-X-BiPΔ-GFP) is cotranslationally cleaved into Ub and X-BiPΔ-GFP. (b) Colocalization analysis between X-BiP-GFP (X= Arg (an arginylated form of Glu-BiP, Arg-Glu-BiP) or Val (a Glu-to-Val mutant, Val-BiP-GFP)) and p62 in HeLa cells. X-BiP-GFP is produced in vivo from a precursor protein, Ub-X-BiP-GFP, which is elaborated in a. Scale bar, 10 μm. (c) An analogous colocalization assay with X-BiP-RFP and LC3. Scale bar, 10 μm. (d) Puncta colocalization analysis of X-BiP19–124-GFP (X-BiPΔ-GFP; X= Arg, Glu, and Val) and p62 in +/+ and p62−/− MEFs. Note that the ability of X-BiPΔ-GFP to form cytosolic puncta not only follows the N-end rule but also requires p62. Scale bar, 10 μm. (e) Puncta colocalization analysis of X-BiPΔ-GFP and LC3 in +/+ and p62−/− MEFs, which shows that the colocalization of R-BiP puncta with LC3 depends on both the N-end rule and p62. Scale bar, 10 μm. (f) Quantitation of d and e indicating that the ability of R-BiP to form cytosolic puncta depends on p62. The graph shows the percentage of BiPΔ-GFP-positive cells that form BiPΔ-GFP punta. Mean +/− s.d. of n=3 independent experiments in which 200 cells were analysed per experimental point. Statistical significance was calculated using a one-way ANOVA test (N.S. p > 0.05; **** p < 0.0001).
Figure 5
Figure 5
The Nt-Arg residue of R-BiP binds to the ZZ domain of p62. (a) The sequences of X-BiP peptides used to pulldown p62. (b) The Nt-Arg residue of R-Bip binds to p62. X-BiP peptides crosslinked to beads were used to pulldown p62 from HEK293 cell extracts transiently expressing p62. (c) Similar to b except that X-nsP4 peptides (X= Arg, Phe, and Val) were used. (d) A diagram showing C-terminally (D1-D4) and N-terminally (D5-D7) deleted p62 mutant proteins. (e) R-BiP peptide pulldown assay with serially deleted p62 mutants. (f) R-nsP4 peptide pulldown assay with serially deleted p62 mutants. (g) A diagram showing wild-type p62 and a ZZ-deletion mutant (p62ZZΔ). (h) R-nsP4 pulldown assay with wild-type p62 and p62ZZΔ. (i) A diagram showing a ZZ-only fragment (p62ZZ(WT), #83–175) and its mutant (p62ZZ(D129A)) in which the conserved Asp129 was mutated to Ala. (j) R-nsP4 peptide pulldown assay using p62ZZ(WT) and p62ZZ(D129A). (k) A GST-pulldown assay combined with X-BiP pulldown assay. A p62-GST fusion, p62ZZ-GST or p62ZZ(D129A)-GST, immobilized on gluthathione beads was incubated with HEK293 extracts expressing Ub-X-BiP-myc/his (X= Arg or Val), followed by centrifugation and immunoblotting analysis of X-BiP-myc/his that bound to p62 ZZ fragments. Note that R-BiP-myc/his binds to wild-type ZZ-only fragment but not to D129A-ZZ fragment. No binding was detected with V-BiP-myc/his. (l) Colocalization assay of X-BiPΔ-GFP (X= Arg or Val) and p62ZZ-RFP coexpressed in p62−/− MEFs. X-BiPΔ-GFP was cotranslationally generated from Ub-X-BiPΔ-GFP using the Ub fusion technique. Note that R-BiPΔ-GFP, but not V-BiPΔ-GFP, forms cytosolic puncta colocalizing with p62ZZ-RFP.
Figure 6
Figure 6
The Nt-Arg residue induces oligomerization and aggregation of p62 in vitro. (a) In vitro oligomerization/aggregation assays of HEK293 cell lysates expressing p62-myc/his. The dipeptide Arg-Ala (type-1) at a final concentration of 20 mM was added to the extracts (1 μg) in comparison with other dipeptides. (b, c) Similar to a. Arg-Ala was compared with Ala-Arg in a time-dependent (b) and dose-dependent (c) manner. (d) The p62-LC3 interaction assay shows that Nt-Arg of Arg-Ala promotes the interaction of p62 with LC3-GST. LC3-GST (3 μg) was immobilized to gluthathione (GSH)-coated wells and incubated with HEK293 cell extracts (20 μg) expressing p62. Following incubation with dipeptides for 2 hrs, bound p62 was detected using anti-p62 antibody. Quantification is of n = 3 independent experiments; bars represent mean ± s.d. Statistical significance was calculated using a one-way ANOVA test (N.S. P > 0.05; ** P < 0.01; **** P < 0.0001. (e) Quantitation of f. Quantification is of n = 3 independent experiments; bars represent mean ± SEM. Statistical significance was calculated using a one-way ANOVA test (* P < 0.05; ** P < 0.01). (f) R-BiPΔ-GST, but not V-BiPΔ-GST, is metabolically stabilized by p62 knockout or pharmaceutical inhibition of autophagy. Shown is immunoblotting of X-BiPΔ-GST (X= Arg or Val) in +/+ and p62−/− MEFs with or without the treatment of 25 μM hydroxychloroquine for 16 hrs. Note that the level of R-BiPΔ-GST (as determined by GST immunoblotting) is lower as compared with V-BiPΔ-GST because of its degradation (lanes 2 vs. 3) and increased in p62-deficient cells (lanes 2 vs. 5) or by the treatment of hydroxychloroquine. (g) R-BiPΔ-GST, but not V-BiPΔ-GST, is metabolically stabilized in ATG5−/− MEFs. Shown is immunoblotting of X-BiP19–124-GST (X-BiPΔ-GST, X= Arg or Val) in +/+ and ATG5−/− MEFs. Note that the level of R-BiPΔ-GST, as determined by GST immunoblotting, is lower than V-BiP19–124-GST (lanes 2 vs. 3) and increased in ATG5-deficient cells (lanes 2 vs. 5). (h) Cycloheximide degradation assay of R-BiP-myc/his generated from Ub-R-BiP-myc/his in +/+ and ATG5−/− MEFs in the absence or presence of 5 μM MG132. Cells were treated with 50 μg/ml cycloheximide, followed by time-course immunoblotting of R-BiP. (i) Quantitation of h. Shown is a representative of two independent experiments.
Figure 7
Figure 7
R-BiP is induced by and associated with cytosolic misfolded proteins, and ATE1-deficient cells are hypersensitive to misregulation of protein quality control. (a) Immunoblotting analysis of R-BiP and Ub conjugates in HeLa cells treated with 0.4 μg/ml poly(dA:dT) for 16 hrs. (b) Colocalization assay with antibodies specific to Ub conjugates (FK2 antibody), p62, and R-BiP in HeLa cells treated with 1 μg/ml poly(dA:dT) for 21 hrs. Scale bar, 10 μm. (c) HeLa cells were treated with various stressors for 24 hrs as described in Methods and subjected to immunoblotting analysis of R-BiP and Ub conjugates using FK1 antibody. (d) HeLa cells were treated with 10 μM MG132 in combination with 200 nM thapsigargin or 200 nM bafilomycin A1 for 16 hrs. The cells were treated with 10 μg/ml cycloheximide, followed by immunoblotting assay. (e) Arginylation of ER proteins is synergistically induced by proteasomal inhibition and ER stress. HeLa cells were treated with 10 μM MG132 and 100 nM thapsigargin for 18 hrs. (f) The treatment of geldanamycin, an inhibitor of the HSP90, for 17 hrs results in co-induction of autophagy with arginylation of ER-residing chaperons. (g) Measurement of the interaction between R-BiP and CL1-YFP, a model substrate that undergoes spontaneous misfolding. See Methods for experimental details. (h) Colocalization assay of YFP-CL1 with R-BiP and p62. MEFs ectopically expressing YFP-CL1 was treated with 1 μg/ml poly(dA:dT) alone for 18 hrs or 10 μM MG132 and 200 nM bafilomycin A1 for 6 hrs, followed by immunostaining of endogenous R-BiP and p62 in comparison with YFP-CL1 fluorescence. Scale bar, 5 μm. (i) Ub-R-BiP was coexpressed with YFP-CL1 or GFP in HeLa cells, followed by fluorescence analysis. (j) Puncta formation assay of p62 in +/+ and ATE1−/− MEFs treated with 10 μM MG132 and/or 0.2 μM bafilomycin A1 for 6 hrs. (k) Control and ATE1-knockdown cells were treated with 10 μM MG132 and/or 0.2 μM bafilomycin A1 for 18 hrs, followed by immunoblotting analysis. (l) MTT assay of control and ATE1-knockdown cells treated with various stressors, including 10 μM MG132. Mean +/− s.d. of n=3 independent experiments in which 10,000 cells in a 24-well plate were analysed per experimental point. Statistical significance was calculated using a two-way ANOVA test (* p < 0.05; ** p < 0.01).
Figure 8
Figure 8
A model illustrating the role of the N-end rule pathway in N-terminal arginylation of ER-residing proteins and the ligand-mediated regulation of autophagy in stressed cells. In this model, ER residents and clients acquire cotranslationally and cotranslocationally the degrons of the N-end rule pathway, which normally remain separated from cytosolic N-end rule machinery. N-terminal arginylation of ER-residing proteins, especially BiP, is induced by various stress signals such as cytosolic misfolded proteins that are initially tagged with Ub but cannot be readily processed by the proteasome (Step 1). One physiological stress signal that induces N-terminal arginylation of ER proteins is the presence of cytosolic foreign dsDNA which triggers innate immune responses. N-terminal arginylation is thought to occur when the N-terminal residue of the substrates is exposed to the cytosolic surface of the ER membrane (Step 2), which may facilitate the cytosolic relocalization of arginylated ER proteins. We do not exclude the possibility that N-terminal arginylation occurs after the substrates complete their cytosolic relocalization. Arginylated ER proteins relocated to non-ER compartments appear to have both shared and distinct functions and metabolic fates. At least a subpopulation of cytosolic R-BiP appears to be associated with its cargoes such as cytosolic misfolded proteins (Step 3). R-BiP, alone or loaded with its cargo, binds to the ZZ domain of p62 through the N-end rule interaction of its Nt-Arg (Step 4). This induces an allosteric conformational change of p62, exposing PB1 and LIR domains. PB1 domain promotes self-oligomerization and aggregation of p62 (Step 5), together with cargoes such as cytosolic misfolded proteins (Step 6). LIR domain mediates the interaction of p62 with LC3 on the autophagic membranes (Step 7). In this model, the Nt-Arg residue acts as an autophagic delivery determinant to autophagosomes for BiP and its cargoes, an activating ligand to p62, and an autophagic degron for BiP and the cargoes of R-BiP and p62.

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