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) ATE1^1A7A 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 (ATE1^1B7A, ATE1^1B7B, or ATE1^1A7A) 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 ATE1^1A7A. (g) N-terminal arginylation of endogenous BiP is not induced by thapsigargin (200 nM) but by overexpressing ATE1^1A7A 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 ATE1^1A7A exhibits a longer half-life. HeLa cells expressing ATE1^1A7A 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 ATE1^1A7A (p), and induced by the transfection of various dsDNAs (q). mNHK-GFP, GFP-fused mouse α1-antitrypsin null Hong-Kong.