Research ArticleStress responses

The endoplasmic reticulum–residing chaperone BiP is short-lived and metabolized through N-terminal arginylation

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Sci. Signal.  02 Jan 2018:
Vol. 11, Issue 511, eaan0630
DOI: 10.1126/scisignal.aan0630
  • Fig. 1 BiP is short-lived.

    (A) Cycloheximide degradation assay. PC3 cells were cultured in the absence or presence of 1 μM thapsigargin for 16 hours. Cells were treated with cycloheximide (30 ng/ml; CHX) for the indicated periods of time and immunoblotted. Representative of three biological replicates. (B) Quantification of (A). Shown are the percentages of BiP amounts relative to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) or actin amounts. (C and D) The same assays as in (A) and (B) using human embryonic kidney–293 (HEK293) cells. Representative of three biological replicates. (E and F) The same assays as in (A) and (B) using HeLa cells. Representative of three biological replicates. (G) Pulse chase degradation assay. HeLa cells were incubated with 0.2 μM thapsigargin in the absence or presence of 10 μM MG132. Newly synthesized proteins were pulsed/labeled with 35S-Met/Cys for 12 min. Cells were incubated with cycloheximide for the indicated periods of time, then subjected to immunoprecipitation and autoradiography of soluble 35S-Met/Cys–labeled BiP. Representative of three biological replicates. (H) Quantitation of (G). Shown are the percentages of BiP signals relative to that at time zero in control cells.

  • Fig. 2 The Nt-arginylation of BiP is induced by the accumulation of misfolded proteins.

    (A) HeLa cells transfected with (plasmid) enhanced green fluorescent protein (pEGFP) or ATE11A7A were treated with 1 μM thapsigargin, tunicamycin (1 μg/μl), 2 mM dithiothreitol (DTT), 5 mM 2-deoxyglucose (2-DG), or 2 μM MG132 and were analyzed by immunoblotting. Representative of three biological replicates. (B) HeLa cells were treated with thapsigargin and/or MG132 for the indicated periods of time and analyzed by immunoblotting. Representative of three biological replicates. (C) HeLa cells were transfected with pEGFP or Null Hong Kong–green fluorescent protein (NHK-GFP) plasmid and cultured for the indicated periods of time and analyzed by immunoblotting. Representative of three biological replicates. (D) HeLa cells transfected with pEGFP or NHK-GFP plasmid were treated with thapsigargin and/or MG132 and subjected to immunoblotting analysis. Representative of three biological replicates. (E) Cells transfected with scrambled or small interfering RNA (siRNA) targeting BiP were treated with thapsigargin and MG132 and analyzed by immunoblotting. Representative of three biological replicates. (F) HeLa cells were transfected with a plasmid encoding GFP or ATE11A7A or treated with thapsigargin and MG132, and then immunoblotted. Representative of three biological replicates. (G) HeLa cells transiently expressing GFP or ATE11A7A were analyzed by quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. Representative of three biological replicates. (H) Quantitative RT-PCR was performed on total RNAs from HeLa cells transfected with siRNAs targeting ATE1. Representative of three biological replicates.

  • Fig. 3 ER stress and proteasomal inhibition–induced R-BiP is located in the cytosol.

    (A and B) Immunostaining analysis of HeLa cells treated with 1 μM thapsigargin and 2 μM MG132. Scale bars, 10 μm. Representative of three biological replicates. (C) HeLa cells were treated with 1 μM thapsigargin and/or 2 μM MG132 and fractionated using differential centrifugation. Total (T) and cytosolic (C) fractions were analyzed using immunoblotting analysis. Representative of three biological replicates. (D) HeLa cells were treated with 1 μM thapsigargin or 2 μM MG132 and separated into cytosolic and microsomal fractions using differential centrifugation. Representative of three biological replicates.

  • Fig. 4 The formation of R-BiP is independent from the ERAD core machinery.

    (A) A diagram showing ERAD components, for which HERP acts as a scaffolding protein. (B) Quantitative RT-PCR was performed on total RNA from HeLa cells transfected with the indicated siRNAs. Representative of three biological replicates. (C) HeLa cells transfected with the indicated siRNAs were treated with 1 μM thapsigargin and analyzed by immunoblotting. Representative of three biological replicates. (D) HeLa cells transfected with the indicated siRNAs were treated with 1 μM thapsigargin and analyzed by immunoblotting. Representative of three biological replicates. (E) HeLa cells treated with thapsigargin and MG132 were incubated with brefeldin A (BFA), followed by immunoblotting. Quantification of the intensities of R-BiP bands relative to those of actin is shown. Representative of three biological replicates. (F) The same assays as in (E) using Exo 1 instead of brefeldin A. Quantification of the intensities of R-BiP bands relative to those of actin is shown. Representative of three biological replicates.

  • Fig. 5 Nt-arginylation is targeted to autophagy and mediates the turnover of BiP.

    (A) Immunostaining analysis of R-BiP and p62 in HeLa cells treated with thapsigargin and MG132. Scale bars, 10 μm. Representative of three biological replicates. (B) Quantitation of R-BiP punctate signals that colocalize with p62 punctate signals in (A). (C) Immunostaining analysis of R-BiP and LC3 in HeLa cells treated with thapsigargin and MG132. Scale bars, 10 μm. Representative of three biological replicates. (D) Quantitation of R-BiP punctate signals that colocalize with LC3 punctate signals in (C). (E) HeLa cells were incubated with thapsigargin and MG132 for the indicated periods of time and analyzed by immunoblotting. Representative of three biological replicates. (F) HeLa cells were incubated with MG132 or bafilomycin A1, treated with cycloheximide for the indicated periods of time, and analyzed by immunoblotting. Representative of three biological replicates. (G) Wild-type (WT) and ATG5−/− MEFs were incubated with thapsigargin and MG132, treated with cycloheximide for the indicated periods of time, and analyzed by immunoblotting. Representative of three biological replicates. (H) HeLa cells transfected with pcDNA or ATE11A7A were incubated with MG132, treated for the indicated periods of time with cycloheximide, and analyzed by immunoblotting analysis. Representative of three biological replicates. (I) HeLa cells were incubated with thapsigargin and MG132, treated with cycloheximide for the indicated periods of time, and analyzed by immunoblotting. Representative of three biological replicates.

  • Fig. 6 The Nt-arginylation of BiP and the increases in HERP amounts are co-induced under ER stress synergistically associated with proteasome inhibition.

    (A) HeLa cells were treated with 1 μM thapsigargin, tunicamycin (1 μg/μl), 1 μM A23187, 1 μM MG132, 100 μM chloroquine, 200 μM H2O2, 300 μM CoCl2, or 1 μM doxycycline and analyzed by immunoblotting. Representative of three biological replicates. (B) HeLa cells were treated with 1 μM thapsigargin for the indicated periods of time and analyzed by immunoblotting. Representative of three biological replicates. (C) HeLa cells were treated with 2 μM MG132 for the indicated periods of time and analyzed by immunoblotting. Representative of three biological replicates. (D) HeLa cells were treated with thapsigargin and/or MG132 for the indicated periods of time and analyzed by immunoblotting. Representative of three biological replicates.

  • Fig. 7 HERP counteracts the Nt-arginylation of BiP.

    (A) HeLa cells were transfected with scrambled or HERP siRNA, treated with cycloheximide for the indicated periods of time, and analyzed by immunoblotting. Representative of three biological replicates. (B) Quantitation of (A). (C) HeLa cells were transfected with scrambled or HERP siRNA, treated with thapsigargin for the indicated periods of time, and analyzed by immunoblotting. Representative of three biological replicates. (D) Quantitation of (C). (E) HeLa cells transfected with pcDNA or HERP-HA were left untreated or treated with cycloheximide for the indicated periods of time and analyzed by immunoblotting. Representative of three biological replicates. (F) Quantitation of (E). (G) HeLa cells transfected with pcDNA or HERP-HA plasmid were treated with thapsigargin for the indicated periods of time and analyzed by immunoblotting. Representative of three biological replicates. (H) Quantitation of (G). (I) HeLa cells transfected with the plasmid encoding HERP-HA, GP78-Flag, HRD1-Flag, or p97-His were treated with thapsigargin and analyzed by immunoblotting. Representative of three biological replicates. (J) HeLa cells were transfected with the plasmid expressing full-length HERP, ΔN114 lacking the N-terminal 114 residues, ΔN200 lacking the N-terminal 200 residues, and ΔC191 lacking the C-terminal 191 residues. Cells were treated with thapsigargin and MG132 and analyzed by immunoblotting. A nonspecific protein band is indicated by an asterisk. Representative of three biological replicates. (K) HeLa cells transfected with scrambled or HERP siRNA were treated with thapsigargin in the absence or presence of brefeldin A and analyzed by immunoblotting. Representative of three biological replicates.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/511/eaan0630/DC1

    Fig. S1. A model for the mechanism underlying the turnover and Nt-arginylation of BiP.

    Fig. S2. The overexpression of ATE1 or knockdown of ATE1 does not induce the UPR.

    Fig. S3. The knockdown of ERAD components does not induce BiP at the mRNA level.

    Fig. S4. Validation of knockdown efficiency for ERAD components.

    Fig. S5. HERP knockdown increases the abundance of R-BiP.

    Fig. S6. The 54-kDa HERP protein is induced and rapidly degraded under ER stress.

    Table S1. Sequences of the primers used in this study.

  • Supplementary Materials for:

    The endoplasmic reticulum–residing chaperone BiP is short-lived and metabolized through N-terminal arginylation

    Sang Mi Shim, Ha Rim Choi, Ki Woon Sung, Yoon Jee Lee, Sung Tae Kim, Daeho Kim, Su Ran Mun, Joonsung Hwang, Hyunjoo Cha-Molstad, Aaron Ciechanover, Bo Yeon Kim,* Yong Tae Kwon*

    *Corresponding author. Email: yok5{at}snu.ac.kr (Y.T.K.); bykim{at}kribb.re.kr (B.Y.K.)

    This PDF file includes:

    • Fig. S1. A model for the mechanism underlying the turnover and Nt-arginylation of BiP.
    • Fig. S2. The overexpression of ATE1 or knockdown of ATE1 does not induce the UPR.
    • Fig. S3. The knockdown of ERAD components does not induce BiP at the mRNA level.
    • Fig. S4. Validation of knockdown efficiency for ERAD components.
    • Fig. S5. HERP knockdown increases the abundance of R-BiP.
    • Fig. S6. The 54-kDa HERP protein is induced and rapidly degraded under ER stress.
    • Table S1. Sequences of the primers used in this study.

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    Citation: S. M. Shim, H. R. Choi, K. W. Sung, Y. J. Lee, S. T. Kim, D. Kim, S. R. Mun, J. Hwang, H. Cha-Molstad, A. Ciechanover, B. Y. Kim, Y. T. Kwon, The endoplasmic reticulum–residing chaperone BiP is short-lived and metabolized through N-terminal arginylation. Sci. Signal. 11, eaan0630 (2018).

    © 2018 American Association for the Advancement of Science

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