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Comparative Study
. 2008 Feb 26;105(8):2877-82.
doi: 10.1073/pnas.0712224105. Epub 2008 Feb 14.

Unfolded protein response and cell death after depletion of brefeldin A-inhibited guanine nucleotide-exchange protein GBF1

Carmen Citterio  1 , Alessandro Vichi, Gustavo Pacheco-Rodriguez, Angel M Aponte, Joel Moss, Martha Vaughan
Affiliations
Comparative Study

Unfolded protein response and cell death after depletion of brefeldin A-inhibited guanine nucleotide-exchange protein GBF1

Carmen Citterio et al. Proc Natl Acad Sci U S A. .

Abstract

Guanine nucleotide-exchange factors (GEFs) activate ADP-ribosylation factor (ARF) GTPases that recruit coat proteins to membranes to initiate transport vesicle formation. Three mammalian GEFs are inhibited by brefeldin A (BFA). GBF1, predominantly associated with cis-Golgi membranes, functions early in the secretory pathway, whereas BIG1 and BIG2 act in trans-Golgi or later sites. Perturbation of endoplasmic reticulum (ER) functions can result in accumulation of unfolded or misfolded proteins that causes ER stress and unfolded protein response (UPR), with accumulation of ER stress response element (ERSE) gene products. BFA treatment of cells causes accumulation of proteins in the ER, ER stress, and ultimately apoptosis. To assess involvement of BFA-sensitive GEFs in the damage resulting from prolonged BFA treatment, HepG2 cells were selectively depleted of BIG1, BIG2, or GBF1 by using specific siRNA. Only GBF1 siRNA dramatically slowed cell growth, led to cell-cycle arrest in G(0)/G(1) phase, and caused dispersion of Golgi markers beta-COP and GM130, whereas ER structure appeared intact. GBF1 depletion also significantly increased levels of ER proteins calreticulin and protein disulfide isomerase (PDI). Proteomic analysis identified ER chaperones involved in the UPR that were significantly increased in amounts in GBF1-depleted cells. Upon ER stress, transcription factor ATF6 translocates from the ER to Golgi, where it is sequentially cleaved by site 1 and site 2 proteases, S1P and S2P, to a 50-kDa form that activates transcription of ERSE genes. Depletion of GBF1, but not BIG1 or BIG2, induced relocation of S2P from Golgi to ER with proteolysis of ATF6 followed by up-regulation of ER chaperones, mimicking a UPR response.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Effect of siRNA-induced depletion of BIG1, BIG2, or GBF1 on HepG2 cell proliferation. (A) Cells were incubated without additions (C), with vehicle alone (M), or with siRNA either nonspecific, NT, or specific for BIG1 (B1), BIG2 (B2), or GBF1 (G1) for 48 or 72 h before samples (30 μg) of total cell proteins were separated by SDS/PAGE and reacted with antibodies against BIG1, BIG2, GBF1, and α-tubulin. (B) Cells were treated as in A, and viable cells (identified by exclusion of 0.4% Trypan blue) were counted. Data are means ± SD of values from three experiments (*, P < 0.05 versus control).
Fig. 2.
Fig. 2.
Effect of GBF1 depletion on PARP cleavage. HepG2 cells were incubated for 48 or 72 h as described in Fig. 1A, before separation of cell proteins (30 μg) by SDS/PAGE and reaction with antibodies against PARP. Representative blots and means ± SD of densitometric values (cleaved PARP) from three experiments are shown (*, P < 0.05 versus C).
Fig. 3.
Fig. 3.
Effect of BIG1 or BIG2 depletion on Golgi complex. Cells depleted of BIG1 or BIG2 by incubation with specific siRNA were washed and fixed with 4% formaldehyde in PBS followed by immunostaining of BIG1 or BIG2 (green), β-COP, or GM130 (red). Findings were similar in three experiments. (Scale bar: 32 μm.)
Fig. 4.
Fig. 4.
Effect of GBF1 depletion on ER and Golgi proteins and structure. (A) Cells were incubated for 48 h with GBF1 siRNA, washed, and reacted with antibodies against GBF1 (green) and β-COP, GM130, calreticulin, or PDI (red). (Scale bar: 32 μm.) (B) Cells were incubated as described in Fig. 4A for 48 h before samples of cell proteins (30 μg) were separated by SDS/PAGE and reacted with antibodies against β-COP, GM130, p115, Golgin84 (Golgi markers), calreticulin, and PDI (ER markers). Representative blots and means ± SD of densitometric values from three experiments are shown (*, P < 0.05 versus C).
Fig. 5.
Fig. 5.
Effect of GBF1 depletion on amounts of ER (BiP) and cytosolic (Hsp90β) chaperones, secreted protein α1-antitrypsin, and ER stress sensor ATF6. Cells were incubated for 48 h with NT, BIG1, BIG2, or GBF1 siRNA before samples of cell proteins (30 μg) were separated by SDS/PAGE and reacted with antibodies against BiP, HsP90β, α1-antitrypsin, or ATF6. Representative blots and means ± SD of densitometric values from three experiments are shown.
Fig. 6.
Fig. 6.
Proteolysis of ATF6 in cells incubated with GBF1 siRNA. (A) After incubation for 48 h with NT or GBF1 siRNA, cells were reacted with antibodies against ATF6 (green) and calreticulin (red). (Scale bar: 20 μm.) (B) Cells were incubated for the indicated time with GBF1 siRNA or for 48 h with NT siRNA before separation of proteins (30 μg) by SDS/PAGE and reaction with antibodies against ATF6, calreticulin, PDI, BiP, or GBF1. (Left) Representative blot showing full-length 90-kDa and activated 50-kDa ATF6. (Right) Representative blots are from the same experiment as that in Left. (Left Lower) Means ± SD of densitometric values from three experiments are shown. (C) Cells were incubated for 48 h with NT, BIG1, BIG2, or GBF1 siRNA before samples of cell proteins (30 μg) were separated by SDS/PAGE and reacted with antibodies against S2P. Findings were similar in three experiments.
Fig. 7.
Fig. 7.
Effect of GBF1 depletion or BFA treatment on localization of S2P and β-COP. Cells were incubated with NT or GBF1 siRNA for 48 h or with 10 μg/μl BFA for 1 h before immunostaining for S2P (green) and β-COP (red). Findings were similar in three experiments. (Scale bar: 32 μm.)
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