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BiP is feed-back regulated by control of protein translation efficiency

Karsten Gülow{ddagger}, Detlef Bienert and Ingrid G. Haas*,§

Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany
{ddagger} Present address: Division of Immunogenetics, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120, Heidelberg, Germany
§ Present address: Max-Planck-Institut für Immunbiologie, Stübeweg 51, D-79108 Freiburg, Germany



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  		Fig. 1. BiP expression is tightly controlled in unstressed cells. (A) Left, detection of mouse BiP in lysates of Bil11. Bil11 cells were cultured in the presence of tetracycline, and lysates were prepared from equal amounts of cells taken at time points indicated after removal of tetracycline. Equivalents of 105 cells were loaded. The western blot was developed with anti-GRP78 antiserum, which detects only heterologous mouse BiP (m-BiP), and with anti-tubulin antibodies. Right, tubulin was used for normalization of the total cellular proteins loaded. Amounts of mouse BiP (black bars) were expressed as a percentage of steady state amounts of mouse BiP present in permanently activated cells. (B) Left, western blot analysis of total BiP (t-BiP) performed on one tenth of the lysates used in A. The blot was developed with monoclonal anti-BiP antibody detecting both human and mouse BiP equally and with anti-tubulin antibodies. Right, tubulin was used for normalization of the cellular proteins loaded. Amounts of total BiP (grey bars) were expressed as a percentage of steady state levels of total BiP present in permanently activated cells. (C) Left, quantification of mouse and total BiP in permanently activated cells. The amounts of lysate indicated were prepared from permanently activated cells and mixed with defined amounts of purified recombinant BiP-GST fusion protein (BiP/GST), as indicated. Mouse or total BiP was detected by using anti-GRP78 antiserum (upper panel) or monoclonal anti-BiP antibody (lower panel), respectively. Note that the BiP-GST fusion protein contains the epitopes for both anti-GRP78 antiserum and monoclonal anti-BiP antibody. Right, calculation of the ratio of mouse BiP according to the amount of total BiP after removal of tetracycline. Amounts of mouse BiP and total BiP giving signals in the linear range were assigned to a corresponding amount of BiP-GST fusion protein, allowing the determination of the ratio of mouse BiP to total BiP in permanently activated cells. The gradual replacement of human BiP (gray bars) by mouse BiP (black bars) after removal of tetracycline was monitored.

 


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  		Fig. 2. BiP is controlled at a translational level. (A) Left, northern blot analysis of poly A+ RNA isolated from Bil11 cells activated for various time periods, as indicated. The probe detecting both mouse (m-BiP) and human BiP (h-BiP) corresponds to a 1.1 kb fragment of the mouse BiP coding sequence (which has more than 92% identity to the human sequence). Actin mRNA was probed with the complete actin coding sequence. Right, quantification was performed by phosphoimaging and normalized for actin signals. Relative amounts of endogenous human BiP mRNA (grey bars) and heterologous mouse BiP mRNA (black bars) are presented. (B) Left, determination of the rate of BiP synthesis in Bilu33 cells expressing only endogenous human BiP (h-BiP) and activated Bil11 cells co-expressing human and mouse BiP (t-BiP). Cells were cultured in the absence of tetracycline, and lysates were prepared at time points indicated after 35S-methionine addition. BiP was immunoprecipitated by monoclonal anti-BiP antibody. To directly compare BiP synthesis rates in the different cells, the respective amounts of cell lysates were normalized according to the amount of TCA-precipitated radioactivity determined in the first samples. Right, gels were exposed for the same time period and relative signal intensities of BiP in Bilu33 (grey bars) and Bil11 cells (black bars) were determined by phosphoimaging. (C) Left, kinetics of BiP degradation in Bilu33 expressing only endogenous human Bip (h-Bip) and activated Bil11 co-expressing human and mouse BiP (t-BiP). Cells were pulse labeled for 1.5 hours with 35S-methionine, and identical volumes of culture were taken to prepare lysates at the time points indicated after initiation of the chase. Total BiP was immunoprecipitated by monoclonal anti-BiP antibody and signals quantified by phosphoimaging. Right, the amount of labeled BiP (grey bars: human BiP in Bilu33; black bars: total BiP in Bil11) is expressed as a percentage of labeled BiP isolated directly after the pulse.

 


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  		Fig. 3. Kinetics of upregulation of BiP expression during UPR. (A) Activated Bil11 cells expressing endogenous human and additional mouse BiP were treated with tunicamycin for 8 hours, and RNA was prepared at the different time points indicated. A northern blot of poly A+ RNA was hybridized with the BiP-specfic probe (also used in Fig. 2A), detecting mouse BiP/luciferase mRNA (mBiP/luc) and endogenous human BiP mRNA. Quantification was performed by phosphoimaging and normalized for actin signals. Black bars represent mouse BiP/luciferase mRNA, and grey bars represent human BiP mRNA. (B) Determination of global protein synthesis. DMSO or tunicamycin (in DMSO) was added to activated Bil11 cells for 1.5 hours prior to addition of 35S-methionine. Lysates were prepared from equivalent amounts of cells harvested at the time points indicated after 35S-methionine addition. For determination of the rate of total protein synthesis, incorporation of 35S-methionine into TCA-precipitated material from lysates of tunicamycin- (black bars) or mock-treated (gray bars) cells was measured. (C) Upper panel, for determination of the rate of BiP synthesis, monoclonal anti-BiP antibody was used for immunoprecipitation of total (human and mouse) BiP (t-BiP). Lower panel, relative signal intensities of total BiP isolated from lysates of tunicamycin- (black bars) or mocktreated (gray bars) cells. (D) Upper panel, for determination of the rate of mouse BiP (m-BiP) synthesis, anti-BiP antiserum was used for immunoprecipitation of mouse BiP. Lower panel, relative signal intensities of mouse BiP isolated from lysates of tunicamycin- (black bars) or mock-treated (gray bars) cells.

 


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  		Fig. 5. Analysis of ribosome distribution in unstressed and stressed HeLa cells. (A) Upper panel, polysome profiles of mock- (left) or tunicamycin-treated (90 minutes; right) HeLa cells. Absorbance at 254 nm (y axis, indicating RNA concentration) is plotted against migration in the 10-50% sucrose gradients (x axis). The positions of polysomes and ribosomal subunits are indicated. The monosomal 80S peak is not resolved from the 60S subunit peak in these gradients. (B) Fractions corresponding to the profile shown in A were pooled as indicated and analyzed by northern blot using a BiP-specific probe (upper panel) that detected endogenous human BiP mRNA (hBiP) or an actin-specific probe (lower panel). Note that after 90 minutes of tunicamycin treatment, increase in BiP mRNA was still below a factor of 1.5.

 


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  		Fig. 4. UPR induction by thapsigargin and effect of actinomycin D on BiP synthesis in activated Bil11 cells. (A) Determination of 35S-methionine incorporation into TCA-precipitated material from lysates of thapsigargin- (black bars) or mock- (DMSO-)treated cells (gray bars) prepared at the time points indicated after 35S-methionine addition. (B) Upper panel, determination of BiP (t-BiP) synthesis rate was performed as described in Fig. 3C. Lower panel, relative signal intensities of total BiP in thapsigargin- (black bars) or mock- (DMSO-)treated (grey bars) cells. (C) Upper panel, determination of mouse BiP synthesis rate was performed as described in Fig. 3D. Lower panel, relative signal intensities of mouse BiP isolated from thapsigargin- (black bars) or mock-treated (gray bars) cells. (D) Determination of 35S-methionine incorporation into TCA-precipitated material from lysates of activated Bil11 cells treated with thapsigargin during starvation (90 minutes) and pulse, and additionally with actinomycin D 5 minutes prior to and during pulse (black-checkered bars). Mock- (DMSO-)treated cells are also shown (gray bars). (E) Upper panel, determination of BiP (t-BiP) synthesis rate was performed as described in Fig. 3C. Lower panel, relative signal intensities of total BiP in thapsigargin and actinomycin D- (black-checkered bars) or mock (DMSO)-treated (grey bars) cells. (F) Upper panel, determination of mouse BiP synthesis rate was performed as described in Fig. 3D. Lower panel, relative signal intensities of total BiP in thapsigargin and actinomycin D- (black-checkered bars) or mock- (DMSO-)treated (grey bars) cells.

 

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