Figure 5. Analysis of Global Glycoprotein Biosynthesis during Acute ER Stress.

(A) Experimental design.

(B) HeLa cells treated with 10 mM DTT for the indicated times were pulse labeled (for 15 min), fractionated, and quantified to determine the glycoprotein-to-cytosolic protein ratio (GCR). The GCR at each time point (normalized to untreated cells) is plotted along with the overall level of protein synthesis (mean ± SD for three experiments).

(C) Radiolabeled cytosolic proteins and glycoproteins from untreated and DTT-stressed (30 min) HeLa cells. Asterisks indicate bands that are minimally attenuated relative to other glycoproteins.

(D) GCR (normalized to untreated cells analyzed in parallel) for HeLa cells acutely treated (for 30 min) with 10 µM Tg, 10 mM DTT, 10 µg/ml BFA, or 10 µM CT. Each point represents an individual experiment, with the gray bar showing the range observed. BFA* indicates treatment for 12 hr.

(E) GCR (solid line) and overall translation (dotted line) was measured in cells pretreated for between 0 and 30 min with 10 mM DTT, or treated for 30 min followed by recovery in normal media for 0–30 min. Samples from time points shaded in gray are shown to the right. Note that during acute stress, many (but not all) glycoproteins are attenuated in their biosynthesis relative to the situation during recovery.

(F) Analysis as in Figure 1A of Frizzled-7 (Fz7), TRAPα, CRF1 receptor (CRFR), and Prl-CRFR by pulse labeling and immunoprecipitation from transiently transfected cells treated for 30 min with 10 µM Tg or 10 mM DTT. Asterisks indicate nonglycosylated forms of each glycoprotein (except Frizzled-7, in which a nonglycosylated form was not detectable). The amount of the translocated and glycosylated form generated under each condition is quantified relative to untreated cells. The decrease in Frizzled-7 and TRAPα paralleled the level of translational attenuation caused by the stress.