Figure 5. COPII is required to export... | Rockefeller University Press

Figure 5.

$Figure 5. COPII is required to export PC1 in vesicles that bud from the ER in a cell-free reaction. (A) Scheme depicting the experimental procedure of the cell-free vesicle budding reactions. In brief, donor membranes prepared from IMR-90 cells were incubated at 30°C with cytosol (from HT1080 or HTPC1.1 cells), nucleotides (an ATP-regenerating system and GTP), and purified recombinant human COPII proteins. Vesicles in 7,000-g supernatant fractions from budding reactions were isolated by flotation. (B) Fractions (12) were taken from the top of an Optiprep gradient after flotation and analyzed by immunoblotting. SEC22B is found in conventional COPII vesicles and serves as a positive control. Vinculin serves as a control for cytosolic proteins that do not float with vesicles. n = 3. (C) Top floated fraction was treated with or without collagenase (0.1 U/µl) in the presence or absence of the detergent Triton X-100 (1%) at the indicated temperature for 10 min. ERGIC53 is found in conventional COPII vesicles and serves as loading control. n = 4. (D) Budding requirements of PC1 and HSP47 were assessed under different incubation conditions. The top fraction after flotation was taken from each sample and analyzed by immunoblotting. Ribophorin I is an ER resident protein that serves as a negative control. ERGIC53 and SEC22B are found in conventional COPII vesicles and serve as positive controls. n = 3. (E) To detect trimeric PC1, we treated donor membrane or floated fractions with nonreducing denaturing sample buffer before gel electrophoresis. Donor membrane or floated fractions treated with reducing denaturing sample buffers were used as controls. n = 2.$

COPII is required to export PC1 in vesicles that bud from the ER in a cell-free reaction. (A) Scheme depicting the experimental procedure of the cell-free vesicle budding reactions. In brief, donor membranes prepared from IMR-90 cells were incubated at 30°C with cytosol (from HT1080 or HTPC1.1 cells), nucleotides (an ATP-regenerating system and GTP), and purified recombinant human COPII proteins. Vesicles in 7,000-g supernatant fractions from budding reactions were isolated by flotation. (B) Fractions (12) were taken from the top of an Optiprep gradient after flotation and analyzed by immunoblotting. SEC22B is found in conventional COPII vesicles and serves as a positive control. Vinculin serves as a control for cytosolic proteins that do not float with vesicles. n = 3. (C) Top floated fraction was treated with or without collagenase (0.1 U/µl) in the presence or absence of the detergent Triton X-100 (1%) at the indicated temperature for 10 min. ERGIC53 is found in conventional COPII vesicles and serves as loading control. n = 4. (D) Budding requirements of PC1 and HSP47 were assessed under different incubation conditions. The top fraction after flotation was taken from each sample and analyzed by immunoblotting. Ribophorin I is an ER resident protein that serves as a negative control. ERGIC53 and SEC22B are found in conventional COPII vesicles and serve as positive controls. n = 3. (E) To detect trimeric PC1, we treated donor membrane or floated fractions with nonreducing denaturing sample buffer before gel electrophoresis. Donor membrane or floated fractions treated with reducing denaturing sample buffers were used as controls. n = 2.