Fig. 7. Tunicamycin hampers secretion and accelerates degradation of the ssYFP-µstp fusion proteins. (A) HEK293 cells expressing a vector encoding ssYFP-µstpCys (Cys) were preincubated for 1 hour with tunicamycin. Cells were pulse-labeled with [³⁵S]methionine-[³⁵S]cysteine and chased for the indicated time with tunicamycin together (+) or without (–) ALLN and MG-132. Cells and medium were separated, cells were lysed and ssYFP-µstpCys was immunoprecipitated (IP) from lysed cells or medium by an antibody against Myc. (B) The graph, representative of three independent experiments, illustrates the amounts of ssYFP-µstpCys remaining in untreated (open symbols) or ALLN- and MG-132-treated (+ Inhib; filled symbols) cells. The amounts recovered from medium (M; squares) or detected in cells as combined unglycosylated and glycosylated forms (C; circles) or as unglycosylated forms (C ung; triangles) were estimated by densitometry of autoradiogram A, calculated as a percentage of the ssYFP-µstpCys level at the end of the pulse (100%), and half-life values (see text) were calculated. (C) HEK293T cells expressing a vector encoding ssYFP-µstpCysNQ (CysNQ) were preincubated for 1 hour and chased with (+) or without (–) tunicamycin (Tm). Cells were pulse-labeled with [³⁵S]methionine-[³⁵S]cysteine and chased for the indicated time with or without tunicamycin together (+) or without (–) ALLN and MG-132. ssYFP-µstpCysNQ was immunoprecipitated (IP) from lysed cells by an antibody against Myc. (D) HEK293 cells expressing ssYFP-µstpSer (Ser) were preincubated for 1 hour and chased with (+) or without (–) tunicamycin (Tm). Cells were pulse-labeled with [³⁵S]methionine-[³⁵S]cysteine and chased for the indicated time with or without tunicamycin, together (+) or without (–) ALLN and MG-132. Cells and medium were separated, cells were lysed and substrates were immunoprecipitated (IP) from lysed cells (upper panel) or medium (lower panel) by an antibody against Myc. Immunoprecipitates were resolved by SDS-PAGE, electroblotted and blots were exposed to autoradiography ([³⁵S]). The unglycosylated and the residual glycosylated substrates are indicated. ssYFP-µstpSer with high-mannose (arrow) or complex (arrowhead) N-glycans are indicated. (E) The graphs, representative of three independent experiments, illustrate the amounts of ssYFP-µstpSer remaining in untreated (open symbols) or ALLN- and MG-132-treated (+ Inhib; filled symbols) cells (upper graph) or recovered from medium (lower graph). Glycosylated forms from untreated cells (gly; circles) or unglycosylated (Tm ung; triangles) and glycosylated (Tm gly; squares) forms from tunicamycin-treated cells were estimated by densitometry of autoradiogram D, calculated as a percentage of the level of ssYFP-µstpSer at the end of the pulse (100%), and half-life values (see text) were calculated.