Fig. 6. Hampered secretion and accelerated degradation of the ssYFP-µstpNQ mutants. (A) HEK293T cells were transfected with an empty vector (mock), with vectors encoding Myc-tagged wild-type versions of ssYFP-µstpCys (Cys) or ssYFP-µstpSer (Ser), or the NQ mutants of ssYFP-µstpCys (CysNQ) or ssYFP-µstpSer (SerNQ). In the NQ mutants, the N residue in the µstp was replaced by a Q residue, abolishing the only glycosylation site in the chimera. Cells were lysed, substrates were immunoprecipitated (IP) by an antibody against Myc, immunoprecipitates were treated with (+) or without (–) endo H, resolved by SDS-PAGE and immunoblotted (IB) with an antibody against Myc. Fully glycosylated WT, unglycosylated NQ and endo-H-treated de-glycosylated substrates are indicated. (B) HEK293T cells expressing ssYFP-µstpCysNQ (CysNQ) or ssYFP-µstpSerNQ (SerNQ) were preincubated for 1 hour and pulse-labeled with [³⁵S]methionine-[³⁵S]cysteine and chased for the indicated time with (+) or without (–) ALLN and MG-132. Cells were lysed, substrates were immunoprecipitated (IP) by an antibody against Myc, resolved by SDS-PAGE, electroblotted and blots were exposed to autoradiography ([³⁵S]). (C) The graph, representative of three independent experiments, illustrates the amounts of ssYFP-µstpCysNQ (CysNQ; circles) or ssYFP-µstpSerNQ (SerNQ; squares) remaining in untreated (open symbols) or ALLN- and MG-132-treated (+ Inhib; filled symbols) cells. The amounts estimated by densitometry of autoradiograms in B were calculated as a percentage of the levels of the NQ mutants at the end of the pulse (100%), and half-life values (see text) were calculated. (D) Hela cells were transfected with an empty vector (mock), with vectors encoding Myc-tagged wild-type versions of ssYFP-µstpCys (Cys) or ssYFP-µstpSer (Ser), or the NQ mutants of ssYFP-µstpCys (CysNQ) or ssYFP-µstpSer (SerNQ). Medium was collected 40 hours post-transfection, and the cells were lysed. Substrates were immunoprecipitated (IP) from the medium by an antibody against Myc, and cell lysates (10%) and immunoprecipitates from the medium were resolved by SDS-PAGE and immunoblotted (IB) with an antibody against Myc. Substrates with complex (arrowhead) or high-mannose (arrow) N-glycans and unglycosylated NQ are indicated. (E) COS-7 cells were transfected with a combination of vectors encoding Myc-tagged ssYFP-µstpSerNQ mutant (SerNQ) and galactosyl transferase-CFP (GalT-CFP). YFP (left panel) and CFP (middle panel) were visualized by confocal fluorescence microscopy individually or as merged images (right panels). Bar, 5 µm.