Figure 1.
Transport of CT B-subunit into the Golgi and the ER. (A) N-Glycosylation and tyrosine sulfation sites added to the C terminals of CTB-subunit and LTIIbB-subunit. (B) Time course of Cl– secretion measured as short circuit current (Isc) induced by 20 nM wt CT (squares) or CT-GS (circles) after application at 0 min to basolateral (filled symbols) or apical membranes (open symbols) of T84 monolayers. Representative of three independent experiments. (C) Phosphorimage showing sulfation (marked by *) and N-glycosylation (marked by **) of CT-GS B-subunit 3 h after apical and basolateral application to T84 cells. Immunoprecipitates were divided into three equal samples and treated with PNGaseF (lane 2), EndoH (lane 3), or mock treated (lane 1) before analysis by SDS-PAGE. Representative of two independent experiments. (D and E) Top, phosphorimages showing time course of 35S sulfation (*) and N-glycosylation (**) of B-subunit of CT-GS applied at 0 min to basolateral (D) or apical membranes (E) of T84 cells. Bottom, immunoblot for CT B-subunit to show equal loading for each lane. (F) Phosphorimage (top) showing effect of 10 μM BFA on sulfation and N-glycosylation of CT-GS B-subunit 3 h after application to T84 cells. T84 cells were treated (lane BFA) or not treated with BFA (lane N), or treated with BFA for 2 h, washed to remove BFA, and incubated again for an additional 3 h (lane BFA-wash). Bottom, immunoblot for CT B-subunit. (G) Phosphorimage (top) showing effect of methyl-β-cyclodextrin on sulfation and N-glycosylation of CT-GS B-subunit 3 h after application to T84 monolayers (lane mβ-CD) or mock-treated control (lane N). Bottom, immunoblot for CT B-subunit. Representative of two independent experiments.