FIGURE 3.

An amino acid stretch within the ADAM10 cytoplasmic tail mediates ER retention. A, amino acid sequences of the ADAM10 C terminus deletion mutants fused to Tac. In bold, arginine-rich motifs located in the ADAM10 cytoplasmic tail. B, left, surface and total expression of TacADAM10 deletion mutants expressed in COS7 cells. Right, quantification of surface/total expression ratios. (*, p < 0.01 versus TacADAM10; n = 8–9 cells per condition). Scale bar, 20 μm. Note that only the Tac721Δ mutant was able to reach the cell surface. C, COS7 cells transfected with Tac721Δ were permeabilized and stained with antibodies against Tac (green) and either calnexin or giantin (red). Scale bar, 20 μm. Inset shows colocalization of Tac721Δ with giantin in a population of cells. D, comparative Western blot analysis of Tac, TacADAM10, and deletion mutants. Note that Tac and Tac721Δ deletion mutant were detected as two species. E, glycosylation analysis reveals that the Tac737Δ doublet and Tac734Δ band are EndoH-sensitive, indicating that they are retained in the ER. The Tac721Δ higher molecular weight species is EndoH-resistant but remains sensitive to PNGase F, indicating that it is able to leave the ER and enter the Golgi. Open circles, EndoH-resistant bands; asterisks, EndoH-sensitive bands.