Figure 4. Retention of β in endoplasmic reticulum.

(a) Cells were grown continuously in tet with or without continuous ouabain to inhibit α1. Blots were cut, and blot pieces were stained separately for α3 and β1. The expression of α3 was reduced for L924P relative to α3WT and D923N, as also seen in Fig. 2. The majority of β subunit migrated at 55 kDa in the α3WT and D923N cells, while more than half migrated at ~40 kDa in L924P, the position of β with high-mannose N-glycans in the ER. (b) On the left, L924P cells did not accumulate immature β until tet induction of mutant α3. On the right, removal of all N-glycans with PNGase F reduced both the mature and immature N-glycan forms of β to the mobility of the intact apoprotein, 30 kDa. (c) When cells were biotinylated with an impermeable reagent, only the mature form of β was recovered with streptavidin beads, not the immature N-glycan form, consistent with its residence in the ER. (d) D742Y but not D743H accumulated immature β when α3 was induced with tet. [gray scale]