Figure 2.
The S113R mutation does not affect the subcellular localization or the orientation of the C terminus of AT-1. A, Subcellular localization of AT-1 assessed in living H4 cells overexpressing a GFP-tagged version of WT (AT-1WT) or mutant (AT-1S113R) AT-1. In both cases, the GFP-tag was inserted at the C terminus so as not to disrupt the N-terminal signal anchor that ensures membrane insertion of AT-1. a, Phase-contrast; b, GFP fluorescence; c, ER tracker; d, merge of a, b, and c; e, phase-contrast; f, GFP fluorescence; g, ER tracker; and h, merge of e, f, and g. B, Predicted topology of WT (left) and SPG42-associated S113R mutant (right) AT-1. The following prediction models were used: a, SOSUI; b, PredictProtein; and c, UniProtKB. C, Schematic view of the experiment reported in D. The myc tag is shown in red. D, ER vesicles from AT-1WT and AT-1S113R expressing cells were incubated with an anti-myc antibody covalently attached to aldehyde-activated agarose beads for immunoprecipitation. After extensive washing, bound proteins were eluted by lowering the pH and analyzed by SDS-PAGE and immunoblotting. Vesicles were used under the sealed (no detergent) or open (with detergent) condition.