Fig. 1

Pro/Gly scan through full-length provasopressin suggests more than one ER aggregating segment. a Domain organization of preprovasopressin. Cysteines are indicated by red dots, disulfide bonds by red lines, and glycosylation by a black diamond. Dots above the sequence indicate distinct mutations causing autosomal dominant neurohypophyseal DI (missense or deletion in black, nonsense or frameshift in pink). The natural mutants used in this study, ∆E47 and C61X, are labeled. The scale indicates the number of the amino acids in provasopressin. In constructs Pro1–Pro10 successive segments of 10 residues in provasopressin were replaced by proline/glycine-rich sequences as illustrated. b The constructs were expressed in HN10 cells for 2 days and analyzed by immunofluorescence staining. Pro1/2/7/9-expressing cells are shown as examples. Nuclei were stained with DAPI (blue). Bar: 10 μm. c The fraction of expressing cells with ER aggregates was quantified and plotted (mean and individual values of three or four independent transfections (as indicated), analyzing ~200 expressing cells per transfection as described in “Methods”). d Immunoblot analysis of transfected cells after separation by reducing sodium dodecyl sulfate (SDS)-gel electrophoresis is shown. Considerable amounts of SDS- and dithiothreitol-resistant provasopressin oligomers were detected for all proteins except Pro1. Molecular weight standards are indicated in kilodaltons