Figure 1.
The C-terminal 16 amino acids of prRDH contain a membrane association signal. A, Alignment of the C termini of vertebrate prRDH and rod and cone pigments. Most vertebrate visual pigments and all prRDH sequences terminate with the... (V/I)XPX motif. Cysteines are shown in red, and the residues at –2 and –4 are colored. B, Shown are 293 cells transfected with GFP fusion proteins containing either a 16 amino acid C-terminal extension from prRDH (top), the same extension but with all three cysteines mutated to alanine (center), or no C-terminal extension (bottom). Nuclei were stained with 4′,6-diamidino-2-phenylindole. In these and all subsequent diagrams of GFP fusion proteins, the GFP portion is represented by a green oval with the N terminus to the left and the C terminus to the right. C, 3H-palmitate labeling of 293 cells transfected with triple-myc-tagged pWL5 (lane 1) or pWL6 (lane 2). GFP fusion proteins were immunoprecipitated with anti-myc mAb for analysis of 3H-palmitate incorporation (left). Anti-GFP immunoblotting shows that the two GFP fusion proteins accumulate to comparable levels (right). The film for detecting the 3H label was exposed for 2 weeks. Close inspection shows two resolvable 3H-labeled bands, suggesting that at least two cysteines can be modified. The arrows indicate the GFP fusion proteins. Molecular mass standards (at left) from top to bottom, are 184, 120, 87, 64, 52, 39, 26, and 21 kDa. D, Hydroxylamine treatment releases prRDH from bovine OS membranes and reveals a C-terminal epitope. Immunoblots were probed with antibodies directed against amino acids 5–312 of bovine prRDH expressed in Escherichia coli (left; anti-FL) or a C-terminal nine amino acid peptide from bovine prRDH (right; anti-C) (Rattner et al., 2000). OS membranes were separated into pellet (P) and supernatant (S) fractions after a 5 hr incubation at room temperature with PBS (–) or 1 m hydroxylamine (HA) and heated to 55°C in SDS sample buffer with 20 mm DTT. The arrow indicates prRDH. Several proteins associated with OS membranes are observed to cross-react with the anti-prRDH C-terminal peptide antibody. Rhodopsin was not released into the supernatant with hydroxylamine treatment, as judged by Coomassie blue staining (data not shown). Molecular mass standards (at left) from top to bottom, are 110, 79, 62, 48, 37, 24, and 19 kDa.