Article Figures & Data
Figures
- 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.
- Figure 2.
The C-terminal 16 amino acids of prRDH targets GFP to the OS of both rods and cones in transgenic Xenopus. A, C, GFP without a C-terminal extension localizes predominantly to the cell bodies of rods (A) and cones (C). B, D, GFP fused to the C-terminal 16 amino acids of prRDH localizes predominantly to the OS in rods (B) and cones (D). Left, GFP (green). Right, Overlay of GFP, ROS labeled with Texas Red-conjugated WGA (red), and 4′,6-diamidino-2-phenylindole (blue). The arrowheads in this and all subsequent figures mark the IS–OS junction.
- Figure 3.
Role of the C-terminal cysteines of prRDH in ROS localization and membrane association. A–D, Subcellular localization in transgenic Xenopus rods of GFP fusion proteins carrying mutated derivatives of the C-terminal 16 amino acids of prRDH. The three prRDH cysteines were mutated to alanine together (A) or individually (B–D). E, Membrane association and electrophoretic mobility of the pWL86–GFP fusion protein from F1 tadpoles. Left and center, Purified ROS were treated with PBS (–), with PBS containing 1% Triton-X 100 (Triton), or with 1 m hydroxylamine (HA) and divided into supernatant (S) and pellet (P) fractions by centrifugation [confirmed by Coomassie staining of rhodopsin (data not shown)]. Right, Mixing the untreated and hydroxylamine-treated samples (M) shows that the shift in apparent molecular mass attributable to hydroxylamine treatment is not an electrophoretic artifact. The arrows at right indicate the GFP fusion protein before and after hydroxylamine treatment. 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 64, 52, 39, 26, and 21 kDa.
- Figure 4.
The... VXPX motif at the C terminus of prRDH facilitates ROS localization of GFP fusion proteins. A, Deletion of the four C-terminal amino acids from the GFP–prRDH C-terminal fusion produces only a small decrease in the efficiency of ROS localization (compare with Fig. 2 B). B, Mutation of the conserved valine at –4 and proline at –2 to alanine results in localization of the majority of the GFP fusion protein to the IS, cell body, and base of the ROS. Bottom center, Endogenous rhodopsin (stained with mAb 1D4; red) is not mislocalized in rods expressing pWL167. We note that the high rhodopsin concentration in the ROS leads to efficient capture of the 1D4 mAb near the ROS surface with inefficient immunostaining in the ROS interior.
- Figure 5.
ROS localization of GFP fusions with N-terminal membrane association domains and C-terminal... (V/I)XPX motifs. A–C, An N-terminal extension derived from Lyn kinase and containing a myristoylation site and a palmitoylation site (G and C, respectively) fused with GFP (A) or with GFP carrying the eight C-terminal amino acids from prRDH (B) or rhodopsin (C). D–F, An N-terminal extension derived from GAP43 and containing two palmitoylation sites (each represented by C) fused with GFP (D) or with GFP carrying the eight C-terminal amino acids from prRDH (E) or rhodopsin (F). In D–F, the fusion protein accumulates most prominently at the base of the ROS. The addition of the prRDH or Xenopus rhodopsin C terminus to the Lyn kinase–GFP fusion leads to an increase in the relative efficiency of ROS localization (A, compare B, C), whereas the addition of the same segment to the GAP43–GFP fusion has little effect (D, compare E, F).
- Figure 6.
Additional palmitoylation sites increase ROS localization of GAP43-GFP fusion proteins with C-terminal... (V/I)XPX motifs. A–C, An N-terminal extension derived from GAP43 and containing three palmitoylation sites (each represented by C) fused with GFP alone (A) or with GFP carrying the eight C-terminal amino acids from prRDH (B) or rhodopsin (C). D–F, An N-terminal extension derived from GAP43 and containing four palmitoylation sites (each represented by C) fused with GFP alone (D) or with GFP carrying the eight C-terminal amino acids from prRDH (E) or rhodopsin (F). Highly efficient ROS localization is observed for the GFP fusion protein with four palmitoylation sites when the... (V/I)XPX motif is added at the C terminus (D, compare E, F).
Tables
