Progressive Rod-Cone Degeneration (PRCD) Protein Requires N-Terminal S-Acylation and Rhodopsin Binding for Photoreceptor Outer Segment Localization and Maintaining Intracellular Stability

Abstract

The light-sensing outer segments of photoreceptor cells harbor hundreds of flattened membranous discs containing the visual pigment, rhodopsin, and all the proteins necessary for visual signal transduction. PRCD (progressive rod-cone degeneration) protein is one of a few proteins residing specifically in photoreceptor discs, and the only one with completely unknown function. The importance of PRCD is highlighted by its mutations that cause photoreceptor degeneration and blindness in canine and human patients. Here we report that PRCD is S-acylated at its N-terminal cysteine and anchored to the cytosolic surface of disc membranes. We also showed that mutating the S-acylated cysteine to tyrosine, a common cause of blindness in dogs and a mutation found in affected human families, causes PRCD to be completely mislocalized from the photoreceptor outer segment. We next undertook a proteomic search for PRCD-interacting partners in disc membranes and found that it binds rhodopsin. This interaction was confirmed by reciprocal precipitation and co-chromatography experiments. We further demonstrated this interaction to be critically important for supporting the intracellular stability of PRCD, as the knockout of rhodopsin caused a drastic reduction in the photoreceptor content of PRCD. These data reveal the cause of photoreceptor disease in PRCD mutant dogs and implicate rhodopsin to be involved in PRCD's unknown yet essential function in photoreceptors.

Figure 1

Membrane topology and post-translational modifications of PRCD. A, Osmotically intact discs were treated with membrane impermeable proteinase K (at 8 μg/ml) followed by Western blotting with antibodies against PRCD and rhodopsin (recognizing its cytoplasmic or intradiscal epitopes). The experiment was performed with two technical repeats for each of two independently obtained biological disc preparations. B, Mouse retina lysates were treated with combinations of DTT (100 mM), hydroxylamine (NH₂OH, 500 mM), and calf intestinal phosphatase (CIP, 10 units/ml) followed by Western blotting with anti-PRCD antibody. The experiment was performed with three individual mouse retinas. C, A cartoon depicting PRCD orientation in the disc membrane and the site of its S-acylation. The C terminus of PRCD is exposed on the cytoplasmic surface of discs, while the N terminus is anchored in the membrane and contains a lipidation attached by S-acylation (red).

Figure 2

PRCD S-acylation is not required for membrane association. A, PRCD-FLAG constructs, either wild type or C2Y mutant, were expressed in mouse retinas by in vivo electroporation and immunoprecipitated using an anti-FLAG antibody (a total of ten injected and expressing mouse retinas were used from ten different mice in one experiment). The constructs were treated by hydroxylamine (NH₂OH, 500 mM) to fully remove S-acylation. The post-treatment shift of the PRCD band (or the lack thereof) on Western blots was documented using anti-FLAG antibody. The dashed lines were drawn to assist in observing the band shift present in the wild type construct and absent in the C2Y mutant construct. B, Bovine discs were treated with hydroxylamine to fully remove PRCD S-acylation, followed by membrane sedimentation. Membranes were re-suspended in the same volume as the initial sample, and equal aliquots from the input material, soluble (Sol), and pellet fractions were analyzed by Western blotting using anti-PRCD antibody. The experiment was performed in two technical repeats for two independently purified disc preparations

Figure 3

Disease-causing PRCD mutant mislocalizes from rod outer segments. Recombinant constructs coding either wild type PRCD or its C2Y mutant behind the rhodopsin promoter were electroporated into the retinas of neonatal mice, and immunostained at P21 with an anti-FLAG antibody (green). Co-transfection with a construct coding soluble mCherry (red) allowed screening for electroporated areas of the retina prior to immunostaining for PRCD (note that the total number of cells expressing WT PRCD was higher than that expressing mCherry because the ratio between the corresponding DNA constructs upon electroporation was 2:1). A merged image is shown on the right. Nuclei are stained by Hoescht. At least three electroporated mice were analyzed for each construct and yielded similar immunolocalization patterns. Scale bar, 10 μm.

Figure 4

Co-chromatography and co-precipitation of PRCD with its binding protein candidates. A, Purified bovine discs were solubilized in 0.1% DDM and subjected to gel filtration chromatography on a Superose 12 column. 400 μl fractions were collected and aliquots were used for Western blotting with antibodies against PRCD, rhodopsin, Gα_t, peripherin and ROM1. B, Proteins from bovine disc membranes were solubilized in 0.1% DDM (input, lane #1) and incubated with anti-rhodopsin antibody 1D4 bound to protein A/G magnetic beads. After incubation, the unbound lysate was collected (lane #2), the beads were washed, and bound proteins eluted (lane #3). In a control experiment, antibody was pre-incubated with its peptide antigen to block the antibody from precipitating rhodopsin, and unbound (lane #4) and bound (lane #5) lysate was collected. All loaded samples were normalized by volume. C, D Bovine discs solubilized in 0.1% DDM (input) were incubated with anti-Gα_t (C) or anti-peripherin (D) antibodies attached to protein A/G magnetic beads. The unbound material was collected (unbound) before washing the beads and eluting bound proteins (bound). Normalized volumes of input, unbound and bound fractions were loaded for Western blotting for PRCD, peripherin, ROM1, and Gα_t using specific antibodies to each of these proteins. E, Streptavidin magnetic beads bound to PRCD peptide were incubated with three concentrations of disc lysate (250, 50 and 8 μg total protein/ml) solubilized in 0.7% CHAPS. Control experiment was performed with empty beads. Bound proteins were eluted and rhodopsin was detected by Western blotting with anti-rhodopsin antibody. Samples containing eluted proteins were normalized by volume; input samples were diluted by 70%. Each figure panel represents an image from at least 3 independent experiments.

Figure 5

PRCD is virtually absent from rhodopsin knockout rods. A, Immunofluorescence staining of PRCD in cross-sections of WT (left) and Rho^−/− (right) mouse retinas collected at P21. Staining of rhodopsin (red), PRCD (green) and peripherin (green) was performed with anti-rhodopsin, anti-PRCD, and anti-peripherin antibodies, respectively. Nuclei are stained by Hoescht. Scale bar, 10 μm. B, Western blot of rhodopsin, PRCD and peripherin in serial dilutions of mouse retinal lysates from WT, Rho^+/− and Rho^−/− mice. The lysates were treated with phosphatase and hydroxylamine to fully remove PRCD post-translational modifications. Representative images are taken from three independent experiments.