Loewen CJ , Moritz OL , Tam BM , Papermaster DS , Molday RS .

EY02422 NEI NIH HHS , EY6891 NEI NIH HHS , R01 EY002422 NEI NIH HHS , R01 EY006891 NEI NIH HHS , F32 EY006891 NEI NIH HHS

	Figure 1. Analysis of Xenopus peripherin-2-GFP fusion protein. (A) Topological model for Xenopus peripherin-2 showing the location of the GFP fusion protein, the epitope for the Xper5A11 mAb, mutations analyzed in this study (white circles), and amino acids that differ from the published sequence (black circles). (B) Western blots of Xenopus peripherin-2-GFP expressed in COS-1 cells. COS-1 membranes were preincubated with or without DTT, solubilized in Triton X-100, treated with or without glutaraldehyde (Glut), and fractionated on SDS gels under reducing (+BME, β-mercaptoethanol) or nonreducing (-BME) conditions. Lanes a–d: WT Xenopus peripherin-2-GFP labeled with an anti-GFP antibody. Lane e: C150S Xenopus peripherin-2-GFP labeled with anti-GFP antibody. Lane f: Coexpression and coimmunoprecipitation of Xenopus peripherin-2-GFP (XPerGFP) and bovine peripherin-2 (BPer) with bovine specific Per2B6-Sepharose. Western blots were labeled with an anti-GFP antibody to detect XperGFP and the Per2B6 antibody to detect BPer.
	Figure 2. Endogenous peripherin-2 and WT peripherin-2-GFP localize to the ROS disk rims and incisures. (A and B) X. laevis retina was labeled with TR-WGA (red) and antiperipherin-2 Xper5A11 antibody (green) and stained with Hoescht 33342 (blue). (A) TR-WGA labeling of glycoproteins in rod (ros) and cone (cos) outer segments; inner segment (is) and nuclei (n) are not labeled; (B) peripherin-2 labeling along the disk rims and incisures (arrowheads) and one edge of the cos (arrow). Bar, 5 μm. (C–E) Cryosections of 4-week-old tadpole eyes expressing WT Xenopus peripherin-2-GFP (green) and stained with TR-WGA (red) and Hoescht 33342 (blue). (C) Retina expressing Xenopus peripherin-2-GFP in ROS. Fusion protein is also present in phagosomes in retinal pigment epithelial cells (arrow). l, lens; r, retina; rpe, retinal pigment epithelium; Bars, 100 μm. (D and E) Micrographs of retinas expressing Xenopus peripherin-2-GFP at higher magnification showing peripherin-2-GFP localized to the ROS disk rims and incisures (arrowheads). Bars, 10 μm (D) and 5 μm (E); (Inset) Cross section of a ROS. Bar, 1 μm. All detectable WT peripherin-2-GFP localized to the ROS. F-H. Electron micrographs of retina labeled for Xenopus peripherin-2-GFP with an anti-GFP antibody and immunogold markers. (F) Longitudinal section (Bar, 0.2 μm) and (G) transverse section (Bar, 0.5 μm) of a rod expressing moderate levels of fusion protein. Peripherin-2-GFP is present on the disk rims (arrows) and incisures (arrowheads). (H) Composite longitudinal section of a ROS expressing high levels of the fusion protein. Some missorting of the fusion protein to the disk lamellae and constriction of the rod outer segment is evident. Bar, 0.5 μm.
	Figure 3. C214S peripherin-2-GFP is retained in the rod inner segment and cell body. (A and B) Confocal micrographs of Xenopus retinas expressing Xenopus C214S-peripherin-2-GFP (green) and labeled with TR-WGA (red) and Hoescht 33342 (blue). All rod photoreceptors expressing C214S peripherin-2-GFP showed localization of the fusion protein in the rod inner segments and cell bodies; in a small number of high expressing cells, however, some fusion protein is also detected at the base of the ROS. (C) Electron micrograph of immunogold labeling of C214S-peripherin-2-GFP showing protein accumulation near the cilium. (D) Confocal micrograph of a Xenopus retina expressing bovine WT-peripherin-2-GFP (green) and labeled for endogenous Xenopus peripherin-2 with Xper5A11 antibody (red). The WT bovine fusion protein, like endogeneous Xenopus peripherin-2 localizes to the ROS. (E) Xenopus retina expressing bovine C214S-peripherin-2-GFP (green) and labeled for endogenous Xenopus peripherin-2 (red). The C214S fusion protein does not localize to ROS or affect the targeting of endogenous peripherin-2 to the ROS. os, outer segment; is, inner segment; n, nucleus; cc, connecting cilium; mi, mitochondrion. Bars: (A, B, D, and E) 5 μm; (C) 0.2 μm.
	Figure 4. P216L peripherin-2 targets to the ROS and induces degeneration. (A) Fluorescence and (B) corresponding DIC image of a retina expressing Xenopus P216L-peripherin-2-GFP (green) and labeled with TR-WGA (red) and Hoescht 33342 (blue). Rods in the peripheral retina (arrow) appear normal while rods in the central retina (arrowhead) have short, highly disorganized outer segments. All detectable P216L fusion protein targets to ROS. Confocal micrographs of peripheral (C) and central (D and E) regions of the same retina show specific targeting of the fusion protein to ROS and degeneration of the central rods. (F–H) Electron micrographs of ROS labeled with an anti-GFP antibody and immunogold particles. (F) ROS from peripheral retina show normal ultrastructure with the P216L mutant protein localized to the disk rims (arrows) and incisures (arrowheads). (G and H) ROS from the central retina appear highly disorganized with whorls of disk membranes with an irregular distribution of fusion protein. ros, rod outer segment; ris, rod inner segment; n, nucleus. Bars: (A and B) 20 μm; (C) 5 μm; (D) 10 μm; (E) 5 μm; (F–H) 0.25 μm.
	Figure 5. Distribution of L185P and C150S peripherin-2-GFP in rod photoreceptors. (A and B) Retina expressing Xenopus L185P peripherin-2-GFP (green) and labeled with TR-WGA (red) and Hoescht 33342 (blue). The L185P mutant is present in ROS in all cells; however, in >25% of the cells the L185P mutant is also present in the inner segment and cell body. (C) Retina expressing Xenopus C150S peripherin-2-GFP in ROS. The fusion protein is exclusively localized to the ROS. The vertical striations characteristic of disk incisure localization are not evident; instead a more mottled labeling of the outer segments is observed with some outer segments showing a single vertical column of labeling (arrows; asterisk marks a ROS in cross section). (D) Electron micrograph showing immunogold labeling of the C150S peripherin-2-GFP. Some missorting to the lamellar region of the disks is apparent. ros, rod outer segment; ris, rod inner segment; n, nucleus. Bars: (A and B) 5 μm; (C) 10 μm; (D) 2.5 μm.
	Figure 6. Expression and biochemical properties of Xenopus peripherin-2-GFP in cultured COS cells. (A) Confocal micrographs of COS cells expressing WT, C214S, or P216L Xenopus peripherin-2-GFP (XPer-GFP) and labeled with an anticalnexin antibody as an ER marker. WT peripherin-2 and a portion of the mutants are present in intracellular vesicles that do not label for calnexin. A significant fraction of the C214S mutant as well as L216P, however, is retained in the ER. (B) Membranes from cells transfected with wild-type (WT), C214S or P216L peripherin-2-GFP were solubilized and incubated in the presence or absence of PNGase. Peripherin-2 was resolved by SDS gel electrophoresis and detected on Western blots labeled with an anti-GFP antibody. A small shift in glycosylated (gly) WT and C214S peripherin-2 and hyperglycosylated (hgly) P216L peripherin-2 to its deglycosylated (dgly) form is observed after treatment with PNGase.
	Figure 7. Velocity sedimentation of P216L and C214S peripherin-2 mutants under nonreducing conditions. COS-1 cells expressing bovine P216L (A) and C214S mutant (B) were solubilized in Triton X-100 and subjected to velocity sedimentation. Western blots of the fractions run on nonreducing SDS gels were labeled with the Per2B6 peripherin-2 antibody. The P216L peripherin-2 exhibits a profile consisting of noncovalent tetramers (a) and higher order disulfide-linked oligomers (b) similar to that observed for WT peripherin-2 (Loewen and Molday, 2000). The C214S peripherin-2 produces a mixture of noncovalent dimers (c), tetramers composed of disulfide-linked dimers (d), and aggregated species that sediment near the bottom of the tube.
	Figure 8. Schematic summarizing the relationship between peripherin-2 subunit assembly, targeting and ADRP. WT (white) and the P216L (shaded) peripherin-2, which form a mixture of core tetramers and disulfide-linked oligomers in association with endogenous X. laevis peripherin-2, target normally to ROS disks (patterned region). The P216L mutant causes ADRP through a dominant negative mechanism. The C150S mutant, which forms core tetramers but not disulfide-linked oligomers, also targets to ROS disks. The L185P mutant forms homodimers and disulfide-linked tetramers, which are retained in the inner segment, and noncovalent tetramers and oligomers with WT peripherin-2, which target to ROS. The C214S mutant forms homodimers, disulfide-linked tetramers and aggregates. These complexes are retained in the cell body, inner segments and cilium. The C214S and L185P mutants cause ADRP through a deficiency in functional core tetramers.
	Figure 9. Model depicting a checkpoint that allows only correctly assembled peripherin-2 tetramers to be incorporated into nascent disks. Peripherin-2 tetramers and a significant fraction of dimers are processed through the ER and Golgi and exit as peripherin-2–containing post-Golgi vesicles in the rod inner segment (RIS). These vesicles are translocated to the base of the connecting cilium. A checkpoint prevents peripherin-2 dimers (C214S and L185P homodimers) from being incorporated into the rod outer segment (ROS) disks. Peripherin-2 tetramers and disulfide-linked oligomers, however, are incorporated into nascent disks and localize to the rims and incisures of mature disks. Interaction of peripherin-2 in the disk rim with the cyclic nucleotide-gated channel (CNGC) in the plasma membrane is also shown (Poetsch et al., 2001).

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