Tam BM, Moritz OL, Hurd LB, Papermaster DS.

EY06891 NEI NIH HHS , EY10992 NEI NIH HHS , F32 EY006891 NEI NIH HHS , R01 EY006891 NEI NIH HHS

	Figure 1. The GFP-fusion proteins. (A) Amino acid sequences of the cytoplasmic tails of X. laevis rhodopsin (rho), the porcine AAR, and the NH2 terminus peptide from human c-src (myr). Numbers refer to the amino acid sequence of X. laevis rhodopsin. The sites of palmitoylation (*), the site of myristoylation (^), and proline353 (@) are indicated. Arrows mark the sites of truncation. The solid line indicates the rho6 sequence. The dashed line indicates the rho8 sequence. (B) Diagrams of the fusion proteins and their cellular localization. Solid bars represent GFP. Open bars represent rhodopsin peptides. Vertically striped bars represent the c-src peptide. Diagonally striped bars represent adrenergic peptides. Straight vertical lines represent myristoyl groups and zigzag lines represent palmitoyl groups. +, membrane association; −, its absence. Inner segment (IS) and/or outer segment (OS) localization of the fusion protein is indicated.
	Figure 2. The cytoplasmic tail of rhodopsin can redirect GFP to the ROS. Confocal micrographs of transgenic retinas expressing GFP and GFP-CT44. Nuclei were stained with Hoescht 33342. (A) GFP distributed predominantly to the cytoplasm of the inner segment (is) and nucleoplasm. GFP resided at low levels in the outer segment (os) and was not associated with membranes. (B) GFP-CT44 localized almost exclusively to the outer segments. GFP (green) and Hoescht 33342 (blue). n, nucleus; m, mitochondria; and s, synaptic terminal. Bar, 5 μm.
	Figure 6. GFP-CT44del25, but not endogenous rhodopsin, accumulates abnormally in the Golgi, RIS plasma membrane, and synapse. EM micrographs of a rod cell expressing GFP-CT44del25. The cell was labeled either with anti-GFP antibody (A) or antirhodopsin antibody, 11D5 (B), followed by a gold-conjugated secondary antibody. The boxed regions are magnified to show detail. Arrows point to GFP (A 1) and rhodopsin (B 1) labeling present in the Golgi. Arrowheads indicate the presence of GFP-CT44del25 (A 2), but not rhodopsin (B 2), on the RIS plasma membrane. Likewise, GFP-CT44del25 (A 3) is abundant in the synaptic terminal; rhodopsin (B 3) is not. Therefore, delocalization of the fusion protein does not result in delocalization of endogenous rhodopsin. os, outer segment; is, inner segment; n, nucleus; m, mitochondria; G, Golgi; and S, synaptic terminal. Bar, 2.5 μm.
	Figure 3. Truncations or mutations of the rhodopsin COOH-terminal domain cause the fusion proteins to partially delocalize to the RIS. Confocal micrographs of transgenic retinas expressing (A) GFP-CT44del25; (B) GFP-CT44del5; (C) GFP-CT44/P353S; and (D) GFP-CT44/P353L. Nuclei were stained with Hoescht 33342. Truncation of the distal 25 (A) or 5 amino acids (B) or mutation of the penultimate proline to either serine (C) or leucine (D) resulted in partial delocalization of the fusion proteins to the lateral plasma membrane, Golgi, and synaptic terminal. GFP (green) and Hoescht 33342 (blue). os, outer segment; is, inner segment; n, nucleus; and s, synaptic terminal. Bar, 5 μm.
	Figure 4. Membrane association is necessary but not sufficient for ROS localization. Fusion proteins are associated with Golgi and post-Golgi membranes. (A–C) Confocal micrographs of a cell expressing GFP-CT44del25 labeled with TR-WGA (red) and Hoescht 33342 (blue). (A) TR-WGA labeling; (B) GFP fluorescence (green); (C) the overlap of the two images. Regions of colocalization are represented by yellow/orange. Together, these demonstrate that the fusion protein colocalizes with membranes, specifically Golgi and post-Golgi membranes (arrows), plasma membrane (arrowheads), and ROS membranes. (D–F) Confocal micrographs of transgenic retinas expressing GFP-CT25 (D), mGFP (E), and mGFP-CT44 (F). Nuclei were stained with Hoechst 33342 dye. GFP-CT25 did not associate with membranes and was not efficiently transported to the ROS. mGFP was membrane associated and was found in both ROS and RIS membranes including mitochondrial membranes found immediately below the inner/outer segment junction (arrows). mGFP-CT44 distributed almost exclusively to the ROS. Together, these demonstrate that both membrane attachment and a targeting signal are required for restricted ROS localization. GFP (green) and Hoescht 33342 (blue). os, outer segment; is, inner segment; n, nucleus; m, mitochondria, and s, synaptic terminal. Bars: (A–C) 2.5 μm; (D–F) 5 μm.
	Figure 5. Rhodopsin's outer segment targeting/retention signal resides within its distal most eight amino acids. Confocal micrographs of transgenic retinas expressing (A) GFP-AAR; (B) GFP-AAR(CC); (C) GFP-AAR(CC)rho8; and (D) GFP-AAR(CC)rho6. GFP-AAR distributed mainly to RIS membranes, especially the synaptic terminal, but was also partially soluble. GFP-AAR(CC) displayed greater membrane association than its single cysteine counterpart and localized to both ROS and RIS membranes. GFP-AAR(CC)rho8 localized exclusively to the ROS while GFP-AAR(CC)rho6 localized to both ROS and RIS membranes. The last eight amino acids of rhodopsin are therefore sufficient to direct ROS localization. GFP (green) and Hoescht 33342 (blue). os, outer segment; is, inner segment; n, nucleus; and s, synaptic terminal. Bar, 5 μm.
	Figure 7. Presentation of the targeting signal affects the efficiency of ROS localization. Confocal micrographs of cells expressing (A) mGFP-CT9; (B) mGFP-CT25; and (C) mGFP-CT44C322/323S. Varying the lengths of the rhodopsin peptides fused to GFP affected the distribution patterns. mGFP-CT9 and mGFP-CT44C322/323, but not mGFP-CT25, were significantly more efficient that mGFP (see Fig. 4 E) in targeting to the ROS. Mitochondria were also labeled to varying degrees by the myristoylated fusion proteins. Arrows indicate the inner/outer segment junction. GFP (green) and Hoescht 33342 (blue). os, outer segment; is, inner segment; n, nucleus; m, mitochondria; and s, synaptic terminal. Bar, 5 μm.
	Figure 8. Overexpression of the fusion proteins is not the cause of delocalization to the RIS. Transgenic eyes expressing GFP-CT44 and GFP-CT44/P353L were excised, fixed, and sectioned in parallel. Frozen sections from each eye were consecutively imaged by confocal microscopy. All image acquisition settings were identical for both samples and postmicroscopy processing was done in parallel to the images. GFP-CT44 (A) was expressed at a much higher level than GFP-CT44/P353L (B) and yet was not found in significant levels in the RIS in contrast to the mutated fusion protein. GFP (green) and Hoescht 33342 (blue). os, outer segment; is, inner segment; n, nucleus; m, mitochondria; and s, synaptic terminal. Bar, 5 μm.

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