Baker SA, Haeri M, Yoo P, Gospe SM, Skiba NP, Knox BE, Arshavsky VY.

EY12859 NEI NIH HHS , EY12975 NEI NIH HHS , EY5722 NEI NIH HHS , P30 EY005722 NEI NIH HHS , R01 EY012859 NEI NIH HHS , R01 EY012975 NEI NIH HHS , R01 EY010336 NEI NIH HHS

	Figure 1. Localization of R9AP and syntaxin 3 in Xenopus photoreceptors. (A) Domain organization shared by R9AP and syntaxin 3. Both contain an N-terminal Habc domain, a SNARE homology domain, and a C-terminal transmembrane domain. (B) Immunofluorescence of R9AP (red) using the antibody against xR9AP2 in rod and cone outer segments of adult Xenopus retina. (C) Immunostaining of syntaxin 3 (green) in adult Xenopus retina. (B and C) The nuclei are counterstained with Hoechsts (blue). RPE, retinal pigmented epithelium; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GC, ganglion cell. No signals were revealed in controls where the primary antibodies were omitted. (D) Immunostaining of syntaxin 3 (green) in two examples of broken rod outer/inner segment preparations double labeled either with phalloidin (red; top), which labels the actin-rich calycal processes (CP), or with acetylated α-tubulin (red; bottom), which labels the axoneme within the connecting cilium and the outer segment. The right panels are the merged image with the shape of the entire cell fragment outlined in white, as seen in the transmitted light channel. Note that multiple calycal processes are seen as the image is a z-stack projection. (E) Cartoon of the rod photoreceptor with the regions containing R9AP in red and the regions containing syntaxin 3 outlined in green.
	Figure 3. Localization of GFP-tagged R9AP and R9AP mutants in transgenic Xenopus rods. The following constructs were expressed in transgenic Xenopus rods: full-length GFP-tagged R9AP (A), GFP-tagged R9AP lacking the Habc domain (B), and GFP-tagged R9AP lacking the SNARE homology (SH) domain (C). (D) GFP-tagged transmembrane domain from R9AP. (E) GFP-tagged R9AP lacking the transmembrane domain. In all panels nuclei are counterstained with Hoechsts (blue). OS, outer segment; IS, inner segment; N, outer nuclear layer; ST, synaptic terminal. Bars, 5 μm.
	Figure 4. Localization of GFP-tagged syntaxin 3 and syntaxin 3 mutants in transgenic Xenopus rods. (A) Full-length GFP-tagged syntaxin 3 (Stx3). The arrow indicates the calycal processes (CP). (B) GFP-tagged syntaxin 3 lacking the Habc domain. (C) GFP-tagged syntaxin 3 lacking the SNARE homology domain. (D) GFP-tagged transmembrane domain from syntaxin 3. (E) Full-length GFP-tagged syntaxin 3 with the FMDE motif mutated to four alanines. OS, outer segment; IS, inner segment; N, outer nuclear layer; ST, synaptic terminal. Bars, 5 μm.
	Figure 5. Localization of soluble and geranylgeranylated GFP-tagged syntaxin 3 mutants in transgenic Xenopus rods. (A) Soluble GFP. (B) GFP-tagged syntaxin 3 lacking both the SNARE and transmembrane domains. (C) GFP-tagged syntaxin 3 lacking the transmembrane domain. (D) Geranylgeranylated GFP. (E) Geranylgeranylated GFP-tagged syntaxin 3 lacking the SNARE homology domain and transmembrane domain. (F) Geranylgeranylated GFP-tagged syntaxin 3 lacking the transmembrane domain. OS, outer segment; IS, inner segment; N, outer nuclear layer; ST, synaptic terminal. Bars, 5 μm.
	Figure 6. Localization of Myc-tagged R9AP/syntaxin 3 chimeras in transgenic Xenopus rods. (A) Full-length Myc-tagged R9AP. (B) Full-length Myc-tagged syntaxin 3 (Stx3). (C) Myc-tagged chimera of R9AP containing the transmembrane domain from Stx3. (D) Myc-tagged chimera of syntaxin 3 containing the SNARE homology domain from R9AP. (E) Myc-tagged chimera of syntaxin 3 containing the transmembrane domain from R9AP. (F) Myc-tagged chimera of R9AP containing the SNARE homology domain from syntaxin 3. OS, outer segment; IS, inner segment; N, outer nuclear layer; ST, synaptic terminal. Bars, 5 μm.
	Figure 7. Localization of a random GFP-tagged transmembrane domain in transgenic Xenopus rods. GFP-tagged transmembrane segment 1 from mGluR1. Bar, 5 μm.
	Figure 8. Localization of ER and mitochondrial-targeted GFP-tagged transmembrane domains and their length mutants in transgenic Xenopus rods. The following constructs were expressed as GFP fusion proteins in Xenopus rods (see Table S2 for sequence details, available at http://www.jcb.org/cgi/content/full/jcb.200806009/DC1): (A) transmembrane domain from microsomal cytochrome b5 (Cyt b5); (B) transmembrane domain from cytochrome b5 elongated by four amino acid residues; (C) transmembrane domain from Bcl XL; (D) transmembrane domain from Bcl XL bearing a point mutation (K233S); (E) transmembrane domain from Bcl XL bearing the K233S mutation and elongated by four amino acid residues. OS, outer segment; IS, inner segment; N, outer nuclear layer; ST, synaptic terminal. Bars, 5 μm.
	Figure 9. Localization of GFP-tagged R9AP transmembrane domain length mutants in transgenic Xenopus rods. (A) GFP-tagged transmembrane domain from R9AP. (B) GFP-tagged transmembrane domain from R9AP shortened by two amino acid residues. (C) GFP-tagged transmembrane domain from R9AP shortened by four amino acid residues. (D) Xenopus tadpole retinas expressing GFP-tagged R9AP transmembrane domains of various lengths as indicated were fractionated into soluble and membrane pools by sonication and sedimentation at 120,000 g. Proteins in each fraction were Western blotted using an antibody against GFP. Lane S, soluble protein fraction; lane M, membrane fraction. OS, outer segment; IS, inner segment; N, outer nuclear layer; ST, synaptic terminal. Bars, 5 μm.
	Figure 10. Patterns of transgenic GFP-R9AP or GFP-syntaxin 3 transmembrane domain expression in ciliated Xenopus tissues. (A) GFP-tagged full-length R9AP expressed in photoreceptors. (B) GFP-tagged transmembrane domain from syntaxin 3 expressed in olfactory cells and in the epidermis (C). (B and C) Cilia are labeled with an antibody against acetylated α-tubulin (red). COS, cone outer segment; ROS, rod outer segment.
	Figure 2. Identification of the photoreceptor-specific R9AP isoform in Xenopus. (A) Phylogeny tree of R9AP protein sequences built using the Neighbor-Joining method. Accession nos. are as follows: bovine, AAM67417; chicken, NP_989836; chimpanzee, XP_512566; dog, XP_855518; human, NP_997274; macaque, XP_001087093; mouse, NP_665839; pufferfish, CAF92756; rat, XP_001079782; zebrafish, XP_687644. Xenopus tropicalis R9AP1 is deduced from genome v4.1, scaffold 94 (920566:921276), and X. tropicalis R9AP2 is deduced from genome v4.1, scaffold 761 (402303:408685). (B) RT-PCR amplification of xR9AP1 and xR9AP2 transcripts from Xenopus retina; omission of reverse transcription (RT) in parallel samples served as a negative control for DNA contamination. (C) Western blot of Xenopus retina extract probed with the antibody specific to xR9AP2. Lane T, total extract; lane S; soluble protein fraction; lane M, membrane fraction. Note that the xR9AP2 band is a doublet, which reflects the previously documented phosphorylation of R9AP in photoreceptors (Martemyanov et al., 2003). (D) xR9AP2 coimmunoprecipitates with RGS9-1 from Xenopus retina. Beads coated with sheep IgG were used for the negative control.

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