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.

Amaya, A method for generating transgenic frog embryos. 1999, Pubmed, Xenbase

Amaya, A method for generating transgenic frog embryos. 1999, Pubmed , Xenbase
Andersen, Bilayer thickness and membrane protein function: an energetic perspective. 2007, Pubmed
Baker, Kinetic mechanism of RGS9-1 potentiation by R9AP. 2006, Pubmed
Batni, Xenopus rod photoreceptor: model for expression of retinal genes. 2000, Pubmed , Xenbase
Besharse, Photoreceptor intersegmental transport and retinal degeneration: a conserved pathway common to motile and sensory cilia. 2003, Pubmed
Chuang, SARA-regulated vesicular targeting underlies formation of the light-sensing organelle in mammalian rods. 2007, Pubmed
Concepcion, The carboxyl-terminal domain is essential for rhodopsin transport in rod photoreceptors. 2002, Pubmed
Deretic, Regulation of sorting and post-Golgi trafficking of rhodopsin by its C-terminal sequence QVS(A)PA. 1998, Pubmed
Deretic, Rhodopsin C terminus, the site of mutations causing retinal disease, regulates trafficking by binding to ADP-ribosylation factor 4 (ARF4). 2005, Pubmed
Deretic, A role for rhodopsin in a signal transduction cascade that regulates membrane trafficking and photoreceptor polarity. 2006, Pubmed
Deretic, Phosphoinositides, ezrin/moesin, and rac1 regulate fusion of rhodopsin transport carriers in retinal photoreceptors. 2004, Pubmed
Fariss, Evidence from normal and degenerating photoreceptors that two outer segment integral membrane proteins have separate transport pathways. 1997, Pubmed
Fu, Novel double promoter approach for identification of transgenic animals: A tool for in vivo analysis of gene function and development of gene-based therapies. 2002, Pubmed , Xenbase
Green, Characterization of rhodopsin mis-sorting and constitutive activation in a transgenic rat model of retinitis pigmentosa. 2000, Pubmed
Hansen, Ultrastructure of the olfactory organ in the clawed frog, Xenopus laevis, during larval development and metamorphosis. 1998, Pubmed , Xenbase
Hay, SNARE complex structure and function. 2001, Pubmed
Hu, R9AP, a membrane anchor for the photoreceptor GTPase accelerating protein, RGS9-1. 2002, Pubmed , Xenbase
Humphries, Retinopathy induced in mice by targeted disruption of the rhodopsin gene. 1997, Pubmed
Kaplan, Lengths of immunolabeled ciliary microtubules in frog photoreceptor outer segments. 1987, Pubmed
Karan, A model for transport of membrane-associated phototransduction polypeptides in rod and cone photoreceptor inner segments. 2008, Pubmed
Kaufmann, Characterization of the signal that directs Bcl-x(L), but not Bcl-2, to the mitochondrial outer membrane. 2003, Pubmed
Keresztes, Expression patterns of the RGS9-1 anchoring protein R9AP in the chicken and mouse suggest multiple roles in the nervous system. 2003, Pubmed
Killian, Hydrophobic mismatch between proteins and lipids in membranes. 1998, Pubmed
Knox, Transgene expression in Xenopus rods. 1998, Pubmed , Xenbase
Koulen, Group I metabotropic glutamate receptors mGluR1alpha and mGluR5a: localization in both synaptic layers of the rat retina. 1997, Pubmed
Kraft, Altered light responses of single rod photoreceptors in transgenic pigs expressing P347L or P347S rhodopsin. 2005, Pubmed
Krispel, RGS expression rate-limits recovery of rod photoresponses. 2006, Pubmed
Kroll, Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. 1996, Pubmed , Xenbase
Lee, Characterization of peripherin/rds and rom-1 transport in rod photoreceptors of transgenic and knockout animals. 2006, Pubmed , Xenbase
Lee, Transport of truncated rhodopsin and its effects on rod function and degeneration. 2007, Pubmed
Lem, Morphological, physiological, and biochemical changes in rhodopsin knockout mice. 1999, Pubmed
Li, Transgenic mice carrying the dominant rhodopsin mutation P347S: evidence for defective vectorial transport of rhodopsin to the outer segments. 1996, Pubmed
Liu, Myosin VIIa participates in opsin transport through the photoreceptor cilium. 1999, Pubmed
Luo, An outer segment localization signal at the C terminus of the photoreceptor-specific retinol dehydrogenase. 2004, Pubmed , Xenbase
Mancias, The transport signal on Sec22 for packaging into COPII-coated vesicles is a conformational epitope. 2007, Pubmed
Marszalek, Genetic evidence for selective transport of opsin and arrestin by kinesin-II in mammalian photoreceptors. 2000, Pubmed
Martemyanov, R7BP, a novel neuronal protein interacting with RGS proteins of the R7 family. 2005, Pubmed
Martemyanov, The DEP domain determines subcellular targeting of the GTPase activating protein RGS9 in vivo. 2003, Pubmed
Morgans, A SNARE complex containing syntaxin 3 is present in ribbon synapses of the retina. 1996, Pubmed
Moritz, A functional rhodopsin-green fluorescent protein fusion protein localizes correctly in transgenic Xenopus laevis retinal rods and is expressed in a time-dependent pattern. 2001, Pubmed , Xenbase
Moritz, Mutant rab8 Impairs docking and fusion of rhodopsin-bearing post-Golgi membranes and causes cell death of transgenic Xenopus rods. 2001, Pubmed , Xenbase
Mossessova, SNARE selectivity of the COPII coat. 2003, Pubmed
Mostov, Polarized epithelial membrane traffic: conservation and plasticity. 2003, Pubmed
Muth, Transport protein trafficking in polarized cells. 2003, Pubmed
Norton, Evaluation of the 17-kDa prenyl-binding protein as a regulatory protein for phototransduction in retinal photoreceptors. 2005, Pubmed
Papermaster, The birth and death of photoreceptors: the Friedenwald Lecture. 2002, Pubmed
Pedrazzini, Mechanism of residence of cytochrome b(5), a tail-anchored protein, in the endoplasmic reticulum. 2000, Pubmed
Pedrazzini, A mutant cytochrome b5 with a lengthened membrane anchor escapes from the endoplasmic reticulum and reaches the plasma membrane. 1996, Pubmed
Peet, Quantification of the cytoplasmic spaces of living cells with EGFP reveals arrestin-EGFP to be in disequilibrium in dark adapted rod photoreceptors. 2004, Pubmed , Xenbase
Ponnambalam, Constitutive protein secretion from the trans-Golgi network to the plasma membrane. 2003, Pubmed
Rodriguez-Boulan, Protein sorting in the Golgi complex: shifting paradigms. 2005, Pubmed
Sen, Immunolocalization of metabotropic glutamate receptors 1 and 5 in the synaptic layers of the chicken retina. 2006, Pubmed
Sharma, Apical targeting of syntaxin 3 is essential for epithelial cell polarity. 2006, Pubmed
Sherry, Distribution of plasma membrane-associated syntaxins 1 through 4 indicates distinct trafficking functions in the synaptic layers of the mouse retina. 2006, Pubmed
Sokolov, Phosducin facilitates light-driven transducin translocation in rod photoreceptors. Evidence from the phosducin knockout mouse. 2004, Pubmed
Steinberg, Disc morphogenesis in vertebrate photoreceptors. 1980, Pubmed
Steinman, An electron microscopic study of ciliogenesis in developing epidermis and trachea in the embryo of Xenopus laevis. 1968, Pubmed , Xenbase
Sung, A rhodopsin gene mutation responsible for autosomal dominant retinitis pigmentosa results in a protein that is defective in localization to the photoreceptor outer segment. 1994, Pubmed
Tai, Rhodopsin's carboxy-terminal cytoplasmic tail acts as a membrane receptor for cytoplasmic dynein by binding to the dynein light chain Tctex-1. 1999, Pubmed
Tam, Identification of an outer segment targeting signal in the COOH terminus of rhodopsin using transgenic Xenopus laevis. 2000, Pubmed , Xenbase
Tam, The C terminus of peripherin/rds participates in rod outer segment targeting and alignment of disk incisures. 2004, Pubmed , Xenbase
Tam, Mislocalized rhodopsin does not require activation to cause retinal degeneration and neurite outgrowth in Xenopus laevis. 2006, Pubmed , Xenbase
Whitaker, Conserved transcriptional activators of the Xenopus rhodopsin gene. 2004, Pubmed , Xenbase
Zhang, Deletion of PrBP/delta impedes transport of GRK1 and PDE6 catalytic subunits to photoreceptor outer segments. 2007, Pubmed
van Meer, Membrane lipids: where they are and how they behave. 2008, Pubmed