Ritter LM , Khattree N , Tam B , Moritz OL , Schmitz F , Goldberg AF .

EY013246 NEI NIH HHS , R01 EY013246-07 NEI NIH HHS , RR017890 NCRR NIH HHS , S10 RR017890-01 NCRR NIH HHS , Canadian Institutes of Health Research , R01 EY013246 NEI NIH HHS , S10 RR017890 NCRR NIH HHS

	Figure 1. A GFP-based BiFC assay reports photoreceptor protein interactions in cultured cells. A, The approach uses fusions of nonfluorescent N- and C-terminal fragments of GFP (or YFP, yellow fluorescent protein) to partners of interest. Interaction of the partners (X, Y) brings the complementary fragments into proximity, promoting fluorophore maturation. B, GFP fragment fusion protein variants encoding photoreceptor proteins—P/rds, rhodopsin, and GARP2. C, Western blot analysis of fusion protein expression in transiently transfected HEK293 cells. The results demonstrate that all constructs can be expressed and that the epitope tag antibodies react specifically. D, Coexpression of complementary (GFPa and GFPb) fragments fused to either the N or C terminus of P/rds produced relatively modest BiFC signals (green) imaged by widefield epifluorescence microscopy. IHC analysis of the individual partners (HA, red; MYC, fuchsia) shows them to be overlapping with, but more broadly distributed than the BiFC signals. A similar assay applied to GARP2 and rhodopsin produced no significant signals, although previous reports suggest that each may engage in weak self-association (Batra-Safferling et al., 2006; Fotiadis et al., 2004). In sum, these results suggest that a BiFC assay based on GFP fragments is sufficient to detect strong, but not weak, interactions.
	Figure 2. BiFC assay based on Venus, a highly fluorescent YFP (yellow fluorescent protein) variant (Nagai et al., 2002), allows detection of additional homotypic interactions by widefield epifluorescence microscopy. A, Coexpression of complimentary pairs of Venus-based fusion proteins generated modest BiFC signals for both GARP2 and rhodopsin, consistent with reports that these proteins can weakly self-associate. IHC analysis of the individual partners (HA, red; MYC, fuchsia) shows them to be overlapping with, but more broadly distributed than the BiFC signals (green). Self-assembly of P/rds produced robust BiFC under similar conditions (data not shown). B, BiFC assay of proposed heterotypic interactions (Poetsch et al., 2001) between GARP2 and P/rds. Coexpression of Va-HA-GARP2 and P/rds-MYC-Vb partners produced a moderate BiFC signal. IHC analysis of the partners shows them to be overlapping with but more broadly distributed than the BiFC signal. In contrast, coexpression of Va-HA-GARP2 with rhodopsin (Rho-MYC-Vb) did not produce significant BiFC, despite robust expression of each partner. IHC epitope tag detection was specific, since reactivity could be blocked by synthetic peptide preincubation. All combinations of complementary GARP2-P/rds partners (such as GARP2-HA-Va and P/rds-MYC-Vb shown) were found to produce similar levels of BiFC.
	Figure 3. A GFP-based BiFC assay detects and localizes P/rds self-assembly in transgenic X. laevis photoreceptor cells. We predicted that P/rds tetramerization, as a model for homotypic protein–protein interaction, could drive robust BiFC in situ. A, Confocal micrographs of ocular cryosections from 14 dpf tadpoles showing GFP (green), WGA-labeled photoreceptor OSs (red), and Hoechst stained nuclei (blue). GFP fluorescence in positive control animals expressing a xP/rds-GFP fusion protein was associated solely with OSs. Scale bars, 100 μm. Insets, Transgenic tadpoles were identified in vivo via widefield epifluorescence screening; GFP was clearly visible through the lens and cornea (arrowheads). B, Western blot analysis of whole eye lysates from 14 dpf tadpoles. Lane 1, Nontransgenic control; lane 2, xP/rds-GFP (predicted 66 kDa); lane 3, bP/rds-GFPa (predicted 58 kDa); lane 4, bP/rds-GFPa (predicted 58 kDa) + bP/rds-GFPb (predicted 49 kDa). The anti-GFP antisera reacted with both N- and C-terminal fragments of GFP (and therefore both fusion protein partners). Partners were expressed at predicted MWs as doublets, likely due to heterologous glycosylation. Proteolytic degradation of bP/rds-GFPa was alleviated by coexpression of bP/rds-GFPb. β-Tubulin reactivity was assayed as a loading control. C, Confocal micrographs of ocular cryosections showing BiFC (green), WGA labeling of OSs (white), anti-GFP labeling of transgenic proteins (red), and Hoechst stained nuclei (blue); Scale bars, 20 μm. Coexpression of complementary partners encoding N- and C-terminal GFP fragments (GFPa, GFPb) individually fused to bP/rds sequences produced strong BiFC in both ISs and OSs, consistent with an initial assembly process within the IS. Expression of a single partner (bP/rds-GFPa) did not generate BiFC. D, Distributions of transgenically expressed GFP fusion proteins in a modestly expressing OS, documented at the ultrastructural level by immunogold transmission electron microscopy (TEM) analysis; Scale bar, 2 μm. The bP/rds fusion proteins (detected with anti-GFP Pab290) were primarily localized at disk rims and incisures; occasional lamellar reactivity was observed. Similar results were seen for xP/rds-GFP transgenic expression, as reported previously (Loewen et al., 2003). These patterns are also similar to that previously reported for endogenous X. laevis P/rds (Kedzierski et al., 1996), detected here with Mab 1G9. E, Wider-view fields showing transgenically expressed bP/rds-GFPa + bP/rds-GFPb fusion proteins in modestly (left) and highly (right) expressing cells. Immunogold TEM analysis used anti-GFP Pab290 for labeling the transgenic proteins. COS, Cone outer segment. Scale bar, 2 μm.
	Figure 4. Self-assembly and trafficking of OS and synaptic photoreceptor proteins fused to complementary Venus fragments produce distinctive patterns of BiFC. Confocal micrographs of tadpole ocular cryosections showing BiFC (green), WGA labeling of OSs (white), anti-GFP labeling of transgenic proteins (red), and Hoechst-stained nuclei (blue). Scale bars, 10 μm. P/rds oligomerization (resulting from Va-HA-P/rds + Vb-MYC-P/rds coexpression) generated robust Venus-based BiFC, essentially similar to that observed when GFP fragments were used (Fig. 2C). The representative cells shown illustrate substantial diffuse complex formation in ISs, with more robust intensities present in OSs (a). CNGB1 homotetramerization (driven by Va-HA-CNGB1 + Vb-MYC-CNGB1 coexpression) also produced BiFC, although the signals generated were far less intense (c). Typical distributions included punctate accumulations within ISs (c, d, arrowheads) and more diffuse localization within OSs that highlighted incisures. Weak synaptic signals were also frequently observed in positive cells. Expression of complementary Venus-rhodopsin partners (RHO-HA-Va + RHO-MYC-Vb) generated the most highly robust BiFC signals observed (e). The most intense BIFC was OS localized; however, substantial signals were present in IS plasma membranes, IS vesicles, and synaptic regions. RIB(B) homotypic interactions (generated by Va-HA-RIB(B) + Vb-MYC-RIB(B) coexpression) were observed to drive robust BiFC (g), seen as diffuse and punctate Venus fluorescence within ISs (g, arrow) and intense accretions within synaptic terminals (g, arrowhead). The BiFC generated by self-assembly of each protein [P/rds, CNGB1, RHO, and RIB(B)] was distributed as a subset of the total transgenic protein present, as visualized by IHC labeling using Pab290 (b, d, f, h) in a given cell/retina.
	Figure 5. Heterotypic interactions assayed by BiFC suggest that GARP variants associate differentially with P/rds. Scale bars, 10 μm. A, Confocal micrographs of tadpole ocular cryosections showing BiFC (green; a, d, g, j), antibody labeling of MYC-tagged (white; b, e, h, k) and HA-tagged (red; c, f, i, l) fusion proteins and Hoechst-stained nuclei (blue; a–l). Coexpression of P/rds with CNGB1 or GARP2, but not rhodopsin or RIB(B), generated significant BiFC; subcellular distributions were dependent on which GARP variant was used. Assembly of Vb-MYC-P/rds with complementary fusion protein Va-HA-CNGB1, but not Va-HA-GARP2, drove BiFC signals that were observed in both ISs and OSs (a vs g). Although Va-HA-GARP2 clearly complemented Vb-MYC-P/rds to produce substantial BiFC, complexes were rarely observed in ISs. Negative controls included combinations of proteins not thought to interact specifically [Va-HA-CNGB1 + RHO-MYC-Vb and Vb-MYC-P/rds+Va-HA-RIB(B)]. In these instances, IHC labeling verified expression of each partner (e, f, k, l). B, BiFC generated by coexpression of Vb-MYC-P/rds with Va-HA-GARP2 was largely restricted to photoreceptor OSs. Confocal micrographs of tadpole ocular cryosections showing, in a–c, BiFC (green), antibody labeling of endogenous X. laevis P/rds (Mab 1G9, white) and ER-resident protein calnexin (SPA-865, red), and Hoechst-stained nuclei (blue) or, in d–f, BiFC (green), antibody labeling of transgenic P/rds (MabC6, white) and Va-HA-GARP2 (HA, red), and Hoechst stained nuclei (blue). Expression of the transgenic proteins resulted in BiFC complexes visualized in OSs (a, d); no effects on distribution of endogenous P/rds (b) or calnexin (b,c) were apparent. We suspected that BiFC complex formation could partially mask the MYC epitope tag (A, b and h). An antibody directed against the unique C terminus of transgenic bP/rds (e) validates the presence of masking, demonstrates a robust expression of V2-MYC-P/rds, and confirms normal trafficking and localization.
	Figure 6. Role of the P/rds C terminus for self-assembly and localization. Scale bars, 10 μm. A, Confocal micrographs of tadpole ocular cryosections showing BiFC (green; a, d, g), Mab 1G9 labeling of endogenous X. laevis P/rds (white; b, e, h), and Hoechst stained nuclei (blue; a–i). Deletion of the P/rds C-terminal domain from one (Va-HA-P/rdsΔC + Vb-MYC-P/rds) or both (Va-HA-P/rdsΔC + Vb-MYC- P/rdsΔC) transgenically expressed proteins did not prevent self-assembly; substantial BiFC was generated in each instance (a, d, g). In contrast, absence of the C-terminal domain tended to favor IS localization (d, arrowheads) in a dose-dependent manner. B, Confocal micrographs of tadpole ocular cryosections showing, in a–c, BiFC (green), antibody labeling of HA-tagged (red) and MYC-tagged (white) fusion proteins, and Hoechst stained nuclei (blue), or d–f, BiFC (green), HA labeling of Va-HA-P/rdsΔC (red), MabC6 labeling of Vb-MYC-P/rds (white), and Hoechst stained nuclei (blue). Labeling of individual transgenic proteins (e and f vs b and c) showed that loss of C-terminal domains produced a somewhat greater effect on BiFC complex than individual partner distributions.
	Figure 7. Immunoprecipitation analyses demonstrate a segregated assembly of transgenic proteins. Reactions were assayed by Western blotting using the indicated antibodies; blots represent quantitative comparison of: detergent lysate (LYS), unbound supernatant (UB), and eluted (ELU) fractions. A, A novel monoclonal antibody, Mab 1G9, was developed to the C terminus of xP/rds (xrds38) and was used to immunoprecipitate (IP) the protein from Triton X-100 tadpole eye extracts under nonreducing conditions. The endogenous protein (most commonly observed as a doublet) could be effectively depleted from lysates (UB) and then recovered following SDS (ELU1), but not acid (ELU2) elution of beads. Importantly, Mab 1G9 showed no cross-reactivity with bP/rds. We found that bP/rds (detected with ortholog-specific MabC6) remained in lysates from which xP/rds was efficiently removed. B, Left, Immunoprecipitation of endogenous xP/rds from transgenic tadpole eyes coexpressing complementary Venus fragments fused to the following: (1) bP/rds lacking its C terminus (Va-HA-bP/rdsΔC); and (2) full-length bP/rds (Vb-MYC-bP/rds). The vast majority of each transgenic fusion protein (detected with polyclonal antibody Pab290) failed to coprecipitate. Right, Immunoprecipitation of endogenous xP/rds from transgenic tadpole eyes expressing bP/rds fused to full-length GFP protein at its C terminus (bP/rds-C150S-GFP). Little or no coprecipitation with endogenous xP/rds was observed. C, Reciprocal immunoprecipitation performed with an anti-GFP matrix confirms segregated assembly. Left, Immunoprecipitation of a xP/rds-GFP fusion protein from transgenic tadpole eyes. Transgenically expressed fusion protein (detected with polyclonal antibody Pab290) was efficiently immunoprecipitated; however, neither endogenous xP/rds (detected with Mab 1G9) or rhodopsin (K62-82) was coprecipitated. Right, What appeared to be a minor amount of coprecipitating xP/rds was in fact, a cross-reacting protein also present in WT tadpole eye lysates. D, Assembly of a P/rds C-terminal deletion mutant. A reciprocal immunoprecipitation performed using the lysate detailed in B above (coexpressed Va-HA-bP/rdsΔC and Vb-MYC-bP/rds). BiFC complexes were collected using an anti-GFP/Venus matrix and blotted for individual fusion protein partners. Although transgenic proteins coprecipitate with each other, endogenous P/rds does not. MabC6 binds the C terminus of bP/rds and therefore is specific to the full-length protein.

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