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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. Image published in: Ritter LM et al. (2011) Copyright © 2011. This image is reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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