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J.J. Janssen, C.A. Driessen, H.J. Winkens, H. Chao; Interactions of RDH5 with RPE Proteins: A Yeast-two-hybrid Approach . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3516.
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Purpose: The visual cycle is the enzymatic pathway by which all-trans-retinal from photoreceptor bleaching is isomerized to 11-cis-retinal in the retinal pigment epithelium (RPE) for visual pigment regeneration. Detailed knowledge of the full network of protein-protein interactions in the visual cycle should provide new insights into its structural layout. Methods: Yeast two hybrid analysis allows the study of protein-protein interactions in an eukaryotic in vivo environment. The CytoTrap yeast two hybrid system was used. In the CytoTrap system proteins are expressed in the cytoplasm and may undergo posttranslational modifications. The system uses a temperature-sensitive yeast strain containing a mutation in the cdc25 gene, the yeast homologue for the human son of sevenless (hSos) gene essential for activation of the Ras pathway. As a result of this mutation cells only grow at 25°C but not at 37°C. This cdc25 mutation can be complemented by the hSos gene product allowing growth at 37°C provided that the hSos protein is localized to the membrane. The pMyr vector is designed for cDNA library construction and proteins expressed from this plasmid are anchored to the plasma membrane. The bait proteins RDH5 are CRALBP are expressed as hSOS fusion proteins.The cDNA library and bait construct are co-transformed into the cdc25H yeast strain. Cells capable of growing at 37°C on galactose medium only, have likely been rescued via a protein-protein interaction. Results: Using CRALBP as a bait protein we were unable to use the CytoTrap two hybrid system successfully. Our original interpretation of the data obtained using CRALBP as a bait protein was that spontaneous temperature revertants present an insurmountable problem in this system. Further analysis, however, indicated that CRALBP, due to intrinsic properties, is targeted to the plasma membrane without any interaction with a target protein being necessary. Using RDH5 as a bait protein we were able to retrieve a number of clones. Clones that were isolated more than once were subject for further investigation. Conclusions: An interaction has been demonstrated between RDH5 and several other RPE/visual cycle proteins. None of the known "interactors" of RDH5, being RGR, RPE65 and CRALBP, have been selected using the CytoTrap yeast two hybrid system. A number of proteins have thus far been isolated more than once. Amongst these were proteins suggested to regulate the activity of other retinal proteins and clones encoding proteins that were previously suggested playing a role in regulating the expression of retinoid metabolizing enzymes.
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