Identification and Confirmation of the Interacting Protein(s) for HRG4 (UNC119)

Abstract

Abstract: : Purpose:We had isolated the novel photoreceptor synaptic protein (HRG4/UNC119), a mutant of which was shown to cause retinal degeneration in human and a transgenic model, by subtractive cDNA cloning and have been continuing to study its functional role in the retina. In the present study, the yeast two-hybrid strategy was used to identify the protein(s) interacting with HRG4, and the putative interactor was confirmed by co-precipitation with expressed HRG4. Methods:An yeast prey library consisting of cells transformed with human retinal cDNA in the pYESTrp2 vector was prepared and large-scale transformed into the bait strain containing the complete coding sequence of HRG4 in the pHybLex/Zeo vector. Positive colonies were detected by selecting on plates lacking the auxotrophic marker (Histidine), followed by a second screen for ß-galactosidase activity by filter lifting assay in addition to the Zeocin selection. The positive clones were identified by sequencing, expressed, and used in a "pull-down" co-precipitation analysis with expressed HRG4-GST-glutathione-Sepharose. Results:2.4 million transformants from the large scale transformation were screened. Additional His+ and ß-galactosidase selection resulted in 13 positives, 9 of which were identified as ADP-ribosylation factor-like protein 2 (ARL2). ARL2 was shown to bind specifically to HRG4 in the pull-down analysis, confirming it as the interactor for HRG4. Conclusion:We have identified and confirmed ARL2 as the interacting protein for HRG4/UNC119. Although the precise function of ARL2 is yet unknown, the known function of ARF suggests that HRG4/UNC119 might have some role in vesicular transport in cooperation with ARL2.

Keywords: 517 photoreceptors • 561 retinal degenerations: cell biology • 528 proteins encoded by disease genes