Abstract
Abstract: :
Purpose: Over expression and loss of function of the dsu gene results in abnormal photoreceptor morphology. Dsu mutant animals show misolocalization of rhodopsin to the outer nuclear layer. In mice that over express dsu, the ROS regions show whorled membrane debris and the RPE is characterized by an increase in vacuole–like structures. In this study we tested the hypothesis that the dsu (dilute suppressor) protein, a 28kDa protein, regulates membrane fusion processes. Methods: Dsu distribution within the retina was determined by confocal microscopy. The distribution of rom–1 and rhodopsin in dsu mutants and dsu over–expressers was determined by confocal microscopy and the structure of the rod cells analyzed by EM. The presence of dsu in retinal fractions was determined by western blot analysis. Dsu was expressed as an MBP –fusion protein using an RTS expression system and purified. The affect of dsu on fusion between ROS plasma membrane and a series of target membranes was determined using a fluorescent cell free assay. Results: dsu in wt animals was found localized to both photoreceptor and RPE cells. In the dsu loss of function mutant, the OS portion of the rod cells showed abnormal structure. Similarly dsu over–expression resulted in whorl–like membrane debris accumulating in the OS space. Dsu was identified in bovine RPE extracts and purified ROS fractions. Dsu inhibited fusion between disk and palm membrane by over 60% and fusion between p/rds– rom–1 containing vesicles by almost 50%. This inhibition was concentration dependent. Conclusions: These result suggest that dsu, is a potent inhibitor of membrane fusion processes in vitro. Furthermore, over–expression or loss of function of this protein results in severe structural abnormalities of the ROS consistent with defects in the fusion process. EY10420 and an E. Matilda Ziegler Vision Award.
Keywords: retina • photoreceptors • cell membrane/membrane specializations