Photoreceptor Peripherin/rds Fusogenic Function Is Inhibited by dsu, Through Direct Binding to the C–terminal Domain

Abstract

Abstract: : Purpose: Alterations in dilute suppressor protein (dsu) expression lead to abnormal morphology in photoreceptor outer segments (OS); over–expression of dsu results in more pronounced changes characterized by whorled membrane debris. We have previously proposed that dsu (dilute suppressor protein) regulates membrane fusion processes. In this study we examined the site of dsu–peripherin/rds interaction and determined the membrane disposition of dsu in both ROS and RPE cells. Methods: Dsu and a series of peripherin/rds C–terminal mutants were expressed using an in vitro expression system and the binding kinetics between peripherin/rds and dsu assessed using surface plasmon resonance (SPR) and co–precipitation studies. Expression levels of dsu in mouse retina were assessed by RT–PCR and the presence of dsu in RPE cells and photoreceptors determined by western blot analysis. The membrane disposition of dsu in RPE cells and, RPE and ROS rafts was evaluated by treatment with hydroxylamine. The effect of dsu on fusion between ROS plasma membrane and a series of target membranes was determined using a fluorescent cell free assay. Results: Dsu inhibits in vitro membrane fusion between OS plasma membrane and both disk membranes and rim specific vesicles in a concentration dependent manner. Dsu binds to the C–terminal domain of peripherin/rds. Further anlysis indicates that it interacts with the terminal ten amino acids; deletion of these terminal ten amino acids enhances fusion and inhibits dsu binding as determined by SPR. Moreover, dsu is membrane anchored in ROS membranes and can be dissociated from the membrane by hydroxylamine. Initial experiments suggest that dsu is in membrane rafts. Conclusions: Dsu inhibition of peripherin/rds dependent membrane fusion requires binding of dsu to the C–terminal domain of peripherin/rds. The relationship between dsu binding and its membrane disposition provides a regulatory mechanism for fusion along the length of the OS.

Keywords: photoreceptors • cell membrane/membrane specializations • protein purification and characterization