Characterization of R9AP, a Membrane Anchor for the Photoreceptor GTPase-Accelerating Protein, RGS9-1

Abstract

The proper recovery of photoreceptor light responses requires timely inactivation of the G-protein transducin (Gt) by GTP hydrolysis. It is now well established that the GTPase-accelerating protein (GAP) RGS9-1 plays an important role in determining the recovery kinetics of photoresponses. RGS9-1 has been found to be anchored to photoreceptor disk membranes by a novel photoreceptor protein, R9AP. R9AP has a single transmembrane domain at its C-terminal region. Membrane tethering by R9AP enhances RGS9-1 GAP activity in vitro and has been hypothesized to be important for the regulation of RGS9-1 function in vivo. In addition, R9AP shows structural similarity to the SNARE complex protein syntaxin and has been shown to be required for the correct targeting and localization of the RGS9-1 protein in photoreceptors. Therefore, R9AP may have additional functions other than that in the phototransduction pathway. This article presents methods and protocols developed for the functional characterization of R9AP in phototransduction, including the immunoprecipitation of the endogenous protein, the expression and purification of recombinant proteins, the reconstitution of proteoliposomes, and assays for its interaction with RGS9-1 and its effects on RGS9-1 GAP activity. These methods may also be applied to the study of R9AP function in other pathways or other cell types or to the studies of other membrane proteins that are structurally similar to R9AP.