Functional Analysis of Membrane–bound Protein Complexes from Mammalian Photoreceptors.

Abstract

Abstract: : Purpose:To isolate and characterize the dynamics of membrane–bound multiprotein complexes in mammalian photoreceptors. Methods:Mammalian photoreceptor outer segments (POS) were isolated from porcine retinae. In order to increase both the efficiency and purity of POS preparation, we compared two standard isolation protocols with samples pre–fractionated by Free Flow Electrophoresis (FFE) and with purified disk membranes. The membranous POS fraction was solubilized with mild detergent and native protein complexes were separated using density gradient centrifugation or Blue Native Gel Electrophoresis. Individual proteins were separated by SDS–PAGE and identified by MALDI–TOF mass spectrometry. Results:The separation of membrane–associated proteins by means of traditional 2–DE methodology has been greatly difficult. Moreover, the denaturing conditions underlying 2–DE prevent isolation of functional protein complexes. Our experiments have therefore combined traditional biochemical native separation methods and improved solubilization and separation conditions to optimize the isolation of stable multiprotein complexes from mammalian photoreceptor outer segments. Initial analysis of crude POS demonstrated high complexity and impurity of the preparations, e.g. sample contamination by protein complexes characteristic for photoreceptor inner segments (phosducin and 14–3–3 proteins). Preparation of intact disk membranes or POS pre–fractionation by FFE greatly reduced complexity and increased sample purity. Using a proteomic approach, we have identified several candidates as novel components of the phototransduction pathway, including small GTPases from Rho and Rab families, and established a preliminary protein interaction map for mammalian photoreceptors. Conclusions:We have detected protein interactions linked to the physiology of vision in mammalian photoreceptors. Protein interaction maps and analysis of their dynamics can be correlated to physiological states and will be used for a better understanding of vision processes and their pathology in diseases causing blindness.

Keywords: photoreceptors • protein purification and characterization • proteomics