Identification of unique rod outer segment plasma membrane proteins

Duke University, Durham NC

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Abstract

Abstract Photoreceptors are retinal neurons producing electrical responses to light. All reactions underlying this process take place in the ciliary outer segment organelle of photoreceptor cells. Outer segments are built from a stack of disc membranes enclosed by a plasma membrane. While protein components of discs are primarily responsible for capturing light and processing the downstream second messenger signal, proteins residing in the plasma membrane are involved in an extraordinary array of biological processes critical for maintaining the functionality and healthy status of photoreceptors. Just a short list of the outer segment plasma membrane functions includes generation of light-evoked electrical responses, membrane transport, structural interactions with discs inside and extracellular matrix outside the cell, disc morphogenesis and outer segment renewal. Despite our deep understanding of the roles which plasma membrane ion channels and exchangers play in generating light responses, we know very little about other functions in which this membrane plays a critical role. In most cased, we do not even know which proteins are involved. The goal of this proposal is to reduce this knowledge gap by obtaining and characterizing the unique proteome of the outer segment plasma membrane. We will employ the cutting edge application of quantitative proteomics, called protein correlation profiling. Unlike old school biochemical techniques, this methodology allows analyzing protein compositions in organelles that can be fractionated but not purified to absolute homogeneity, just as in the case of the outer segment plasma membrane. In Aim 1, membranes isolated from bovine rod outer segments will be subjected to fractionation steps designed to enrich their plasma membrane content. The relative abundances of all identified proteins will be then compared to the corresponding abundances of known plasma membrane markers. This will enable us to identify candidate proteins residing exclusively in this membrane and those which are highly enriched. In Aim 2, we will confirm the outer segment plasma membrane localization of selected candidate proteins using a combination of immunohistochemistry, immuno-electron microscopy and transgenic expression of recombinant constructs encoding tagged proteins. This is a resource- and hypothesis- generating study which will undoubtedly facilitate the efforts of many laboratories addressing a broad array of questions related to photoreceptor biology and disease.