Purification of cone outer segment for proteomic analysis on its membrane proteins in carp retina.

Takashi Fukagawa,Kazuaki Takafuji,Shuji Tachibanaki,Satoru Kawamura

doi:10.1371/journal.pone.0173908

Abstract

Rods and cones are both photoreceptors in the retina, but they are different in many aspects including the light response characteristics and, for example, cell morphology and metabolism. These differences would be caused by differences in proteins expressed in rods and cones. To understand the molecular bases of these differences between rods and cones, one of the ways is to compare proteins expressed in rods and cones, and to find those expressed specifically or dominantly. In the present study, we are interested in proteins in the outer segment (OS), the site responsible for generation of rod- or cone-characteristic light responses and also the site showing different morphology between rods and cones. For this, we established a method to purify the OS and the inner segment (IS) of rods and also of cones from purified carp rods and cones, respectively, using sucrose density gradient. In particular, we were interested in proteins tightly bound to the membranes of cone OS. To identify these proteins, we analyzed proteins in some selected regions of an SDS-gel of washed membranes of the OS and the IS obtained from both rods and cones, with Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) using a protein database constructed from carp retina. By comparing the lists of the proteins found in the OS and the IS of both rods and cones, we found some proteins present in cone OS membranes specifically or dominantly, in addition to the proteins already known to be present specifically in cone OS.

Highlights

In the vertebrate retina, there are two types of photoreceptors, rods and cones
All experiments with carp (Cyprinus carpio) and with mice in this study were performed in accordance with the Osaka University Guidelines for the Welfare and Use of Laboratory Animals, and approved by the Committee on Animal Care of the Graduate School of Frontier Biosciences of Osaka University
It would be ideal to detect proteins expressed in purified cone outer segment (OS) and compare them with proteins expressed in cone inner segment (IS), rod OS and rod IS

Summary

Introduction

There are two types of photoreceptors, rods and cones. Both photoreceptors consist of four regions: outer segment (OS), inner segment (IS), cell body and synaptic terminal. Carp (Cyprinus carpio) is so far the only animal from which we can obtain purified cones in a quantity large enough to do biochemical studies [4] Using these purified carp rods and cones, we have been testing the above possibility, and found that the signal amplification is lower, and termination of each reaction is much faster in cones than in rods [5,6,7,8]. It is still possible that proteins other than known ones are present in rod OS (ROS) or cone OS (COS) and contribute to the differences in the response characteristics between rods and cones. Our analysis was made not in the whole region of the molecular masses of proteins in a gel, we found that overall, protein expression profiles are similar between ROS and COS in carp under the conditions employed in this study

Ethics statement

Results and discussion

ATPase β subunit