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Nikolai P Skiba, Laurie Molday, Robert S Molday, Vadim Y Arshavsky; Identification of protein components of the rod outer segment plasma membrane by label-free protein correlation profiling. Invest. Ophthalmol. Vis. Sci. 2018;59(9):605.
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© ARVO (1962-2015); The Authors (2016-present)
Vertebrate photoreceptors respond to light by producing an electrical response originating in the outer segment plasma membrane harboring cGMP-gated channels. In addition to this function, plasma membrane plays a central role in continuous process of outer segment renewal, disc morphogenesis and communication with neighboring cells. Despite our deep understanding of visual signal transduction, we know surprisingly little about other functions of the outer segment plasma membrane. One impeding factor is our incomplete knowledge of the protein components residing within this membrane. This study aims to comprehensively characterize the proteome of this membrane using protein correlation profiling
We first obtained a crude outer segment preparation from bovine retinas using density gradient centrifugation and then enriched plasma membrane by immunoprecipitation with antibodies against its unique protein component, the Na/Ca-K exchanger. The relative content of each protein identified in the crude and enriched plasma membrane fractions was determined using label-free quantitative mass spectrometry
Our proteomic analysis assessed the relative abundances of >800 proteins identified in crude and enriched outer segment plasma membrane preparations. Using ANOVA method of fold-change values in correlated sets, we identified a small group of transmembrane proteins whose relative abundance in these preparations closely correlated with those of plasma membrane markers – the subunits of cGMP gated channel and Na/Ca-K exchanger. This behavior suggests that they are unique plasma membrane proteins. A larger group of ~40 proteins displayed a pattern of plasma membrane enrichment, suggesting that they are shared between the outer segment plasma membrane and other subcellular compartments. Proteins identified in both groups include cell adhesion molecules, receptors, transporters, SNARE proteins, small GTPases and several other poorly characterized transmembrane and peripheral membrane proteins
Our data demonstrate that protein correlation profiling of outer segment plasma membrane preparations of increasing purity is an efficient approach to identify the proteome of this cellular compartment. Knowledge of this proteome will facilitate understanding of the detailed mechanisms underlying photoreceptor morphogenesis, maintenance and signaling
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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