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Nycole A Maza, Peter D Calvert; Intercompartmental transport of peripheral membrane proteins in rod photoreceptors. Invest. Ophthalmol. Vis. Sci. 2017;58(8):633.
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© ARVO (1962-2015); The Authors (2016-present)
In retinal photoreceptors, peripheral membrane proteins are synthesized in the myoid region and then partition amongst the major photoreceptor compartments that subserve vision; the ciliary outer segment and the synapse. The mechanisms underlying translocation to and enrichment within these compartments have been the subject of intense investigation, but remain unclear. We examined the hypothesis that lipidated proteins enriched in the outer segment are in fact in constant turnover, and thus are capable of transport through the connecting cilium to the cell body.
A myristoyl (MYR) and/or farnesyl (FAR) transferase motif was added to photoactivatable GFP (PAGFP) with a 15aa polybasic (+), acidic (-), or neutral (0) charge domain in between. Each probe was placed under the control of the XOP promoter and expressed in Xenopus laevis rod photoreceptors via REMI transgenics. Retinas were harvested and protein dynamics were analyzed via live cell confocal microscopy and multiphoton fluorescence relaxation after photoactivation (mpFRAPa). Diffusion coefficients and equilibration times were calculated from mpFRAPa data. Two-tailed student T-test was used for statistical analysis.
Photoactivation of lipidated PAGFP probes in the rod outer segment revealed that probes previously shown to be enriched in the outer segment are continuously transported into the cell body of the rod photoreceptor. Diffusion from the outer segment to the cell body was quantified and revealed that lipidated proteins equilibrate throughout the rod photoreceptor on the order of hours.
Despite clear enrichment within the major photoreceptor compartments, peripheral membrane proteins are not constrained to those compartments. This indicates that local enrichment is established through a dynamic equilibrium between local membrane binding and release into a soluble pool that is able to transport between compartments, and suggests that lipidated proteins are enriched in rod compartments on the basis of affinity for specific membrane compositions.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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