Purchase this article with an account.
Nycole A Maza, Mehdi Najafi, Ivayla Geneva, Peter D Calvert; Surveying the electrostatic binding potential of rod photoreceptor membranes and its role in setting peripheral membrane protein distribution. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):444.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The mechanisms underlying signal-dependent translocation of key phototransduction proteins in the rod photoreceptor are not understood. We propose a systematic study of peripheral membrane proteins and their interaction with cell structures and membranes, in live photoreceptors, in order to determine the roles of electrostatic charge and posttranslational lipidation on peripheral membrane protein localization and distribution in dark- and light- adapted rods.
Live cell electrostatic membrane probes were created by attaching genetic sequences with lipid transferase motifs and amino acid linkers bearing systematically varied degrees of charge to E/PAGFP. Probes in which the lipidation motif is mutated such that no lipid modification occurs were used to assess the impact of charge alone, whereas charge neutral linkers with lipid transferase motifs reported the impact of lipidation alone. The sequence for each probe was inserted behind the XOP promoter, and introduced to Xenopus sperm nuclei in vitro using restriction enzyme mediated integration. Retinas were harvested, and the distribution patterns and mobilities of each probe were assessed in rods using live cell confocal microscopy and multiphoton fluorescence relaxation after photoactivation (mpFRAPa). Five tadpoles were required to reach statistical significance for each probe.
Addition of a myristoylated, poly-basic targeting sequence to the N-terminus of EGFP results in a concentration distribution that differs from both EGFP alone as well as the Myr-EGFP construct. Since the inner segment is less crowded than the outer segment, soluble EGFP fluorescence in the inner segment is approximately twice as bright as that of the outer segment. However, the concentration distribution of a myristoylated EGFP probe with a +8 linker is localized primarily to the outer segment, with a small amount of fluorescence found in the ellipsoid region of the inner segment.
Our results indicate that positive charge, in addition to post-translational lipidation, is important for outer segment localization of peripheral membrane proteins. This study suggests that electrostatic charge and post-translational lipidation are both intimately involved in the distribution and localization of photoreceptor proteins.
This PDF is available to Subscribers Only