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E. Demers, S. Champagne, C. Salesse; Membrane Interactions of Human Retinitis Pigmentosa 2 Protein, RP2. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1683. doi: https://doi.org/.
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Retinitis pigmentosa (RP) is the major form of heritable blindness which results in progressive retinal degeneration. A severe form of RP, X-linked retinitis pigmentosa, is linked to mutations of the 350 residues protein RP2. Although RP2 is ubiquitously expressed, mutations of this protein are only known to cause an ocular disease phenotype. This protein has dual acylation sites (including one on ser6) and localizes predominantly to the plasma membrane. The preferential targeting of RP2 to the plasma membrane has been clearly demonstrated when the deletion of Ser6 of RP2 prevented membrane localization and resulted in RP. The objectives of this research work were to overexpress and purify RP2, and to characterize its membrane binding properties using phospholipids monolayers as model membranes.
The complete sequence of RP2 (amino acids 1-350) in the expression vector pGEX-4T-3 was transformed and expressed in E. coli. Purification was accomplished by affinity chromatography. RP2 was injected into the subphase underneath phospholipid monolayers with different fatty acyl chains (length and unsaturation) and polar headgroups. RP2 binding was monitored by surface pressure measurements.
The injection of RP2 underneath phospholipid monolayers led to an increase in surface pressure which clearly indicates its membrane binding. The surface pressure data demonstrate that the adsorption kinetics of RP2 remains unchanged at different pH but is strongly affected by the ionic strength of the subphase as well as by the type of phospholipid fatty acyl chain (length and unsaturation) and headgroups. For example, RP2 shows a stronger binding to the negatively charged phosphatidylserine than phosphatidylcholine and phosphatidylethanolamine.
The parameters responsible for the modulation of RP2 binding to membranes are still largely unknown and its function is subject of discussion. The present work demonstrates that non-acylated RP2 strongly binds membranes. This interaction will be further studied by spectroscopic methods.
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