The dual roles of RPE65 S-palmitoylation in membrane association and visual cycle function

Sheetal Uppal,Susan Gentleman,Tingting Liu,Eugenia Poliakov,T Michael Redmond

doi:10.1038/s41598-019-41501-w

Sheetal Uppal, Susan Gentleman + Show 3 more

Open Access

https://doi.org/10.1038/s41598-019-41501-w

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Abstract

Association with the endoplasmic reticulum (ER) membrane is a critical requirement for the catalytic function of RPE65. Several studies have investigated the nature of the RPE65-membrane interaction; however, complete understanding of its mode of membrane binding is still lacking. Previous biochemical studies suggest the membrane interaction can be partly attributed to S-palmitoylation, but the existence of RPE65 palmitoylation remains a matter of debate. Here, we re-examined RPE65 palmitoylation, and its functional consequence in the visual cycle. We clearly demonstrate that RPE65 is post-translationally modified by a palmitoyl moiety, but this is not universal (about 25% of RPE65). By extensive mutational studies we mapped the S-palmitoylation sites to residues C112 and C146. Inhibition of palmitoylation using 2-bromopalmitate and 2-fluoropalmitate completely abolish its membrane association. Furthermore, palmitoylation-deficient C112 mutants are significantly impeded in membrane association. Finally, we show that RPE65 palmitoylation level is highly regulated by lecithin:retinol acyltransferase (LRAT) enzyme. In the presence of all-trans retinol, LRAT substrate, there is a significant decrease in the level of palmitoylation of RPE65. In conclusion, our findings suggest that RPE65 is indeed a dynamically-regulated palmitoylated protein and that palmitoylation is necessary for regulating its membrane binding, and to perform its normal visual cycle function.

Highlights

RPE65 is a critical player in the visual cycle that continuously regenerates 11-cis retinal, the chromophore of rhodopsin, and is the retinol isomerase that converts all-trans retinyl esters to 11-cis retinol[1,2,3]
Quantitative analysis of palmitoylation revealed that only a small fraction of RPE65 (~25%) was modified by S-palmitoylation compared to rhodopsin with ~70% palmitoylation (Fig. 1C)
These results suggest that RPE65 palmitoylation is a dynamic or transitory process, i.e., with the concurrent existence of both palmitoylated and non-palmitoylated states

Summary

Introduction

RPE65 is a critical player in the visual (retinoid) cycle that continuously regenerates 11-cis retinal, the chromophore of rhodopsin, and is the retinol isomerase that converts all-trans retinyl esters to 11-cis retinol[1,2,3]. Www.nature.com/scientificreports native and expressed RPE65 suggested the presence of significant post-translational modification of the membrane-associated form of RPE6517; this would be consistent with palmitoylation. A reversible and dynamic process, involves the attachment of a saturated palmitic (C16) fatty acid to a cysteine residue This post-translational modification (PTM) has been implicated in protein localisation, regulation of protein stability and activity, and promotion of stable membrane binding[18]. From structural considerations, and by biochemical observations, C112 was identified as the actual palmitoylation site[13,19] This mode of membrane binding has, been challenged by another study that suggested that no post-translational palmitoylation is involved in anchoring RPE65 to the membrane, but rather that membrane association only occurs via electrostatic interactions[14]. We find that LRAT influences the dynamics of RPE65 palmitoylation, and that this may be important for normal visual cycle function

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