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T. Michael Redmond, Sheetal Uppal, Eugenia Poliakov, Susan Gentleman; Mapping and analysis of S-palmitoylation sites on RPE65. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4518.
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S-palmitoylation, a post-translational lipid modification of cysteine thiols, is the major form of protein acylation in eukaryotic cells. Reversible and highly dynamic in nature, it is known to regulate protein function and localization. RPE65, the visual cycle all-trans- to 11-cis- retinoid isomerase, is indispensable for regenerating the 11-cis retinal chromophore of opsins. RPE65 associates with membranes though it lacks membrane targeting motifs. It is proposed that S-palmitoylation of RPE65 at Cys112 controls its membrane association. However, the existence, identity and role of RPE65 palmitoylation still remain a matter of debate.
Acyl-exchange labeling experiments, both acyl-resin assisted capture (acyl-RAC) and acyl-biotin exchange (ABE) assays, were used to map S-palmitoylation sites on RPE65. This was done using both wildtype and cysteine mutants generated by site-directed mutagenesis. Palmitoylation of native RPE65 was analyzed by mass spectrometry. We also performed acyl-RAC assays in conjunction with lecithin:retinol acyltransferase (LRAT) and LRAT mutants to investigate how RPE65 palmitoylation may be integrated into the overall visual cycle.
We detect palmitoylation of wildtype RPE65 by acyl-exchange labeling, but unlike structurally palmitoylated rhodopsin (100% palmitoylated), RPE65 shows incomplete palmitoylation. By site-directed mutagenesis we have interrogated each of the cysteine residues of RPE65. Subsequent acyl-RAC analysis demonstrates that that three (Cys112, Cys146, and Cys195) out of RPE65’s 12 cysteine residues are potential sites for palmitoylation; a finding confirmed by mass spectrometry analysis. Additionally, experiments in HEK293F cells showed reduced levels of RPE65 palmitoylation when co-expressed with LRAT, whereas co-expression with the less active LRAT/Cys161Ser mutant did not result in any significant decrease in RPE65 palmitoylation levels.
We conclude that RPE65 is dynamically palmitoylated with significant turnover of the acylation. Our findings indicate that a structural role for RPE65 palmitoylation, such as in rhodopsin, is not likely. In addition, our data question whether Cys112 is the only palmitoylation site of RPE65. Furthermore, our results suggest that palmitate shuttles between RPE65 and LRAT and thus regulates the dynamics of RPE65 palmitoylation, warranting further study.
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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