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Tingting Liu, Eugenia Poliakov, Susan Gentleman, T. Michael Redmond; Probing RPE65 palmitoylation by acyl-exchange labeling. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1733.
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© 2017 Association for Research in Vision and Ophthalmology.
Early studies showed that RPE65 is a membrane-associated protein. This membrane association was later partly attributed to S-palmitoylated cysteine residues. However, the existence, site and role of palmitoylation(s) in RPE65 are controversial. Recent mass spectrometric studies indicated that Cys112 is the required palmitoylation site for membrane association. Controversially, another study, also using mass spectrometry methods, denied RPE65 palmitoylation and concluded that membrane association occurred via electrostatic interactions with acidic phospholipid headgroups as earlier suggested. To resolve this contradiction, we used an independent method to determine the existence and relevance of palmitoylation in RPE65 structure and function.
To assay RPE65 palmitoylation, we used the acyl-biotinyl exchange (ABE) and acyl-RAC (resin assisted capture) methods. In both methods, free thiols are blocked by thiol reagent followed by cleavage of cysteine-palmitoyl linkages by hydroxylamine (HA). In ABE, the newly free thiols are labeled with biotin-HPDP followed by avidin-agarose affinity-purification; while for acyl-RAC, the HA-treated protein with free thiols is directly applied to thiopropyl sepharose 6B resin for pull-down. Eluates from both approaches were analyzed by western blotting and mass spectrometry.
We found that both the ABE and acyl-RAC methods were sensitive enough to detect palmitoyl modification of RPE65. However, we found that RPE65 was incompletely palmitoylated, showing only 3-5% palmitoylation by either method, and depended on whether RPE65 was co-expressed with LRAT or not. We confirmed that the structurally palmitoylated rhodopsin was fully palmitoylated, while CRALBP, a negative control, was not palmitoylated. Mass spectrometric analysis of ABE-labeled RPE65 peptides confirms that Cys231 is not the site of palmitoylation; other Cys-peptides are being sought. We also found that RPE65 binds to palmitoyl-CoA-agarose and is eluted by 10 mM palmitoyl-CoA, but not by 10 mM CoA, further suggesting RPE65’s affinity for the palmitoyl moiety.
We conclude that RPE65 is dynamically palmitoylated, with significant turnover in acylation. It is evident, as previously suggested, that RPE65 is not palmitoylated at all times. The functional importance of this putative dynamic palmitoylation is being studied. It is possible it may play a heretofore unappreciated role in the visual cycle.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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