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Updated: Jan 27, 2026

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins
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Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

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

Sheetal Uppal1, Tingting Liu1,2, Eugenia Poliakov1

  • 1Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD, 20892, United States.

Scientific Reports
|March 28, 2019
PubMed

Insights

Retinal pigment epithelium 65 (RPE65) is palmitoylated, a modification essential for its endoplasmic reticulum membrane association and visual cycle function. This palmitoylation is regulated by the lecithin:retinol acyltransferase (LRAT) enzyme.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Vision Science

Background:

  • Endoplasmic reticulum (ER) membrane association is crucial for RPE65 catalytic activity.
  • The exact mechanism of RPE65 membrane binding, particularly S-palmitoylation, remains debated.
  • Understanding RPE65's membrane interaction is key to deciphering its role in the visual cycle.

Purpose of the Study:

  • To re-examine RPE65 S-palmitoylation and its functional consequences in the visual cycle.
  • To identify the specific sites of RPE65 palmitoylation.
  • To investigate the regulation of RPE65 palmitoylation by LRAT.

Main Methods:

  • Post-translational modification analysis to detect RPE65 palmitoylation.
  • Extensive site-directed mutagenesis to identify palmitoylation sites (C112, C146).
  • Inhibition of palmitoylation using specific chemical inhibitors (2-bromopalmitate, 2-fluoropalmitate).
  • Assays to measure RPE65 membrane association in wild-type and mutant forms.
  • Enzyme activity assays involving LRAT and all-trans retinol.

Main Results:

  • RPE65 is confirmed to be S-palmitoylated, though not universally (approx. 25% of protein).
  • Palmitoylation sites were mapped to cysteine residues C112 and C146.
  • Inhibition of palmitoylation abolished RPE65 membrane association; C112 mutants showed impaired membrane binding.
  • Lecithin:retinol acyltransferase (LRAT) regulates RPE65 palmitoylation, decreasing it in the presence of all-trans retinol.

Conclusions:

  • RPE65 is a dynamically regulated palmitoylated protein.
  • Palmitoylation is essential for RPE65's proper membrane binding and visual cycle function.
  • LRAT activity modulates RPE65 palmitoylation levels, impacting its membrane association.

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