Abstract
Purpose: :
Rpe65 is an abundant protein in the retinal pigment epithelium (RPE) that converts all trans retinyl esters to 11 cis retinol. Rpe65 associates with phospholipid membranes but contains no membrane-spanning domains. Rpe65 was previously reported to undergo S palmitoylation at residues C231, C329, and C330. However, recent results from our and other labs suggest that these Cys residues are not modified and are not required for isomerase catalytic activity or the association of Rpe65 with membranes. In this work we used mass spectrometry (MS) to identify other modified residues that may explain the affinity of Rpe65 for membranes.
Methods: :
RPE microsomes were prepared from the eyes of freshly slaughtered cattle. One aliquot was incubated with dithiothreitol (DTT) to remove any S acyl groups on Cys residues in Rpe65. Another was incubated with tributylphosphine (TBP) to reduce disulfides without removing S acyl modifications in Rpe65. The hydrophobicities of both Rpe65 forms were studied by reverse-phase liquid chromatography (LC) followed by immunoblotting. The molecular mass of both Rpe65 forms were measured by electrospray-ionization (ESI) MS. Rpe65 in the two aliquots were isotopically tagged with acrylamide and subjected to LC-MS analysis following proteolytic digestion. Possible post-translational modification sites were identified by studying the relative ratio of the isotopic mass peaks.
Results: :
DTT-treatment of RPE microsomes yielded a single eluted peak of Rpe65 by reverse-phase LC and immunoblotting. In contrast, the TBP-treated sample eluted broadly, suggesting a DTT-sensitive modification. ESI-MS analysis of the DTT-treated sample yielded a molecular mass that was within 10 Da of the predicted mass, indicating no non-Cys post-translational modifications of Rpe65. LC MS/MS analysis of proteolytic peptides permitted identification of all 12 Cys residues in Rpe65. Residue C396 showed the highest degree of modification at ~20%. No other Cys residues showed detectable modification.
Conclusions: :
Bovine Rpe65 is partially S acylated on residue C396. However, the strong association of Rpe65 with membranes is unlikely caused by this modification, since C396 is not conserved in Rpe65 from other vertebrate species, and since only ~20% of bovine Rpe65 is S acylated on C396. A more likely explanation for the membrane affinity of Rpe65 is unidentified structural elements within the protein that interact with phospholipid headgroups.
Keywords: protein modifications-post translational