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M. Jin, S. Li, G. H. Travis; The Isomerase Activity and Association of Rpe65 With Membranes Is Not Dependent on LRAT. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3262.
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Rpe65 is a membrane-associated protein with no predicted membrane-spanning segment. It has been hypothesized that Rpe65 is S-palmitoylated on Cys231, Cys329, and Cys330 by LRAT, and that these modifications are responsible for its membrane-association. The purpose of this study was (1) to determine if LRAT is required for the membrane-association of Rpe65; (2) to test the role of palmitoylation of the three Cys; (3) to explore significance of the membrane-association of Rpe65.
RPE cells were collected from eyes of cattle, wild-type and lrat -/- mice (from K. Palczewski lab at Case Western Univ.). We replaced the three Cys in bovine Rpe65 with Ala or Ser in all combinations by site-directed mutagenesis. These substituted Rpe65’s were expressed in 293T cells alone and in combination with LRAT (293T-LRAT cells). Membrane-free supernatants were prepared from cell homogenates by ultracentrifugation. Rpe65-content was determined by quantitative immunoblotting. Isomerase activity was measured by monitoring 11-cis retinol (11cROL) formation from all-trans retinol (atROL) or all-trans retinyl palmitate (atRP) added into cell homogenates, membrane-free supernatants, or medium of the culture cells.
Approximately 80% of Rpe65 in RPE homogenates from lrat -/- mice was associated with membrane, similar to the fraction from wild-type mice. Consistent with this result, approximately 20% of Rpe65 expressed in 293T and 293T-LRAT cells was in the membrane-free supernatants. We observed similar membrane fractionation for single-, double-, and triple-substituted Rpe65’s. The single-substituted Rpe65’s had significant isomerase activity, while triple-substituted Rpe65 possessed no isomerase activity. Similar to wild-type Rpe65, triple-substituted Rpe65 was still associated with membrane. Homogenates from bovine and wild-type mice RPE synthesized 11cROL from both atROL and atRP substrates. However, homogenates from lrat -/- mice RPE only synthesized 11cROL from atRP. Finally, the isomerase activities in the membrane-free supernatants were tightly correlated with the amount of Rpe65.
(1) The membrane-association of Rpe65 is not dependent on LRAT. (2) Palmitoylation of Cys231, Cys329, and/or Cys330 is not required for Rpe65 to associate with membranes. (3) The substrate for Rpe65-isomerase in lrat -/- mouse RPE is atRP. (4) The isomerase specific-activities of soluble and membrane-associated Rpe65 are similar. Dissociation of Rpe65 from membranes in vivo however, would separate the enzyme from its source of atRP substrate and therefore may serve as a mechanism to down-regulate isomerase activity.
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