May 2007
Volume 48, Issue 13
ARVO Annual Meeting Abstract  |   May 2007
The Isomerase Activity and Association of Rpe65 With Membranes Is Not Dependent on LRAT
Author Affiliations & Notes
  • M. Jin
    Jules Stein Eye Institute, UCLA School of Med., Los Angeles, California
  • S. Li
    Jules Stein Eye Institute, UCLA School of Med., Los Angeles, California
  • G. H. Travis
    Jules Stein Eye Institute, UCLA School of Med., Los Angeles, California
  • Footnotes
    Commercial Relationships M. Jin, None; S. Li, None; G.H. Travis, None.
  • Footnotes
    Support NIH Grant EY015844
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 3262. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose:: 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.

Methods:: 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.

Results:: 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.

Conclusions:: (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.

Keywords: retinoids/retinoid binding proteins • retinal pigment epithelium • proteins encoded by disease genes 

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