Role of LRAT on the retinoid isomerase activity and membrane association of Rpe65

Abstract

Absorption of a photon by a vertebrate opsin pigment induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the bleached opsin requires chemical re-isomerization of the chromophore via an enzyme pathway called the visual cycle. The retinoid isomerase in this pathway is Rpe65, a membrane-associated protein in the retinal pigment epithelium (RPE) with no predicted membrane-spanning segments. It has been suggested that Rpe65 is S-palmitoylated by lecithin:retinol acyl transferase (LRAT) on Cys(231), Cys(329), and Cys(330), and that this palmitoylation is required for isomerase activity and the association of Rpe65 with membranes. Here we show that the affinity of Rpe65 for membranes is similar in wild-type and lrat(-/-) mice. The isomerase activity of Rpe65 is also similar in both strains when all-trans-retinyl palmitate is used as substrate. With all-trans-retinol substrate, isomerase activity is present in wild-type but undetectable in RPE homogenates from lrat(-/-) mice. Substitution of Cys(231), Cys(329), and Cys(330) with Ser or Ala did not affect the affinity of Rpe65 for membranes. Further, these Cys residues are not palmitoylated in Rpe65 by mass spectrometric analysis. Global inhibition of protein palmitoylation by 2-bromopalmitate did not affect the solubility or isomerase activity of Rpe65. Finally, we show that soluble and membrane-associated Rpe65 possesses similar isomerase specific activities. These results indicate that LRAT is not required for isomerase activity beyond synthesis of retinyl-ester substrate, and that the association of Rpe65 with membranes is neither dependent upon LRAT nor the result of S-palmitoylation. The affinity of Rpe65 for membranes is probably an intrinsic feature of this protein.

FIGURE 1. Visual cycle in RPE cells

Absorption of a photon (hv) by a rhodopsin pigment molecule induces isomerization of 11-cis-RAL to all-trans-RAL resulting in activated metarhodopsin II. Decay of metarhodopsin II yields apoopsin and free all-trans-RAL, which is reduced to all-trans-ROL by one of several all-trans-ROL dehydrogenases (all-trans-RDH). The all-trans-ROL is released by photoreceptors and taken up by RPE cells where it is esterified to a fatty acid from phosphatidylcholine by LRAT. Rpe65 isomerase uses all-trans-RE as substrate to form 11-cis-ROL plus a free fatty acid. 11-cis-ROL is oxidized to 11-cis-RAL by one of several 11-cis-ROL dehydrogenases (11-cis-RDH). 11-cis-RAL is released by the RPE and taken up by the photoreceptor where it combines with apo-opsin to re-form rhodopsin.

FIGURE 2. Isomerase kinetics in RPE from WT and lrat^-/- mice

A, Michaelis-Menten plot showing the initial rates of 11-cis-ROL synthesis (V₀) versus concentrations of all-trans-RP substrate using RPE homogenates from WT mice as an enzyme source. B, similar plot as in Fig. 2A except RPE homogenates from lrat^-/- mice were used as an enzyme source. Eadie-Hofstee transformations of the data in A and B yielded the kinetic parameters, V_max and K_m. C, 11-cis-ROL synthesis from all-trans-ROL substrate by WT and lrat^-/- mouse RPE homogenates. Error bars show S.D. (n = 3). D, representative HPLC chromatogram of retinoids extracted following an in vitro isomerase assay of a WT mouse RPE-homogenate using all-trans-ROL substrate. The 11-cis-, 13-cis-, 9-cis-, and all-trans-ROL peaks are labeled. Inset shows the UV-spectrum acquired from the 11-cis-ROL peak. E, representative HPLC chromatogram of retinoids extracted following an in vitro isomerase assay of an lrat^-/- mouse RPE-homogenate using all-trans-RP substrate. Peaks showing all-trans-RP and 11-cis-, 13-cis-, 9-cis-, and all-trans-ROL are labeled. Inset shows the UV-spectrum acquired from the 11-cis-ROL peak.

FIGURE 3. LRAT-independent association of Rpe65 with membranes in mouse RPE

A and B, representative immunoblot analysis of Rpe65 in total RPE homogenates and membrane pellets. Lanes of the gel contained 1.0 μl of total homogenate (homog) (1.0 μg/μl), and 1.0 or 1.5 μl of suspended membrane pellet (memb) from WT (A) or lrat^-/- (B) RPE. C and D, representative immunoblot analysis of Rpe65 in 5.0 μl of membrane-free supernatants (sup), and 4.0, 2.0, 1.0, or 0.5 μl of total homogenates (homog) (1.0 μg/μl) from WT (C) or lrat^-/- (D) RPE. The Rpe65 bands were visualized by ECL detection.

FIGURE 4. Membrane association of Rpe65 after substitution of the putatively S-palmitoylated Cys residues

Rpe65 immunoblots of total homogenates and washed membranes from 293T (A) and (B) and 293T-L cells (C) and (D) expressing normal and the indicated Cys-to-Ser substituted Rpe65. Negative control samples were prepared from 293T or 293T-L cells transfected with non-recombinant pRK5 vector. Each lane contains 10 μg of protein. Rpe65 bands were visualized by ECL detection. Note the similar Rpe65 immunoreactivities in membrane samples from cells expressing unsubstituted and substituted Rpe65.

FIGURE 5. Fraction of normal and C231A/C329A/C330A-substituted Rpe65 in membrane-free supernatants

Rpe65 immunoblots of normal (A) and (B) or C231A/C329A/C330A-substituted (C) and (D) Rpe65 expressed in 293T (A) and (C) or 293T-L (B) and (D) cells. Lanes contain the indicated amounts of total protein from membrane-free supernatants (sup) or cell homogenates (homog). Note the similar immunoblot signal-intensities of normal Rpe65 in lanes containing 5.0 μl of supernatant and 1.0 μl of total homogenate (1.0 μg/μl) (A) and (B), and the similar signal intensities of C231A/C329A/C330A-substituted Rpe65 in lanes containing 15 μl of supernatant and 3.0 μl of total homogenate (C) and (D). Thus, for both forms of Rpe65, the soluble fraction is ∼20%.

FIGURE 6. Strategy to determine palmitoylation status of Cys²³¹, Cys³²⁹, Cys³³⁰ in Rpe65

A, flow chart for the preparation of Rpe65 peptide samples for LC-MS analysis. One aliquot of bovine RPE membranes are incubated with DTT, which reduces disulfides and de-palmitoylates Cys residues in all proteins including Rpe65. The resulting free sulfhydryl groups are alkylated by incubating the microsomes with non-deuterated acrylamide (AA-d0). A second aliquot of RPE microsomes are incubated with TBP to reduce disulfide without de-palmitoylating Cys residues. The resulting free sulfhydryls are alkylated by incubating with triple-deuterated acrylamide (AA-d3). The microsomal samples are combined and the constituent proteins separated by SDS-PAGE. The band containing Rpe65 is digested with trypsin or endopeptidase Glu-C, and the resulting peptides are analyzed by LC-MS/MS. B, schemes of possible mass spectra. If a Cys-containing peptide is not palmitoylated, it would incorporate equal amounts of AA-d0 and AA-d3. Therefore, two sets of native isotopic peaks that differ by three atomic mass units would be equally abundant in the spectrum. If the Cys residue is partially palmitoylated, the triple-deuterated ions would be correspondingly less abundant than the nondeuterated ions. If the Cys residue is fully palmitoylated, no triple-deuterated ions would be represented in the spectrum.

FIGURE 7. ESI-MS data showing similar abundance of AA-d0 and AA-d3 modified peptides containing residues Cys²³¹, Cys³²⁹, and Cys³³⁰

A, mass chromatogram of ions with m/z range 1450.3–1451.3. This range contains the single charged tryptic peptide, T223–234 from bovine Rpe65 after modification of Cys²³¹ with AA-d0. NL indicates the ion current of the base peak for abundance normalization. B, mass chromatograms of ions with m/z range 1453.3–1454.3 containing singly charged peptide T223–234 after modification of Cys²³¹ with AA-d3. C, zoom-scan spectrum of the indicated m/z values acquired at 32.13 min. Note duplication of the three native isotopic peaks (dark numbers)at +3 m/z (light numbers). The similar abundance of the corresponding AA-d0 and AA-d3-labeled ions indicates that Cys²²⁹ is not palmitoylated. D, mass chromatogram of ions with m/z range 1300.8–1301.8. This range contains the triple charged GluC peptide, G323–352 from bovine Rpe65 after modification of Cys³²⁹ and Cys³³⁰ with AA-d0. E, mass chromatogram of ions with m/z range 1302.4–1303.4, which contains triple charged peptide G323–352 after modification of Cys³²⁹ and Cys³³⁰ with AA-d3. F, zoom-scan spectrum of the indicated m/z values acquired at 47.99 min. The native-isotopic peaks (dark numbers) differ by ∼0.3 m/z due to the triple charge on peptide G323–352. Note the duplication of these four peaks at +2 m/z (light numbers). The similar abundance of the corresponding AA-d0 and AA-d3-labeled ions indicates that Cys³²⁹ and Cys³³⁰ are not palmitoylated.

FIGURE 8. Isomerase activities of normal and substituted Rpe65 that eliminate the putative S-palmitoylation sites

Synthesis of 11-cis-ROL by 293T-LC cells expressing the indicated Rpe65 from all-trans-ROL substrate added to the medium. Isomerase activities are expressed as the percent 11-cis-ROL synthesized by unsubstituted Rpe65 assayed in 293T-LC cells (pmol of 11-cis-ROL per mg protein). No synthesis of 11-cis-ROL by 293T-LC cells transfected with nonrecombinant pRK5 (negative control). Error bars show S.D. (n = 3).

FIGURE 9. Isomerase activities of soluble and membrane-associated Rpe65

A, immunoblot analysis of Rpe65 in total homogenates (homog) (1.0 μg/μl) and membrane-free supernatants (sup) of bovine RPE. The amount of total protein loaded is shown for each lane. Note the similar Rpe65 immunoreactivity in lanes containing 0.2 μl of homogenate and 1.2 μl of supernatant, indicating that ∼17% of Rpe65 is soluble in bovine RPE. B, isomerase activities of bovine RPE homogenates and membrane-free supernatants, shown as percent of the isomerase activity in bovine RPE homogenates. C, isomerase activities of homogenates from 293T-LC cells expressing Rpe65 that were untreated (control) or incubated with 100 μM 2BP in the culture medium for eight hours (2BP). Shown is percent of the isomerase activity in control cells. Error bars show S.D. (n = 3). D, isomerase activities of normal or Y368H-substituted Rpe65 following extraction from expressing 293T-cell membranes with the indicated concentrations of sodium cholate. Activities are expressed as pmol of 11-cis-ROL synthesized per mg of protein during a 2-h incubation. The substrate was all-trans-RP. E, immunoblot analysis of Rpe65 in the same supernatant samples as D. Note the strong correlation of isomerase activity and Rpe65 immunoreactivity in the supernatant fractions of cells expressing normal Rpe65.