Rpe65 isomerase associates with membranes through an electrostatic interaction with acidic phospholipid headgroups

Article abstract of DOI:10.1074/jbc.M109.025643

Opsins are light-sensitive pigments in the vertebrate retina, comprising a G protein-coupled receptor and an 11-cis-retinal-dehyde chromophore. Absorption of a photon by an opsin pigment induces isomerization of its chromophore to all-trans-retinal-dehyde. After a brief period of activation, opsin releases all-trans-retinaldehyde and becomes insensitive to light. Restoration of light sensitivity to the apo-opsin involves the conversion of all-trans-retinaldehyde back to 11-cis-retinal-dehyde via an enzyme pathway called the visual cycle. The critical isomerization step in this pathway is catalyzed by Rpe65. Rpe65 is strongly associated with membranes but contains no membrane- spanning segments. It was previously suggested that the affinity of Rpe65 for membranes is due to palmitoylation of one or more Cys residues. In this study, we re-examined this hypothesis. By two independent strategies involving mass spectrometry, we show that Rpe65 is not palmitoylated nor does it appear to undergo other post-translational modifications at significant stoichiometry. Instead, we show that Rpe65 binds the acidic phospholipids, phosphatidylserine, phosphatidylglycerol, and cardiolipin, but not phosphatidic acid. No binding of Rpe65 to basic phospholipids or neutral lipids was observed. The affinity of Rpe65 to acidic phospholipids was strongly pH-dependent, suggesting an electrostatic interaction of basic residues in Rpe65 with negatively charged phospholipid headgroups. Binding of Rpe65 to liposomes containing phosphatidylserine or phosphatidylglycerol, but not the basic or neutral phospholipids, allowed the enzyme to extract its insoluble substrate, all-trans- retinyl palmitate, from the lipid bilayer for synthesis of 11-cis-retinol. The interaction of Rpe65 with acidic phospholipids is therefore biologically relevant.

Full text of DOI:10.1074/jbc.M109.025643

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 2, pp. 988–999, January 8, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Rpe65 Isomerase Associates with Membranes through an
Electrostatic Interaction with Acidic Phospholipid Headgroups*
Received for publication, May 26, 2009, and in revised form, October 27, 2009 Published, JBC Papers in Press, November 5, 2009, DOI 10.1074/jbc.M109.025643
Quan Yuan‡, Joanna J. Kaylor‡, Anh Miu‡, Sara Bassilian§, Julian P. Whitelegge§, and Gabriel H. Travis‡¶1
From the ‡Jules Stein Eye Institute, §Pasarow Mass Spectrometry Laboratory, Neuropsychiatric Institute-Semel Institute, and
¶Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California 90095
RAL),2 which is covalently coupled to a Lys residue in the opsin
protein via a protonated Schiff-base linkage. Absorption of a
photon by 11-cis-RAL induces its isomerization to all-trans-
Opsins are light-sensitive pigments in the vertebrate retina,
comprising a G protein-coupled receptor and an 11-cis-retinal-
dehyde chromophore. Absorption of a photon by an opsin pig-
ment induces isomerization of its chromophore to all-trans- retinaldehyde (all-trans-RAL), transiently activating the opsin
pigment. After a brief period, the photobleached pigment
decays to yield apo-opsin and free all-trans-RAL. Recombina-
tion of apo-opsin with another 11-cis-RAL regenerates the vis-
ual pigment. To maintain light sensitivity, the all-trans-RAL
released following photobleach of an opsin pigment is con-
verted back to 11-cis-RAL by a multistep enzymatic pathway
called the visual cycle (Fig. 1). Most steps of the visual cycle take
place within cells of the retinal pigment epithelium (RPE), an
epithelial monolayer adjacent to photoreceptor outer segment.
The critical step in this pathway is all-trans to 11-cis re-isomer-
ization of the retinoid, catalyzed by Rpe65 isomerase (1–3).
Consistently, mice with a knock-out mutation in the rpe65 gene
contain no 11-cis-retinoids in their ocular tissues and are blind
(4). Similarly, mutations in the human RPE65 gene cause a
severe, recessively inherited blinding disease called Leber con-
genital amaurosis (5).
retinaldehyde. After a brief period of activation, opsin releases
all-trans-retinaldehyde and becomes insensitive to light. Resto-
ration of light sensitivity to the apo-opsin involves the conver-
sion of all-trans-retinaldehyde back to 11-cis-retinaldehyde via
an enzyme pathway called the visual cycle. The critical isomer-
ization step in this pathway is catalyzed by Rpe65. Rpe65 is
strongly associated with membranes but contains no mem-
brane-spanning segments. It was previously suggested that the
affinity of Rpe65 for membranes is due to palmitoylation of one
or more Cys residues. In this study, we re-examined this hypoth-
esis. By two independent strategies involving mass spectrome-
try, we show that Rpe65 is not palmitoylated nor does it appear
to undergo other post-translational modifications at significant
stoichiometry. Instead, we show that Rpe65 binds the acidic
phospholipids, phosphatidylserine, phosphatidylglycerol, and
cardiolipin, but not phosphatidic acid. No binding of Rpe65 to
basic phospholipids or neutral lipids was observed. The affinity
of Rpe65 to acidic phospholipids was strongly pH-dependent,
suggesting an electrostatic interaction of basic residues in
Rpe65 with negatively charged phospholipid headgroups. Bind-
ing of Rpe65 to liposomes containing phosphatidylserine or
phosphatidylglycerol, but not the basic or neutral phospholip-
ids, allowed the enzyme to extract its insoluble substrate, all-
trans-retinyl palmitate, from the lipid bilayer for synthesis of
11-cis-retinol. The interaction of Rpe65 with acidic phospholip-
ids is therefore biologically relevant.
Rpe65 is an abundant 61-kDa protein in RPE cells. Rpe65 is
homologous to ␤-carotene 15,15Ј-oxygenase in mammals (6)
and apo-carotene oxygenase in cyanobacteria (7). By x-ray dif-
fraction analysis, Rpe65 is a seven-bladed ␤-propeller with an
Fe2ϩ-His4 arrangement at its axis (8). The four His residues that
define the Fe2ϩ-binding site are conserved in all members of
this protein family (Fig. 2). Rpe65 catalyzes the isomerization
and hydrolysis of an all-trans-retinyl ester (all-trans-RE), such
as all-trans-retinyl palmitate (all-trans-RP), to 11-cis-retinol
(11-cis-ROL), and a free fatty acid (9–11). In this reaction, the
energy of ester hydrolysis is used to drive the energetically unfa-
vorable conversion of a planar all-trans-retinoid to the strained
11-cis configuration (12). All-trans-REs are insoluble in water
and stored in RPE internal membranes. Rpe65 strongly associ-
ates with membranes (13), providing access to its all-trans-RE
substrate. The basis for the association of Rpe65 with mem-
branes is unresolved. Rpe65 has no predicted transmembrane
segments. It was suggested that Rpe65 is reversibly S-palmitoy-
Visual perception in vertebrates is mediated by two types of
photosensitive cells, rods and cones. Both contain a process
called the outer segment that consists of a stack of flattened
membranous discs loaded with rhodopsin or cone-opsin visual
pigment. Opsin pigments are photo-sensitive members of the G
protein-coupled receptor superfamily. The light-absorbing
ligand of most opsin pigments is 11-cis-retinaldehyde (11-cis- 2 The abbreviations used are: 11-cis-RAL, 11-cis-retinaldehyde; all-trans-RAL,
all-trans-retinaldehyde; TBP, tributylphosphine; PG, phosphatidylglycerol;
PS, phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanol-
amine; RPE, retinal pigment epithelium; CL, cardiolipin; DTT, dithiothreitol;
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grants EY01584 and EY11713 from NEI.
PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatogra-
phy; ESI, electrospray ionization; MS/MS, tandem mass spectrometry; ER,
endoplasmic reticulum; all-trans-RP, all-trans-retinyl palmitate; all-trans-
RE, all-trans-retinyl ester; 11-cis-ROL, 11-cis-retinol; S-palm-N-Ac-Cys-Me,
S-palmitoyl-N-acetyl-L-cysteine methyl ester; LC, liquid chromatography;
AA, acrylamide.
1 Charles Kenneth Feldman Professor of Ophthalmology. To whom corre-
spondence should be addressed: Jules Stein Eye Institute, UCLA School of
Medicine, 100 Stein Plaza, Los Angeles, CA 90095. Tel.: 310-267-2673;
E-mail: travis@jsei.ucla.edu.
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Association of Rpe65 with Membranes
lated on residues Cys-231, Cys-329, and Cys-330 (14). A “palmi-
toylation-switch” mechanism for the regulation of isomerase
activity was proposed whereby binding of Rpe65 to its all-
trans-RP substrate is mediated by reversible S-palmitoylation
of these three residues (14). However, it was subsequently
shown that Cys-231, Cys-329, and Cys-330 are not modified in
Rpe65 (15). More recently, it was suggested that Rpe65 is pal-
mitoylated on Cys-112 (16). In this study, we examined all 12
Cys residues in bovine Rpe65 and found no evidence for signif-
icant S-palmitoylation at any site. However, we found that
Rpe65 interacts directly with three acidic phospholipids but not
with basic phospholipids or neutral lipids, explaining the affin-
ity of Rpe65 for membranes. We show that this interaction is
functionally relevant, as it permits Rpe65 to extract all-trans-
REs from the lipid bilayer and use them as substrate for synthe-
sis of 11-cis-ROL.
FIGURE 1. Visual cycle in RPE cells. Absorption of a photon (hv) by a rhodop-
sin pigment molecule induces isomerization of 11-cis-RAL to all-trans-RAL
resultinginactivatedmetarhodopsinII. DecayofmetarhodopsinIIyieldsapo-
opsin and free all-trans-RAL, which is reduced to all-trans-ROL by one or more
all-trans-ROL dehydrogenases (all-trans-RDHs). The all-trans-ROL is released
EXPERIMENTAL PROCEDURES
Materials—Unless stated otherwise, all chemicals and sol-
by photoreceptors into the extracellular space and taken up by RPE cells vents were purchased from Sigma. 2,3,3-Acrylamide-d3 was
where it is esterified to a fatty acid from phosphatidylcholine by lecithin:
retinol acyltransferase (LRAT). Rpe65 isomerase uses all-trans-RE as substrate
toform11-cis-ROLplusafreefattyacid. 11-cis-ROLisoxidizedto11-cis-RALby
one of several 11-cis-ROL dehydrogenases (11-cis-RDHs). Both 11-cis-ROL and
purchased from Cambridge Isotope Laboratories. Modified
trypsin was purchased from Promega. Endopeptidase Glu-C
and Asp-N were purchased from Roche Diagnostics. Phospho-
11-cis-RAL are bound to cellular retinaldehyde-binding protein in RPE cells.
lipids (PC and PE from egg yolk, PG from egg yolk lecithin, PS
11-cis-RAL is released by the RPE and taken up by the photoreceptor where it
from bovine brain, and PI from bovine liver) were purchased
combines with apo-opsin to re-form rhodopsin.
from Sigma as chloroform solutions. The membrane lipid strip
FIGURE 2. Annotated sequence alignment of Rpe65 from several vertebrate species. Bovine Rpe65 contains 12 Cys residues (red boxes). Eight are con-
served in all species, and the remaining four, including Cys-396, are not conserved. The four conserved His residues that form the Fe2ϩ-coordination sphere are
indicated by green boxes. The two highly basic sequences that map to the surface of Rpe65 are indicated by blue boxes. The possible hydrophobic membrane-
anchor region (residues 103–123) is indicated by the purple box.
JANUARY 8, 2010•VOLUME 285•NUMBER 2
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Association of Rpe65 with Membranes
was purchased from Echelon Bioscience Inc. CompleteTM trifugal vacuum concentrator and dissolved into 10 ␮l of 0.1%
EDTA-free proteinase inhibitor mixture tablets were pur- formic acid, 5% acetonitrile in water.
chased from Roche Diagnostics. Protein concentration was
Synthesis of S-Palmitoyl-N-acetyl-L-cysteine Methyl Ester—
detected by a mini BCA kit from Pierce (part of Thermo Fisher In a glass flask equipped with a stir bar, 17.7 mg (0.1 mmol) of
Scientific). GelCode Coomassie Blue was also purchased from N-acetyl-L-cysteine methyl ester (S-palm-N-Ac-Cys-Me) was
Pierce. The Millipore Montage In-Gel Digestzp kit was pur- incubated with palmitoyl chloride (20 eq), N-hydroxybenzo-
chased from Fisher.
triazole (20 eq), dicyclohexylcarbodiimide (20 eq), and 4-di-
Preparation of Bovine RPE Homogenates and Microsomes— methylaminopyridine (22 eq) in 2 ml of anhydrous tetrahydro-
Bovine eyes were obtained from a local slaughterhouse and furan for 15 h. After filtration, the reaction was stopped with
used within 4 h. After dark adaptation for 1 h, bovine RPE cells addition of 5 ml of 0.1 M HCl, and the mixture was extracted
were collected by gentle brushing from dissected cow eyeballs twice with CH2Cl2. The pooled extracts were washed with brine
after removal of the retina. RPE cells were suspended in 30 ml and dried with anhydrous Na2SO4. Pure S-palm-N-Ac-Cys-Me
of 20 mM HEPES (pH 7.4), 150 mM NaCl with addition of was separated by reversed phase HPLC and characterized by
CompleteTM EDTA-free proteinase inhibitor and homoge- tandem mass spectrometry (MS/MS).
nized by nitrogen cavitation at 1000 p.s.i. The supernatant was
Confirmation of Cysteine De-palmitoylation by DTT but Not
collected after centrifugation at 3000 ϫ g as a bovine RPE TBP—To 3 aliquots of purified S-palm-N-Ac-Cys-Me in 50 mM
homogenate. The homogenate was lyophilized to a dry powder ammonium bicarbonate (pH 8.4) were added 20 ␮l of 50 mM
and resuspended in 2 ml of distilled H2O and stored at Ϫ80 °C. DTT, 20 ␮l of 50 mM TBP in 50 mM ammonium bicarbonate
Bovine RPE microsomes were collected by centrifugation of the (pH 8.4), and 20 ␮l of 50 mM ammonium bicarbonate (pH 8.4),
supernatant mentioned above at 100,000 ϫ g, and the pellet was respectively. The aliquot with DTT was incubated at 70 °C for
resuspended in HEPES or Tris-HCl buffer and stored in 3 h, and the other 2 aliquots were incubated at 37 °C for 0.5 h.
Ϫ80 °C.
The 3 aliquots were then incubated with 20 ␮l of 1:1 mixture of
Preparation of Acrylamide-modified Bovine Rpe65—Two ali- 250 mM acrylamide (AA-d0/d3) in 50 mM ammonium bicarbon-
quots of bovine RPE microsomal protein solutions (225 mg of ate (pH 8.4) at 25 °C for 3 h. The final samples were subjected to
protein in each) were dissolved in 50 ␮l of 50 mM Tris-HCl capillary LC/MS analysis.
buffer (pH 8.0) containing 0.1% SDS. To one aliquot, 20 ␮l of 50
Liquid Chromatography with Electrospray Ionization-Mass
mM dithiothreitol (DTT) in 50 mM ammonium bicarbonate (pH Spectrometry (LC-MS) of Intact Rpe65 Protein—Proteins were
8.4) was added, and the solution was incubated at 70 °C for 3 h. fractionated by reverse-phase liquid chromatography as
To another aliquot, 20 ␮l of 50 mM tributylphosphine (TBP) in described below. The eluent was split between a fraction collec-
50 mM ammonium bicarbonate (pH 8.4) was added, and the tor, collecting 1-min fractions, and an electrospray ionization
ϩ
solution was incubated at 37 °C for 0.5 h. The DTT sample was (ESI) mass spectrometer, API III mass spectrometer (PE
incubated with 20 ␮l of 250 mM acrylamide (AA-d0) in 50 mM Sciex, Applied Biosystem, Foster City, CA), as described previ-
ammonium bicarbonate (pH 8.4) at 25 °C for 3 h, and its TBP ously (17, 18). For HPLC fractionation, a PLRP-S polymeric
counterpart was incubated with triple deuterium-labeled acryl- column was used with a pore size of 300 Å, bead size of 5 ␮m,
amide (AA-d3) instead. The 2 aliquots were pooled and sepa- and dimensions of 150 ϫ 2 mm (Polymer Laboratories,
rated by SDS-PAGE. The protein bands representing purified Amherst, MA). The column was maintained at 40 °C and ini-
bovine Rpe65 were dissected and subjected to in-gel proteolytic tially equilibrated in solvent A (0.1% trifluoroacetic acid in
digestion (described below). A control sample incubated with water) at 95% and solvent B (0.1% trifluoroacetic acid in aceto-
a 1:1 acrylamide-d0/d3 solution was prepared in the same nitrile) at 5%. Rpe65 protein was eluted at a flow rate of 100
manner.
␮l/min using a program of 5 min at 5% B, followed by a 25-min
In-gel Proteolytic Digestion—Following the manufacturer’s gradient from 5 to 30% B, and ending with a 100-min gradient
procedure in the Montage kit, gel slices containing purified from 30 to 100% B, for a total gradient time of 130 min. The
ϩ
Rpe65 were diced and placed in a 96-well plate. 100 ␮l of destain API III mass spectrometer was tuned and calibrated as
solution (50:50 acetonitrile/water) was added to each well and described previously (19) to yield a mass accuracy of 0.01%
incubated for 20 min. After removal of the destaining solution (1.0 Da at 10 kDa). The 50-␮l fractions collected during chro-
by vacuum, 100 ␮l of 100% acetonitrile was added to dehydrate matography were stored at Ϫ20 °C. The data were analyzed using
the gel slices. After incubation for 10 min, the solution was BioMultiview 1.3.1 software (Applied Biosystems).
removed by aspiration. The gel slices were re-hydrated in a
Nano-liquid Chromatography with Data-dependent Tandem
25–30 ␮l of solution containing the selected protease (weight Mass Spectrometry—Samples were analyzed by nano-liquid
ratio of substrate to enzyme, 100:1 for trypsin and 200:1 for chromatography-MS/MS with data-dependent acquisition (Q
endoproteinase Glu-C and Asp-N) in 50 mM ammonium bicar- STAR XL, Applied Biosystems, Foster City, CA) as described
bonate (pH 8.0), and incubated at 37 °C for 15 h. The digested previously (20). After dissolution in 10 ␮l of 0.1% formic acid
peptides were extracted from the gel slice by addition of 200 ␮l and 5% acetonitrile (v/v), samples were injected onto a trapping
of extraction solution (50 mM ammonium bicarbonate) and column (3 cm, 75 ␮M, C18, Micro-Tech) previously equili-
incubated for 30 min. After washing with 100 ␮l of washing brated in 100% solvent A (0.1% formic acid, 5% acetonitrile in
buffer (0.1% formic acid), the peptides were recovered by addi- water) at a flow rate of 2 ␮l/min. Following 10 min of washing,
tion of elution solution (50:50 0.1% formic acid/methanol) with the trapping column was eluted through a pre-equilibrated ana-
vacuum. The final peptide solutions were lyophilized in a cen- lytical column (15 cm, 75 ␮M, C18, Micro-Tech) at a flow rate of
990 JOURNAL OF BIOLOGICAL CHEMISTRY
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Association of Rpe65 with Membranes
300 nl/min using the compound linear gradient of solvents A
Effect of pH on the Association of Rpe65 with Membranes—
and B (0.1% formic acid in acetonitrile) as follows: 3 min at 100% Ten-ml aliquots containing 2–3 mg/ml bovine RPE homoge-
A; 80% A, 20% B at 8 min; 65% A, 35% B at 13 min; 25% A, 75% nate (see above) were dissolved in 990 ml of PBS (pH 7.0, 8.0,
B at 23 min; and 90% A, 10% B at 23.1 min. The column eluent 10.0, and 11.0). The aliquots were homogenized by nitrogen
was directed to an uncoated, pulled silica nanospray tip (Picotip cavitation at 1000 p.s.i. and subjected to centrifugation at
FS360-20-10-N-5-C12, New Objective) at 2.4 kV for ionization 100,000 ϫ g. The pellets and the lyophilized powders of the
without nebulizer gas. The mass spectrometer was operated in supernatants were dissolved in 50 ml of denatured SDS-PAGE
“information-dependent acquisition” mode with a survey scan sample buffer. The samples were separated by a 10% SDS-
(350–1600 m/z), data-dependent MS/MS on the two most PAGE, and Rpe65 was visualized by GelCode Coomassie Blue
abundant ions with exclusion after two MS/MS experiments. stain and by immunoblotting.
The Cys peptide sequences were detected using BioAnalyst
program (Applied Biosystem) with help of the characteristic were heated for 10 min at 75 °C, separated by SDS-PAGE in a
doublet isotopic tag. 10% polyacrylamide gel, and transferred to an Immobilon-P
Immunoblot Analysis—Proteins in Laemmli sample buffer
Algorithm for Calculation of d3/d0 from the MS Data—The membrane (Millipore). The membrane was incubated in block-
relative isotopic peak abundances of Cys peptides were ing buffer (PBS (pH 7.4), 5% nonfat milk) for 2 h at 37 °C and
obtained via integrated peak areas of the isotopic mass peaks by then overnight at 4 °C with rabbit polyclonal antiserum against
Analyst QS (version 1.1, Applied Biosystems, Carlsbad, CA). the Rpe65 peptide, NFITKINPETLETIK (residues 150–164).
Starting from the first highest isotopic peak, their relative abun- After washing three times in PBS/Tween 20 for 10 min per
dances were assigned as I0, I1, I2, …, etc. According to the iden- wash, the membrane was incubated with horseradish peroxi-
tities of the peptides analyzed by tandem mass, their theoretical dase-conjugated goat anti-rabbit IgG (Jackson Immuno-
(native) isotopic abundance of peptide isotopic peaks was cal- Research) for 1 h and washed again. Rpe65 that reacted with
culated as I*0, I1*, I*2, …, etc., based on their molecular formulas. the antibodies was visualized with the enhanced ECL-Plus
The following were generated thereafter: d3/d0 ϭ x; I0 ϭ I*0, I1 ϭ Western blot detection system (Amersham Biosciences).
I*1, I2 ϭ I*2; I3 ϭ I3* ϩ xI*0, I4 ϭ I*4 ϩ xI1*, I5 ϭ I5* ϩ xI2*; I6 ϭ I*6 ϩ xI3*.
In Vitro Isomerase Assay in the Presence of Liposomes—For
I*6 Х 0, in most of the cases, then I6 ϭ xI*3 as shown in Equa- assays that used all-trans-ROL substrate, the isomerase assay
tion 1,
mixtures contained 20 mM HEPES (pH 7.5), 150 mM NaCl, 6%
bovine serum albumin, 10 ␮M all-trans-ROL, and 50–100 ␮g of
bovine RPE homogenate. For assays that used all-trans-RP sub-
strate, the isomerase assay mixtures contained 20 mM HEPES
(pH 7.5), 150 mM NaCl, 6% bovine serum albumin, 10 ␮M all-
trans-RP, and 50–100 ␮g of bovine RPE homogenate. The final
volume for each reaction was 400 ␮l. To make liposomes, 20 ␮l
of 10 mg/ml phospholipid in chloroform was dried under a
stream of nitrogen gas. HEPES buffer without substrate and
proteins in the isomerase assay mixture were added, and the
mixture was sonicated on ice under nitrogen for 20 min. For
all-trans-RP-containing liposomes, 10 ␮l of 400 mM all-
trans-RP in hexane was added to the phospholipids before dry-
d3/d0 ϭ x
*
*
*
I0 ϭ I0, I1 ϭ I1, I2 ϭ I2
*
*
*
*
*
*
I3 ϭ I3 ϩ xI0, I4 ϭ I4 ϩ xI1, I5 ϭ I5 ϩ xI2
*
*
I6 ϭ I6 ϩ xI3
*
I6 Х 0
I3 Ϯ I32 Ϫ 4I0I6
2I0
ͱ
d3/d0 ϭ x ϭ
(Eq. 1)
The d3/d0 ratios were calculated as the product of the equation ing. Incubations were started by adding the bovine serum albu-
involving I0, I3, and I6. The final d3/d0 ratios were normalized to min and bovine RPE homogenate. In some assays, all-trans-RP
the peptide containing Cys-231, which was shown in a previous substrate was dissolved in sodium cholate at 6 mM final concen-
study to be nonpalmitoylated (15).
tration in the assay mixture. After incubation for 1 h in the dark
Membrane Lipid Binding Assay—The membrane lipid strips at 37 °C, the reactions were quenched by adding 0.2% SDS and
were blocked by incubation in PBS ϩ 3% nonfat dry milk and 2 volumes of methanol. Retinoids were extracted with hexane
gently agitated for 1 h at 25 °C. After discarding the block solu- and analyzed by HPLC, as described below. Isomerase-specific
tion, the strips were incubated with samples of bovine RPE activities were calculated after subtracting endogenous 11-cis-
homogenates containing the indicated total protein concentra- ROL, determined by hexane extraction of representative reac-
tions (20, 40, and 80 mg/ml) in PBS ϩ 3% nonfat-dry milk for 1 h tion mixtures before addition of substrate. Bovine RPE homog-
at 25 °C. The protein solutions were removed, and the strips enates were bleached by UV light for 5 min to remove the
were washed with three changes of PBS/Tween 20 for 10 min endogenous retinoids.
per wash with gentle agitation. The strips were then incubated
Cloning of Chicken Rpe65—Chicken Rpe65 was cloned by
with antisera against Rpe65 (see below) diluted 1:2000 in block- primer amplification of the gene from chicken retina cDNA
ing solution for 1 h at 25 °C. The strips were washed three times based on the previously reported sequence (NCBI accession
for 10 min with PBS/Tween 20 and incubated with horseradish NM 204884). Briefly, RNA was prepared from freshly dissected
peroxidase-conjugated goat anti-rabbit IgG (Jackson Immuno- chicken retinas (Absolutely RNA, Stratagene) and used as a
Research) for 1 h at 25 °C. Rpe65 that reacted with the antibod- template for first-strand cDNA synthesis (Superscript III,
ies was visualized with the enhanced ECL-Plus Western blot Invitrogen). The complete Rpe65 coding region was amplified
detection system (Amersham Biosciences).
by PCR using primers 5Ј-GAGACAATGTACAGCCAGGTG
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Association of Rpe65 with Membranes
TABLE 1
Observed peptides in the proteolytic digestion of AA-d0/d3-treated bovine Rpe65
The cysteine residues are underlined. T, trypsin; G, endoproteinase Glu-C; AN, Asp-N.
Peptide
Mass d0/d3
Cys no.
Sequence
T223–234
1448.68/1451.70
3260.76/3263.78
2578.27/2581.29
1893.89/1896.91
4468.21/4471.23
1832.87/1835.89
3610.71/3616.75
3902.81/3908.85
3884.06/3887.08
5461.76/5464.78
2687.33/2690.35
2481.20/2484.22
2291.05/2294.07
2122.96/2125.98
3225.64/3228.66
1717.85/1720.87
1920.83/1923.85
2102.08/2105.10
2442.26/2445.28
231
SEIVVQFPCSDR
G22–51
45
LSSPLTAHVTGRIPLWLTGSLLRCGPGLFE
TIKQVDLCNYVSVNGATAHPHIE
LCNYVSVNGATAHPHIE
G162–184
G168–184
G185–224
G218–233
G321–348
G323–352
G365–399
G418–462
AN87–109
AN110–129
AN142–160
AN167–185
AN186–214
AN218–232
AN277–292
AN378–397
AN445–463
169
169
195
NDGTVYNIGNCFGKNFSIAYNIVKIPPLQADKEDPISKSE
DPISKSEIVVQFPCSD
231
329, 330
329, 330
396
HEFLIVDLCCWKGFEFVYNYSYLANLRE
FLIVDLCCWKGFEFVYNYSYLANLRENWEE
VRRYVLPLNIDKADTGKNLVTLPNTTATAILCSDE
FPQINYQKYGGKPYTYAYGLGLNHFVPDRLCKLNVKTKETWVWQE
DAYVRAMTEKRIVITEFGTCAFP
DPCKNIFSRFFSYFRGVEVT
448
106
112
146
DYYACTETNFITKVNPETL
169
DLCNYVSVNGATAHPHIEN
195
DGTVYNIGNCFGKNFSIAYNIVKIPPLQA
DPISKSEIVVQFPCS
231
278
DCFESNETMGVWLHIA
396
DTGKNLVTLPNTTATAILCS
448
DRLCKLNVKTKETWVWQEP
(forward) and 5Ј-GTGCTTGCAAATGGAGACTG (reverse) ing under a stream of nitrogen, samples were dissolved in 100 ␮l
under the following conditions: 25 cycles of 94 °C for 30 s; 60 °C of hexane, and retinoids were separated by chromatography on
for 30 s; and 72 °C for 60 s in a PTC-200 gradient thermocycler a silica column (Supelcosil LS-SI 5 ␮m, 4.6 ϫ 250-mm inner
(MJ Research). The amplified fragment was cloned into the diameter) by gradient elution (mobile phase 0.2–10% dioxane
Topo-TA cloning vector (Invitrogen). The chicken Rpe65 in hexane, 2.0 ml per min flow rate) in an Agilent 1100 liquid
cDNA insert was released by restriction digestion and sub- chromatography equipped with a UV photodiode-array detec-
cloned into the pcDNA3.1 mammalian expression vector tor. Identified peaks were confirmed by spectral analysis and
(Invitrogen). The full coding region was checked by DNA co-elution with authentic retinoid standards.
sequencing (DYEnamic ET Terminator Cycle Sequencing, ABI
Surface Electrostatic Potential Map—Electrostatic surface
3100 DNA Sequencer, GE Healthcare). HEK-293T cells were potential of bovine Rpe65 was calculated and viewed using
transfected with the expression plasmid for protein expression DeepView/Swiss-PDBViewer version 4.01 (21). The Protein
(Polyfect, Qiagen).
Data Bank file of bovine Rpe65 (code 3FSN) was obtained from
Preparation of Homogenates from Cells Expressing Chicken RCSB. The surface electrostatic potential computation was
Rpe65—HEK-293T cells expressing chicken Rpe65 were sus- performed using the Poisson-Boltzmann equation method
pended in 5 ml of 20 mM HEPES (pH 7.4), 150 mM NaCl with (22).
addition of CompleteTM EDTA-free proteinase inhibitor and
RESULTS
homogenized by nitrogen cavitation at 1000 p.s.i. The superna-
tant was collected after centrifugation at 3000 ϫ g and stored at
Ϫ80 °C.
Identification of 19 Peptides Containing the 12 Cys Residues
in Bovine Rpe65—Based on the amino acid sequence, we
In Vitro Isomerase Assay of Chicken Rpe65 Using All- defined a set of 19 proteolytic peptides in bovine Rpe65 that
trans-RP as Substrate in Phospholipid Liposomes—The isomer- collectively contain the 12 Cys residues (Table 1). To identify
ase assay mixtures contained 20 mM HEPES (pH 7.5), 150 mM these Cys-containing peptides, we incubated a sample of bovine
NaCl, 6% bovine serum albumin, 10 ␮M all-trans-RP, and RPE microsomes with dithiothreitol (DTT) under conditions
50–100 ␮g of chicken Rpe65 cell homogenate. The final vol- that reduce disulfides and deacylate modified Cys residues (23).
ume for each reaction was 500 ␮l. To make all-trans-RP-con- Next, we incubated the sample with a 1:1 mixture of triple deu-
taining liposomes, 20 ␮l of 10 mg/ml mixture of phospholipids terium-labeled acrylamide (AA-d3) and nondeuterated acryl-
in chloroform were mixed with 10 ␮l of 400 ␮M all-trans-RP in amide (AA-d0). Under the conditions used, acrylamide effi-
hexane and dried under a stream of nitrogen gas. HEPES buffer ciently S-alkylates free thiol groups (24). The sample was
in isomerase assay mixture (without proteins) was added and separated by SDS-PAGE, and gel slices containing Rpe65 were
sonicated on ice under nitrogen for 20 min in the dark. Incuba- excised. The purified Rpe65 was subjected to in-gel digestion
tions were started with addition of the chicken Rpe65 cell using trypsin, endoproteinase Glu-C, and/or Asp-N . Every Cys
homogenate. After incubation for 0.5 h in the dark at 37 °C, the residue in the resulting peptide mixtures should then be mod-
reactions were quenched by adding 0.2% SDS and 2 volumes of ified with AA-d3 and AA-d0 at a 1:1 ratio. We separated these
methanol. Retinoids were extracted with hexane and analyzed peptide mixtures by reverse-phase liquid chromatography (LC)
by HPLC, as described below. Isomerase-specific activities were and analyzed by mass spectrometry (MS). Fig. 3A shows the
calculated after subtracting endogenous 11-cis-ROL, deter- chromatogram for quadruple-charged ion of m/z ϭ 621.7059,
mined by hexane extraction of representative reaction mixtures corresponding to the Cys-112-containing, AA-d0-modified
before addition of substrate.
peptide, AN110–129 (Table 1). Fig. 3D shows the chromato-
HPLC Analysis of Retinoids—Retinoids were analyzed by gram for a double-charged m/z ϭ 860.2878 ion, corresponding
normal-phase HPLC as described previously. In brief, after dry- to the Cys-231-containing, AA-d0-modified peptide, AN218–
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FIGURE 3. ESI-MS/MS data showing similar abundance of AA-d0- and AA-d3-modified peptides containing residues Cys-112 and Cys-231.
A, selected ion monitoring chromatogram of an ion with m/z 621.7059. This ion corresponds to tetra-charged Asp-N peptide AN110–129 from bovine
Rpe65 after modification of Cys-112 with AA-d0. B, tandem mass spectrum of the 621.7059 m/z ion with the corresponding observed b- and y-ions
indicated. C, zoom spectrum of the indicated m/z values acquired at 35 min. Note the deviation from the ideal d0/d3 distribution shown on Fig. 4 due to
the overlap of native isotopic d0 ions on the d3 ion species. The similar abundance of the corresponding AA-d0 and AA-d3-labeled ions indicates that
Cys-112 is not palmitoylated. D, selected ion monitoring chromatogram of an ion with m/z 860.2878 corresponding to the double-charged Asp-N
peptide AN218–232 from bovine Rpe65 after modification of Cys-231 with AA-d0. E, tandem MS chromatogram of the m/z 860.2878 ion with the
corresponding b- and y-ions indicated. F, zoom spectrum of the indicated m/z values acquired at 24 min. The similar abundances of the corresponding
AA-d0- and AA-d3-labeled ions indicate that Cys-231 is not palmitoylated. Note that the b- and y-ions containing Cys residues show similar d0/d3 doublet
peaks, whereas ions lacking Cys show only singlet peaks.
232 (Table 1). Similar chromatograms were acquired for ions of observed masses were in good agreement with the predicted
m/z ϭ 622.4657 and 861.7873, corresponding to the AA-d3- masses for all 19 AA-d0- and AA-d3-modified peptides.
modified AN110–129 and AN218–232 peptides, respectively
To confirm identification of these peptides, we performed
(data not shown). Using the protease combinations indicated in MS/MS analysis following in-gel digestion of Rpe65 with the
Table 1, we performed LC/MS analysis of the remaining 17 indicated proteases. The resulting y- and b-ion series are shown
peptides (data not shown). After correcting for charge, the in Fig. 3B for peptide AN110–129 and Fig. 3E for AN218–232.
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dues. Together, these results vali-
date our identification of the 19
Cys-containing Rpe65 peptides.
Rpe65 Is Not S-Palmitoylated—
We tested for palmitoylation of the
12 Cys residues in bovine Rpe65 by
the strategy shown in Fig. 4. This
strategy employs two disulfide-re-
ducing agents. As discussed, DTT
reduces disulfides and deacylates
Cys residues (23). TBP reduces di-
sulfides but does not react with ac-
ylated Cys residues (25). To confirm
these activities of DTT and TBP on
palmitoylated Cys residues, we syn-
thesized S-palm-N-Ac-Cys-Me as a
model reagent. We incubated ali-
quots of S-Palm-N-Ac-Cys-Me with
DTT, TBP, or without any reducing
agent under the same conditions as
those used with Rpe65. Next, we
incubated the samples with a 1:1
mixture of AA-d0/d3, and subjected
them to LC/MS analysis. Virtually
no m/z ϭ 416 ion corresponding to
S-palm-N-Ac-Cys-Me was detect-
able in the sample treated with DTT
FIGURE 4. Strategy to determine palmitoylation status of Cys residues in Rpe65. A, flow chart for the
preparation of Rpe65 peptide samples for LC-MS analysis. One aliquot of bovine RPE membranes is 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 with nondeuterated acrylamide (AA-d0). A second
aliquot is incubated with TBP to reduce disulfide without de-palmitoylating Cys residues. Next, the nonpalmit-
oylated sulfhydryls are alkylated by incubating with triple-deuterated acrylamide (AA-d3). These two aliquots
are pooled and the constituent proteins separated by SDS-PAGE. The Rpe65 band is excised and digested with
trypsin, endopeptidase Glu-C, and/or Asp-N, and the resulting peptides are analyzed by LC-MS/MS. B, diagram
showing predicted spectral patterns for the different potential palmitoylation states of a Cys residue. If a
Cys-containing peptide is not palmitoylated, it would incorporate equal amounts of AA-d0 and AA-d3. There-
fore, the 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 (Fig. 5A). Instead, this sample con-
less abundant than the nondeuterated ions. If the Cys residue is fully palmitoylated, no triple-deuterated ions
tained m/z ϭ 249 and 252 ions
would be present in the spectrum.
of approximately equal intensities,
corresponding to S-AA-d0-N-Ac-
Cys-Me and S-AA-d3-N-Ac-Cys-
Me. In contrast, samples incubated
with TBP or no reducing agent con-
tained only the m/z ϭ 416 ion
(S-palm-N-Ac-Cys-Me) with virtu-
ally no detectable m/z ϭ 249 or 252
ions (Fig. 5A). These data suggest
that incubation with DTT should
fully de-palmitoylate Rpe65 were it
so modified, whereas incubation
with TBP should have no effect on
the palmitoylation state of Rpe65.
FIGURE 5. Analysis of Rpe65 S-palmitoylation. A, effects of DTT and TBP on Cys palmitoylation. S-Palm-N-Ac-
Cys-MewastreatedwithDTT, TBP, orwithoutareducingagent(control)followedbyreactionwitha1:1mixture
of AA-d0/d3. This histogram shows the ion intensities of S-palm-N-Ac-Cys-Me (m/z ϭ 416), S-AA-d0-N-Ac-
Cys-Me (m/z ϭ 249), and S-AA-d3-N-Ac-Cys-Me (m/z ϭ 252) in the different samples. Note the virtual absence
of palmitoylated-Cys in the sample incubated with DTT and the presence of only palmitoylated Cys in the
samples incubated with TBP or no reducing agent (control). B, AA-d3/d0 ratio for each Cys residue in bovine
Rpe65. This ratio is equivalent to the fraction of nonpalmitoylated Cys in each peptide. The values were nor-
malized to the peptide containing Cys-231, which is known to be nonpalmitoylated. Except for Cys-396, the
We prepared bovine RPE micro-
somes and split them into 2 equal
aliquots. One aliquot was incubated
with DTT, to reduce and fully de-
palmitoylate Rpe65, and then with
other11CysresiduesshowAA-d3/d0 valuescloseto1, indicatingnonpalmitoylationoftheseresiduesinbovine AA-d0, to alkylate all the Cys resi-
Rpe65. Error bars show standard deviations (n ϭ 3).
dues in Rpe65. The other aliquot
was incubated with TBP, to reduce
These data yielded the partial amino acid sequences, identifying disulfides without altering any palmitoylated Cys residues, and
both peptides. We performed similar MS/MS analysis on the then with AA-d3, to alkylate the free but not palmitoylated Cys
remaining Rpe65 peptides. For each of the 19 peptides, the ob- residues in Rpe65. The 2 aliquots were pooled and separated by
served and predicted sequences were in full agreement. The ion SDS-PAGE, and gel slices containing Rpe65 were excised. The
peaks from Cys-containing peptide fragments were doublets purified Rpe65 was subjected to in-gel digestion using trypsin,
versus singlets from non-Cys-containing fragments (Fig. 3, B endoproteinase Glu-C, and/or Asp-N (Table 1) for MS analysis.
and E) due to AA-d0 plus AA-d3 modifications of the Cys resi- The zoomed in spectrum for peptide AN110–129 is shown in
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FIGURE 6. ESI-MS deconvolution analysis to determine the molecular mass of intact Rpe65. After incubation with DTT or TBP, undigested Rpe65 was
separated by reverse-phase HPLC and analyzed by ESI-MS. A and B show the deconvoluted molecular mass spectra for Rpe65 treated with DTT. C and D show
the deconvoluted spectra for Rpe65 treated with TBP. A and C show the full mass range, and B and D show a zoomed in mass range. Note the absence of an ion
peak at 61,143 Da, which would indicate palmitoylation of Rpe65.
Fig. 3C. Here, the 621.4511, 621.7059, and 621.9554 m/z These m/z values are similar to the calculated molecular mass
peaks correspond to the quadruple-charged AA-d0, AA-d0ϩ1, of unmodified bovine Rpe65 (60,905). The mass accuracy of this
and AA-d0ϩ2 isotopic ions, respectively. The 622.2069 peak method is 100 ppm or ϳ6 Da. By comparison, the mass of a
contains the AA-d0ϩ3 and AA-d3 ions. The 622.4657 peak con- single palmitoyl group is 238. These observations establish that
tains the AA-d0ϩ4 and AA-d3ϩ1 ions; the 622.7065 peak con- the maximum possible stoichiometry for palmitoylation of
tains the AA-d0ϩ5 and AA-d3ϩ2 ions, and the 622.9534 peak Rpe65 is 20%. A similar argument can be applied for phosphor-
contains the AA-d0ϩ6 and AA-d3ϩ3 ions. Fig. 3E shows the ylation and most other post-translational modifications. Low
spectrum for peptide AN218–232. The 860.2878, 860.7849, mass modifications of Rpe65 cannot be ruled out by these data.
and 861.2850 m/z peaks correspond to double-charged AA-d0,
Rpe65 Binds to Phospholipids with Negatively Charged
AA-d0ϩ1, and AA-d0ϩ2 isotopic ions, respectively. The Headgroups—An alternative explanation for the association of
861.7873 peak contains the AA-d0ϩ3 and AA-d3 ions. The Rpe65 with membranes is a direct interaction between the pro-
862.2876 peak contains the AA-d0ϩ4 and AA-d3ϩ1 ions, tein and phospholipid headgroups. To test this possibility, we
the 862.7862 peak contains the AA-d0ϩ5 and AA-d3ϩ2 ions, incubated RPE homogenates with membrane strips containing
and the 863.2789 peak contains the AA-d0ϩ6 and AA-d3ϩ3 immobilized lipids and the major phospholipids. After washing,
ions. We performed similar analysis of the remaining 17 pep- the strips were incubated with antisera against bovine Rpe65
tides (data not shown). The ratio of AA-d3 to AA-d0, which is and developed as immunoblots. We observed Rpe65 immuno-
equivalent to the fraction of unmodified Cys, was calculated reactivity associated with spots containing three acidic phos-
from these MS data for each peptide using the algorithm pholipids, PS, PG, and cardiolipin (CL) (Fig. 7A). We observed
described under “Experimental Procedures.” These ratios are no binding of Rpe65 to phosphatidic acid, nor to the positively
plotted in Fig. 5B. The only residue in bovine Rpe65 that charged phospholipids, phosphatidylethanolamine (PE) and
showed greater than 5% modification was Cys-396. However, PC, nor to any neutral lipids (Fig. 7A).
Cys-396 is not conserved within mammals (Fig. 2), suggesting
Association of Rpe65 with Membranes Is pH-dependent—We
that modification of this residue is not required for the associ- incubated bovine RPE microsomes in buffers of pH 7–11. Next,
ation of Rpe65 with membranes. It is notable that Cys-231, we pelleted the microsomes and performed immunoblot anal-
Cys-329, and Cys-330, which were suggested to be palmitoy- ysis of Rpe65 in the pellet and supernatant fractions. Rpe65
lated in an earlier study (14), and Cys-112, which was suggested remained tightly associated with membranes up to pH 10 (Fig.
to be palmitoylated more recently (16), show no evidence of 8, A and B). At pH 11, we observed significant solubilization of
palmitoylation in this study.
Rpe65. These results are consistent with an electrostatic inter-
Rpe65 Does Not Undergo Post-translational Modifications at action between a basic region on the surface of Rpe65 and acidic
Significant Stoichiometry—To test further for Rpe65 post- headgroups on the membrane surface.
translational modifications, we performed ESI-MS analysis on
Nonsubstrate-containing PS, PG, and CL Liposomes Inhibit
the intact (nonproteolyzed) protein purified from bovine RPE Rpe65 Isomerase Activity—Addition of all-trans-ROL to an RPE
microsomes after reduction with TBP or DTT. For both sam- homogenate results in synthesis of all-trans-REs by lecithin:
ples, greater than 80% of total Rpe65 immunoreactivity eluted retinol acyltransferase in ER membranes (15). These mem-
from the PLRP-S polymeric column in a single peak (data not brane-bound all-trans-REs serve as substrate for Rpe65 isomer-
shown). The major ion in this peak fraction was 60,921 m/z for ase. To confirm binding of Rpe65 to PS, PG, and CL, we pre-
the TBP and 60,915 m/z for the DTT sample (Fig. 6, A–D). pared liposomes from these and other phospholipids, added the
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liposomes to Rpe65 assay mixtures containing bovine RPE and restricting its access to the substrate-containing ER
homogenates and all-trans-ROL, and assayed for synthesis of membranes.
11-cis-ROL. Compared with control assay mixtures with no
PS, PG, and CL Liposomes Containing All-trans-RP Serve as a
added liposomes, we observed significant inhibition of 11-cis- Source of Substrate for Rpe65—To test the functional signifi-
ROL synthesis in assays containing PS, PG, and CL but not PC cance of Rpe65 binding to phospholipids, we prepared lipo-
or PE liposomes (Fig. 9A). Here, the PS, PG, and CL-containing somes from PC, PE, PS, PG, and CL that also contain all-trans-
liposomes are inhibiting isomerase activity by binding Rpe65 RP. These were added to bovine RPE homogenates stripped of
endogenous retinoids by exposure to UV light. We assayed for
synthesis of 11-cis-ROL. As a positive control for substrate
delivered in liposomes, we used all-trans-RP solubilized in
sodium cholate. We observed virtually no isomerase activity
when all-trans-RP was delivered in liposomes containing PC or
PE (Fig. 9B). However, delivery of all-trans-RP in PS, PG, or CL
liposomes resulted in significant 11-cis-ROL synthesis (Fig. 9B).
These results suggest that the interaction of Rpe65 with acidic
phospholipid headgroups is functionally relevant, providing
access to its all-trans-RP substrate within the bilayer.
Negatively Charged Phospholipids Are Required for Mem-
brane Association and Isomerase Activity of Rpe65—We inves-
tigated the effect on isomerase activity of increasing PG or PS
concentrations in liposomes that otherwise contain the same
molar ratios of phospholipids as human RPE membranes (PC/
PE/PI ϭ 2:6:1) (26). We prepared liposomes containing PC, PE,
and PI at these molar ratios plus 0, 10, or 20% PG or PS. The
liposomes also contained 10 ␮M all-trans-RP. We incubated
these liposomes with homogenates of HEK-293T cells express-
ing chicken Rpe65 and measured 11-cis-ROL synthesis. Inter-
estingly, we observed a 10.5-fold increase in isomerase activity
when the HEK-293T cell homogenate was incubated with
PC/PE/PI liposomes that also contained 10% PG versus lipo-
somes with no added PG (Fig. 9C). No further change in
isomerase activity was seen when the PG content was increased
from 10 to 20% (Fig. 9C). We observed a similar pattern with PS.
Addition of 10% PS to PC/PE/PI liposomes increased isomerase
activity by 8.5-fold (Fig. 9C). No further increase in 11-cis-ROL
synthesis was seen when the PS content of the substrate lipo-
FIGURE 7. Binding of Rpe65 to negatively charged phospholipids.
A, membranes containing the indicated immobilized lipids were incubated
with 80, 40, or 20 ␮g of bovine RPE homogenates. After washing, bound
Rpe65 was detected by immunoblotting. Note the Rpe65 immunoreactivity
somes was increased from 10 to 20%. These data show that
Rpe65 isomerase activity is strongly stimulated by addition of a
relatively small fraction of the acidic phospholipids, PG or PS,
on the spots containing PS, PG, and CL. B, molecular structures of PS, PG, and
to liposomes containing the same major phospholipids as RPE
CL. Note that CL contains two net negative charges, versus one negative
membranes.
charge for PS and PG.
DISCUSSION
Lecithin:retinol acyltransferase,
Rpe65, and 11-cis-RDH act sequen-
tially in RPE cells to convert all-
trans-ROL into 11-cis-RAL (Fig. 1).
These enzymes are located in the
ER of RPE cells. Lecithin:retinol
acyltransferase and 11-cis-RDH are
integral membrane proteins (27,
28). The mechanism whereby Rpe65
associates with membranes is the
subject of this study. We per-
formed MS analysis on Rpe65 from
FIGURE 8. Rpe65 dissociates from microsomal membranes at high pH. Bovine RPE microsomes were incu-
bated in buffers at the indicated pH values and then disrupted by nitrogen cavitation. After centrifugation, the
resulting high speed pellets and supernatants were separated by SDS-PAGE. A, Coomassie-stained gel of the
pellet and supernatant fractions. B, Rpe65 immunoblot of the same fractions. Note the release of Rpe65 from
bovine RPE microsomes and found
that it is not significantly palmitoy-
membranes into the supernatant between pH 10 and 11.
lated on any Cys residue. We ob-
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thermore, Cys-231, Cys-329, and
Cys-330 were found by MS analysis
not to be palmitoylated. The affinity
of Rpe65 for membranes was shown
to be similar in wild-type and
؊/؊
lrat
knock-out mice that lack
the proposed palmitoyltransferase
(15). Finally, no change in the cata-
lytic activity of Rpe65 or its affinity
for membranes was seen after treat-
ment of expressing cells with the
global inhibitor of protein palmitoy-
lation, 2-bromopalmitate (15). The
results presented here provide fur-
ther evidence against the palmitoy-
lation-switch hypothesis.
FIGURE9. InhibitionofRpe65isomeraseactivityanddeliveryofall-trans-RPsubstratetoRpe65byacidic
liposomes. A, synthesis of 11-cis-ROL (in milli-absorbance units at 318 nm) from all-trans-ROL by bovine RPE
homogenates containing either no liposomes (positive control) or liposomes constituted from the indicated
phospholipids. Note the partial inhibition of 11-cis-ROL synthesis by liposomes containing PS and PG, com-
plete inhibition by liposomes containing CL, and no inhibition by liposomes containing PC and PE. B, synthesis
of 11-cis-ROL from all-trans-RP by bovine RPE homogenates that had been stripped of endogenous retinoids
byexposure toUV light. Theall-trans-RPsubstrate wasdelivered in sodium cholate detergent(positive control)
or in liposomes constituted from the indicated phospholipids. Note the synthesis of 11-cis-ROL when all-
trans-RP was delivered in PS, PG, or CL liposomes, but not liposomes containing PC or PE. C, isomerase activities
of expressed chicken Rpe65 with liposomes of different phospholipid composition that also contain all-
trans-RP substrate. Liposomes in the experiments labeled 0% contained PC, PE, and PI at the same molar ratios
as human RPE membranes with no added PG or PS. Liposomes in the experiments labeled 10% contained the
same phospholipids plus 10% PG or PS. Liposomes in the experiments labeled 20% contained the same phos-
pholipids plus 20% PG or PS. Note the dramatic activation of isomerase activity (11-cis-ROL synthesis) with
addition of PG or PS. Error bars shown show standard deviations (n ϭ 3).
More recently, it was suggested
that Cys-112 is palmitoylated in
Rpe65, based on MS/MS data and
the observation that C112A-substi-
served minor modification of Cys-396 by our MS assay. How- tuted Rpe65 was not present in the membrane fraction of
ever, because Cys-396 is not conserved (Fig. 2), palmitoy- expressing cells, whereas the unsubstituted protein was present
lation of this residue is unlikely to explain the affinity of Rpe65 (16). These data are in disagreement with the results presented
for membranes. Furthermore, the extent of Cys-396 modifica- here (Figs. 5 and 6). How can this discrepancy be resolved? The
tion (ϳ16%) is insufficient to account for the ϳ85% association MS/MS spectra of the Cys-112-containing peptide presented
of Rpe65 with membranes (15). The starting material for our by Takahashi et al. (16) had very high backgrounds. In such
MS analysis was RPE microsomes. Accordingly, we analyzed noisy spectra, a series of ions can be found that appear to sup-
the membrane-associated form of Rpe65. If palmitoylation is port a nonexistent post-translational modification, especially
required for Rpe65 to associate with membranes, we should when the peptides are large (Ͼ2 kDa). To make their data fit the
have observed this modification at a stoichiometry approaching proposed palmitoylation of Cys-112, Takahashi et al. (16) had
100%. As a further test for palmitoylation or other post-trans- to assume that within the same 21-residue peptide Lys-113 was
lational modifications, we determined the mass of undigested acetylated and that Thr-101, Thr-105, and Ser-117 were phos-
Rpe65 by ESI-MS following reduction with DTT or TBP. The phorylated and to accept cross-correlation (Xcorr) scores
resulting masses were 60,915 and 60,921 Da, respectively, and below 2.0 (16). Furthermore, in experiments where either Cys-
the calculated mass of unmodified bovine Rpe65 is 60,905 Da. 106 or Cys-112 was substituted with Ala, Takahashi et al. (16)
The error in these determinations is ϳ6 Da. Given the close then had to assume that Lys-113 was no longer acetylated and
agreement between the measured mass of Rpe65 from bovine that Thr-105 and Ser-117 or Thr-101 and Ser-117 were phos-
eyes and the calculated mass of unmodified Rpe65, this protein phorylated, respectively. No independent evidence for acetyla-
is unlikely to undergo palmitoylation, which would increase its tion or phosphorylation of Rpe65 was ever presented. Finally,
mass by 238 Da per modification, at a net stoichiometry of C112A-substituted Rpe65 was not present in the soluble frac-
greater than 20%. Fatty acylation does not significantly alter tion of expressing HEK-293T cells, as would be expected if
ionization efficiency under the conditions used. Other post- palmitoylation of Cys-112 was responsible for the association of
translational modifications, such as phosphorylation, which Rpe65 with membranes. Instead, C112A Rpe65 was present in
would increase the mass by 80 Da per modified residue, are also the insoluble cytoskeletal pellet (16), suggesting that the substi-
unlikely to occur at greater than 20% stoichiometry. Low mass tuted protein was misfolded. These results call into question
modifications of non-Cys residues in Rpe65, such as N-methyl- the conclusion of Takahashi et al. (16) that Cys-112 is palmit-
ation or N-acetylation, may occur at high stoichiometry. How- oylated in Rpe65.
ever, none of these possible modifications could explain the
association of Rpe65 with membranes.
The crystal structure of bovine Rpe65 was recently published
(8). Kiser et al. (8) found evidence against palmitoylation of
In a previous study, a palmitoylation-switch mechanism Cys-231, Cys-329, or Cys-330 within the crystal structure.
was proposed whereby Rpe65 undergoes reversible palmi- Unfortunately, Cys-112 was present within a disordered region;
toylation by lecithin:retinol acyltransferase on residues Cys- hence, their crystallographic data provided no information
231, Cys-329, and Cys-330 (14). Palmitoylation of Rpe65 was about the palmitoylation state of Cys-112. Based on a low res-
suggested as a mechanism to regulate isomerase catalytic activ- olution MS analysis, Kiser et al. (8) suggested that Cys-112 is
ity. The data supporting this hypothesis were challenged in sub- palmitoylated. However, the data supporting this conclusion
sequent studies that showed no change in the catalytic activity are problematic. First, if the Cys-112-containing peptide were
of Rpe65, or its affinity for membranes, when Cys-231, Cys-329, palmitoylated, it should have eluted much later from the C18
and Cys-330 were substituted with Ala or Ser (15, 29). Fur- column than the observed 14 min (8). Furthermore, ESI-MS
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analysis of a Cys-palmitoylated peptide should include an ion
resulting from neutral loss of palmitoyl group (238 Da). No such
neutral loss ions were visible in the published MS spectra of
Kiser et al. (8). Finally, the MS/MS spectrum of the purported
Cys-112-containing peptide only showed clear b7 ions. These
ions, due to loss of Leu/Ile, are present in many peptides of a
given m/z following digestion by pepsin; therefore, they provide
little useful sequence information. The other b-and y-series
ions, which would have identified this peptide, were either weak
or noninformative. Accordingly, it appears that the peptide
analyzed by Kiser et al. (8) was misidentified. In this study, the
MS/MS spectrum for the Cys-112-containing peptide showed a
clear y-ion series that permitted its unambiguous identification
(Fig. 3B).
In a previous study, intact Rpe65 was purified from mem-
brane and soluble fractions of expressing Sf9 cells and analyzed
by matrix-assisted laser desorption ionization-MS (30). The
molecular mass of Rpe65 from the membrane fractions was
0.7–1.0 kDa higher than the mass of Rpe65 from the soluble
fractions (30). These results are also in disagreement with the
results presented here. The mass spectra of intact Rpe65 in the
previous study had peak widths of ϳ10 kDa (30), indicating very
low mass resolution. At such low resolution, a mass difference
of 1 kDa in a 61-kDa protein cannot be reliably determined. The
MS peak width of intact Rpe65 in this study was ϳ50 Da (Fig. 6).
Thus, the mass resolution here was ϳ200-fold higher than in
the previous study. Rpe65 was prepared from microsomal
membranes in this study, which increased the likelihood of
detecting any palmitoylated forms. However, the measured
mass of Rpe65 after TBP reduction (60,921 Da) was within 16
Da of the predicted mass for unmodified Rpe65 (60,905 Da),
further suggesting that Rpe65 is not significantly palmitoylated.
The affinity of Rpe65 for membranes appears instead to be
mediated by a direct interaction between the protein and the
negatively charged phospholipids PS, PG, and CL. We demon-
strated this interaction by showing direct binding of Rpe65 to
FIGURE 10. A, electrostatic surface potential of bovine Rpe65. Regions with
negative electrostatic potential (acidic) are shown in red, neutral regions are
shown in white, and regions with positive electrostatic potential (basic) are
showninblue. Theleftandrightimagesarefromoppositesidesoftheprotein.
Note the strongly basic (blue) regions, corresponding to residues 294–298,
354–359, and other basic residues scattered throughout the sequence.
B, hypothesized interactions of visual cycle proteins on the ER membrane of
anRPEcell. Lecithin:retinolacyltransferase(LRAT)and11-cis-RDHareintegral-
membrane proteins, whereas both Rpe65 and CRALBP (40) are soluble pro-
teinswithaffinityforacidicphospholipidheadgroups. Followingsynthesisby
lecithin:retinol acyltransferase, Rpe65 extracts all-trans-RP from the mem-
brane and isomerizes it to 11-cis-ROL. 11-cis-RDH oxidizes it to 11-cis-RAL,
which binds to CRALBP and is transferred to the apical plasma membrane of
the RPE cell for export to the photoreceptors.
immobilized phospholipids (Fig. 7A). We also showed that no further stimulation with addition of 20% PG or PS (Fig. 9C).
PS-, PG-, or CL-containing, but not PC-or PE-containing, lipo- Consistently, native RPE membranes contain less than 10%
somes added to RPE homogenates inhibited synthesis of 11-cis- acidic phospholipids (26). These results suggest that the stim-
ROL by Rpe65 from all-trans-REs synthesized by lecithin:reti- ulation effect of acidic phospholipids on isomerase activity and
nol acyltransferase in the endogenous ER membranes (Fig. 9A). Rpe65 binding to membranes occurs with normal RPE phos-
Finally, we showed that Rpe65 can utilize all-trans-RP con- pholipid composition.
tained within PS, PG, or CL liposomes, but not PC or PE lipo-
Rpe65 is located in ER membranes (31). Although PS and PG
somes, as substrate for the synthesis of 11-cis-ROL (Fig. 9B). are minor phospholipids of the ER, CL is thought to be present
The latter observation establishes the physiological relevance of exclusively in mitochondria (32–37). Thus, the observed in
the interaction between Rpe65 and acidic phospholipids.
vitro affinity of Rpe65 for CL liposomes (Fig. 7A) may be due to
We investigated the effect of adding PG or PS to liposomes the net double-negative charge of CL versus the single-negative
that otherwise contain the same phospholipid composition as charges of PS and PG (Fig. 7B), and therefore may not reflect the
human RPE membranes (PC/PE/PI ϭ 2:6:1) (26) plus all- in vivo situation. The observed affinity of Rpe65 for PS and PG
trans-RP substrate. Here, we used chicken Rpe65 expressed in probably does reflect the in vivo situation, especially consider-
HEK-293T cells as an enzyme source. When the liposomes ing the results presented in Fig. 9C. These interactions are likely
were constituted with the addition of 10% PG, synthesis of to involve positively charged residues on the surface of Rpe65.
11-cis-ROL increased 10-fold compared with PC/PE/PI lipo- We generated an electrostatic surface-potential map for Rpe65
somes without PG (Fig. 9C). Addition of 10% PS to the same based on its structure (8). Most of the protein surface is pre-
RPE/membrane phospholipid mixture yielded an 8.5-fold dicted to be neutral or negatively charged (Fig. 10A). However,
increase in 11-cis-ROL synthesis (Fig. 9C). This stimulation of two strongly basic segments, KKRKK (residues 294–298) and
isomerase activity appeared to saturate at or below 10% PG or KKNARK (residues 354–359) (Fig. 2) are also present on the
PS in the substrate-containing liposomes, because we observed surface and may represent interacting structural elements. The
998 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 2•JANUARY 8, 2010

Association of Rpe65 with Membranes
Science 308, 267–269
predominant basic residue in these segments is Lys, which has a
pKa of 10.5. Interestingly, Rpe65 dissociated from RPE mem-
branes between pH 10 and 11 (Fig. 8), suggesting titration of
these Lys residues. Rpe65 can also be dissociated from mem-
branes by incubating in a buffer of high ionic strength (38). If
the affinity of Rpe65 for membranes was due to palmitoylation,
its association with membranes would be stronger, not weaker,
at high ionic strength. These observations provide further evi-
dence for an electrostatic interaction between membranes and
Rpe65. We observed no binding of Rpe65 to phosphatidic acid
(Fig. 7A), suggesting that the affinity of Rpe65 for acidic phos-
pholipids is not exclusively electrostatic but may involve hydro-
gen bonding as well.
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of a predicted hydrophobic channel (8). The Fe2ϩ-His4 coordi-
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JANUARY 8, 2010•VOLUME 285•NUMBER 2
JOURNAL OF BIOLOGICAL CHEMISTRY 999

Enzyme Catalysis and Regulation:
Rpe65 Isomerase Associates with
Membranes through an Electrostatic
Interaction with Acidic Phospholipid
Headgroups
Quan Yuan, Joanna J. Kaylor, Anh Miu, Sara
Bassilian, Julian P. Whitelegge and Gabriel H.
Travis
J. Biol. Chem. 2010, 285:988-999.
doi: 10.1074/jbc.M109.025643 originally published online November 5, 2009
Access the most updated version of this article at doi: 10.1074/jbc.M109.025643
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