(Received for publication, June 27, 1995; and in revised form, August 2, 1995 )
From the
Choroideremia, an X-linked form of retinal degeneration, results from defects in the Rab escort protein-1 (REP-1) gene. REP-1 and REP-2 assist in the attachment of geranylgeranyl groups to Rab GTPases, a modification essential for their action as molecular switches regulating intracellular vesicular transport. If Rabs that depend preferentially on REP-1 for prenylation exist, they will accumulate unprenylated in choroideremia cells. Using recombinant Rab geranylgeranyl transferase and REPs to label unprenylated cytosolic proteins, we identified one unprenylated protein in choroideremia lymphoblasts that was prenylated in vitro more efficiently by REP-1 than by REP-2. This protein was purified and identified as Ram (renamed Rab27), a previously cloned Rab of unknown function. Immunohistochemistry of rat retina showed that Ram/Rab27 is expressed in the pigment epithelium and choriocapillaris, the two retinal cell layers that degenerate earliest in choroideremia. These results raise the possibility that the retinal degeneration in choroideremia results from the deficient geranylgeranylation of Ram/Rab27 or a closely related protein.
Choroideremia (CHM) ()is a form of X-linked retinal
degeneration that can be classified under the broad group of hereditary
peripheral retinal dystrophies or retinitis pigmentosa(1) .
Affected hemizygous males develop slowly progressive retinal
degeneration leading to night blindness in their teens, loss of
peripheral vision, and complete blindness by middle age. Carrier
females generally retain retinal function but exhibit characteristic
focal areas of degeneration and pigmentation upon fundoscopic
examination, which likely represent clonal areas of disease due to
random X inactivation. Pathologic examination of choroideremic eyes
show profound atrophy of three adjacent retinal layers, the
photoreceptors, the retinal pigment epithelium (RPE), and the
choriocapillaris. Studies of carriers and early diseased eyes suggest
that the RPE and/or the choriocapillaris are the primary site of
degeneration, with secondary involvement of photoreceptors(1) .
The gene responsible for CHM was localized to chromosomal band Xq21 and
isolated by positional cloning techniques(2, 3) . An
insight into the function of the CHM protein came with the finding that
it encodes a subunit of Rab geranylgeranyl transferase, an enzyme that
modifies Rab proteins with the covalent attachment of a lipid
moiety(4, 5) .
Rab proteins are low molecular weight GTPases of the Ras superfamily involved in the regulation of intracellular vesicular traffic in exocytic and endocytic pathways(6, 7, 8, 9) . More than 30 different Rabs have been identified, each with a characteristic intracellular distribution suggesting that each Rab functions in a distinct transport event, e.g. Rab1 functions in endoplasmic reticulum to Golgi transport while Rab5 functions in plasma membrane to endosome transport. A current model for the function of Rab proteins in vesicular transport is based on studies in yeast and mammalian cells. It predicts that Rabs cycle between a cytosolic, inactive, GDP-bound state and a membrane-bound, active, GTP-bound state. The cytosolic form is recruited to the cytoplasmic leaflet of a donor organelle membrane and activated by exchange of the bound GDP for GTP. Once on a transport vesicle, Rabs regulate docking and fusion events that lead to the release of lumenal contents from the transport vesicle into the lumen of the appropriate acceptor organelle. After fusion has occurred, GTP is hydrolyzed to GDP and Rabs are resolubilized by Rab GDP dissociation inhibitor (Rab GDI), which delivers Rabs back to the donor organelle membrane, completing the cycle.
In order for Rabs to interact with
intracellular membranes, they must bear C20 geranylgeranyl (GG) groups
attached to cysteines at or near the carboxyl terminus of the proteins.
Rab proteins that cannot undergo GG modification remain cytosolic and
are inactive(10, 11, 12, 13) .
Geranylgeranylation of two carboxyl-terminal cysteines in Rab proteins
via thioether bonds is a complex reaction that requires two components (4, 14, 15, 16) . Rab GG transferase
(Rab GGTase, previously designated Component B) or GGTase-II is an
/
heterodimer with structural homology to another prenyl
transferase, Ras farnesyl transferase(17) . However, unlike
farnesyl transferase, Rab GGTase is inactive by itself and requires an
accessory protein, designated Rab escort protein (REP, previously
designated Component A) that binds to unprenylated Rab, presents it to
Rab GGTase, and thereby facilitates GG transfer. After
geranylgeranylation, REP remains bound to Rab in a stable complex that
can be released in vitro by detergents(18) . In
vivo, REP delivers geranylgeranylated Rab to its target donor
membrane(19) . The structural and functional homology between
REP and RabGDI suggests that both use similar mechanisms to deliver
Rabs to membranes. It was hypothesized that whereas REP delivers newly
synthesized Rabs to target membranes, RabGDI acts in the recycling of
Rabs(19) .
Peptide sequences derived from purified rat REP-1 and later cDNA cloning revealed that it is the counterpart of the human protein encoded by the CHM gene(4, 18) . A partial deficiency of Rab GGTase activity was demonstrated in extracts of CHM-derived lymphoblasts(5) . The loss of Rab GGTase activity could be restored by addition of purified REP-1, but not Rab GGTase(5) , consistent with a defective REP-1 gene in CHM. In yeast, both Rab GGTase (encoded by the BET2 and BET4 genes) and REP (encoded by the MRS6/MSI4 gene) activities are essential for cell viability(20, 21, 22, 23) , and a complete deficiency of REP activity would likely be lethal in humans. The fact that CHM patients have no detectable defect other than the retinal lesion and that CHM cells retain partial Rab GGTase activity suggests that the deficiency of REP-1 is compensated in most tissues of CHM patients. The identification of REP-2 (or CHM-like protein)(24) , a protein that is 75% identical to REP-1 (or CHM protein) raised the possibility that REP-2 could compensate for the loss of REP-1 function in CHM. Indeed, recombinant REP-2 can assist in the geranylgeranylation in vitro of different Rabs(25) .
In search of the molecular mechanisms underlying retinal degeneration in CHM, we hypothesized that the reason why REP-2 is only partially effective in compensating for the loss of REP-1 in CHM retinal cells might be due to the existence of one or more Rabs that depend preferentially on REP-1 for prenylation. In this study, we demonstrate that one Rab protein selectively accumulates unprenylated in the cytosol of CHM lymphoblasts. This protein was purified, sequenced, and identified as the human homolog of a previously identified rat Rab protein, designated Ram(26) , which we rename Rab27. Ram/Rab27 is predominantly membrane bound in normal lymphoblasts, whereas it is predominantly cytosolic in CHM lymphoblasts, suggesting that its function is compromised. Furthermore, rat Ram/Rab27 is expressed in normal retinas, specifically in the two cell layers that characteristically degenerate in CHM, the RPE and the choriocapillaris. These results raise the possibility that the retinal degeneration in CHM is due to the defective function of Ram/Rab27 (or a closely related protein) in the RPE and choriocapillaris.
Seven of the nine cell lines used in this study
were described previously(5) . The cell lines are designated as
follows: control cell lines, control-1 (culture designation L389, male,
age 38), control-2 (culture L430, male, age 26), control-3 (culture
L423, male, age 30), control-4 (culture L429, female, age 24), and
control-5 (culture L411, male, age 33); Choroideremia cell lines, CHM-1
(culture L659, male, age 26), CHM-2 (culture L660, male, age 45), CHM-3
(culture L665, male, age 54), and CHM-4 (culture L669, male, age 35).
All CHM cell lines contained no detectable REP-1 by immunoblot analysis
(data not shown). Large scale production of CHM-1 cells was achieved by
growth at 37 °C in 1-liter plastic roller bottles (Falcon)
containing RPMI 1640 medium supplemented with glutamax-I (Life
Technologies, Inc.), 25 mM sodium HEPES, pH 7.4, and 10%
heat-inactivated fetal bovine serum (Life Technologies, Inc.). Initial
cultures at a density of 2 10
cells/ml were aerated
with 10% CO
, 90% air, and fed every other day with the same
medium. Cells at a density of 1
10
cells/ml were
collected by centrifugation at 1,500 revolutions/min for 15 min at 4
°C, washed once with ice-cold phosphate-buffered saline,
recentrifuged as before, and stored frozen at -80 °C.
The assay to detect
unprenylated Rab proteins present in lymphoblasts used the same
principle, with the following modifications. Samples to be assayed in a
volume of 45 µl were added to reaction mixtures containing 500
mM sodium HEPES (pH 7.2), 50 mM MgCl, 10
mM DTT, 10 mM Nonidet P-40, 10 µM [
H]GG pyrophosphate (33,000
disintegrations/min/pmol), and 200-800 ng each of Rab GGTase and
REP-1 or REP-2 prepared in a volume of 5 µl and incubated for 15
min at 37 °C. The reactions were stopped with the addition of 15
µl of 4
SDS-sample buffer(27) , and aliquots were
subjected to SDS-PAGE. Following SDS-PAGE, the gel was either treated
with Entensify (DuPont NEN), dried, and exposed to Kodak XAR film at
-70 °C or transfered to a nitrocellulose filter and exposed
on a PhosphorImager (Fuji). For purification of p27, the same assay
containing REP-1, not REP-2, was used.
Figure 4:
Isolation of peptides from p27 by C-18
reverse phase chromatography. p27 was radiolabeled with
[H]geranylgeranyl by in vitro prenylation, followed by Triton X-114 partitioning and acetone
precipitation as described under ``Experimental Procedures.''
The protein pellet was incubated with either endoproteinase Lys-C (panel A) or Arg-C (panel B), and the resulting
peptides were isolated on a reverse-phase µRPC column using a
SMART
System chromatography instrument. The column was
equilibrated in MiliQ water with 0.1% trifluoroacetic acid at 24 °C
at a flow rate of 200 µl/min, the samples were injected, and the
column was washed with 1 ml of the same solution. The peptides were
eluted with a 3.5-ml linear gradient between 0 and 70% acetonitrile
with 0.1% trifluoroacetic acid, followed by a 2.5-ml linear gradient
between 70 and 100% of the same solution. One-hundred µl fractions
were collected after 3.5 ml of elution, and an aliquot was counted in a
scintillation counter (
). The absorbance detected at 214 nm is
shown as a straight line.
Method Two used a conventional method(30) . The
sample was subjected to in vitro prenylation, Triton X-114
partitioning, and acetone precipitation (approximately 500 pmol of
[H]GG groups incorporated), and then was
subjected to electrophoresis on a 12% SDS-PAGE and transferred to
nitrocellulose. The nitrocellulose filter was stained with Ponceau S
(Sigma) and exposed to a PhosphoImager. The fluorograph was aligned
with the nitrocellulose filter, and the corresponding protein band was
excised and subjected to solid phase digestion with endoproteinase
Lys-C. Peptides released from the filter were purified by high pressure
liquid chromatography and sequenced as described
previously(4) .
For immunoblot analysis, samples were
subjected to SDS-PAGE, transferred to nitrocellulose filters, and
stained with 0.2% Ponceau S in 5% acetic acid. The nitrocellulose
filters were incubated in solution A (50 mM Tris-HCl (pH 7.4),
150 mM NaCl, 0.2% Tween-20, and 5% nonfat dry milk) for 30 min
at room temperature with agitation, followed by incubation in solution
1 containing the primary antibody for 1 h. The blots were washed for 2
10 min in solution B (solution A + 1% Nonidet P-40),
followed by incubation for 30 min in solution B containing donkey
anti-rabbit IgG conjugated to horseradish peroxidase (Amersham Corp.).
The blots were then washed for 2
10 min with solution C (50
mM Tris-HCl (pH 7.4), 1% SDS, 0.5% cholic acid, and 1% Nonidet
P-40). Bound IgG was visualized by the ECL method (Amersham). All blots
were calibrated with prestained molecular weight markers (Bio-Rad).
For immunohistochemistry, Sprague-Dawley rats were perfused with
phosphate-buffered saline (PBS) (pH 7.4) prior to dissection of the eye
cup, which included the retina, pigment epithelium, choroid, and
sclera. The tissue was immediately placed in 4% paraformaldehyde in PBS
(pH 7.4) for 12 h at 4 °C. The tissue was infiltrated with 30%
(w/v) sucrose in PBS (pH 7.4) at 4 °C, embedded in O.C.T compound
(Tissue-Tek), and frozen in this media on dry ice. Longitudinal frozen
sections (10-12 µm) of the eye cup were placed on
Superfrost/Plus microscope slides (Fisher). Immunohistochemical
staining of these sections was performed using the Vectastain ABC Elite
kit following the instructions of the manufacturer (Vector
Laboratories). Normal goat serum (1.5%) was added to the incubation
solution (PBS (pH 7.4) and 0.3% Triton X-100) during incubation of
tissue sections with IgG and avidin-biotin complexes. Bound antibodies
were detected using an alkaline phosphatase substrate (Vector
Laboratories Alkaline Phosphatase Substrate Kit III), resulting in a
blue colorimetric reaction. Competition of N103 antibody immunostaining
was performed by incubation of diluted N103 antibody (0.5 µg/ml)
with AminoLink Coupling Gel (Pierce) beads cross-linked with 10
molar excess of either His-Ram/Rab27 or His-Rab1a prepared as described
above, at 22 °C with agitation for 1 h. The beads were then
sedimented by centrifugation, and the resulting supernatant was
utilized for immunohistochemistry.
To determine if CHM lymphoblasts efficiently prenylate all
their cellular Rabs in vivo, we prepared cytosolic extracts
from CHM and normal lymphoblasts and subjected them to in vitro prenylation using [H]GG pyrophosphate as the
lipid donor, Rab GGTase, and either REP-1 or REP-2. Under these
conditions, any unprenylated Rab present in the cytosolic extract will
be covalently modified by [
H]GG groups and may be
detected upon SDS-PAGE and autoradiography. When five different normal
extracts were analyzed in this manner, virtually no radiolabeled
proteins were detected (Fig. 1, lanes C-L), suggesting
that newly synthesized Rabs are rapidly and efficiently prenylated in
normal cells. When the same cells were grown in the presence of
compactin, an inhibitor of the synthesis of mevalonate, the precursor
of prenyl groups, a large number of cytosolic proteins with apparent
molecular mass between 20 and 30 kDa were labeled (Fig. 1, lanes A and B), indicating that unprenylated
endogenous Rabs can be detected using this method. A similar pattern of
bands was obtained when using either recombinant REP-1 or REP-2,
consistent with the idea that either REP-1 or REP-2 can efficiently
process most Rabs in vitro(25) .
Figure 1:
REP-dependent transfer of
[H]geranylgeranyl to lymphoblast proteins.
Cytosolic extracts were prepared as described under ``Experimental
Procedures'' in buffer containing 20 mM sodium HEPES (pH
7.2), 10 mM NaCl, 0.1 mM Nonidet P-40, and 1
mM DTT, from human normal lymphoblasts (controls, lanes A-L) or choroideremia lymphoblasts (CHM, lanes M-T) grown in the presence (+) or absence(-)
of compactin as indicated. Extracts (50 µg for lanes A and B and 125 µg for lanes C-T) were subjected to in vitro prenylation, in the presence of 1 µM
[
H]GG pyrophosphate, 200 ng of Rab GG
transferase, and 800 ng of either REP-1 or REP-2 as indicated. Reaction
mixtures were stopped after 30 min at 37 °C with SDS-sample buffer,
subjected to SDS-PAGE on 10-20% precast minigels (Bio-Rad), the
gel was enhanced with Entensify (DuPont NEN) and exposed to Kodak XAR
film for 14 days at -70 °C. Gels were calibrated with the
indicated molecular weight standards. Arrow at right denotes the position of migration of
p27.
When extracts from four different CHM cell lines were subjected to in vitro prenylation, a strikingly different result was obtained. Two discrete bands of approximately 27 and 23 kDa were radiolabeled in all four CHM-derived extracts (Fig. 1, lanes M-T). The 27 kDa band appears to be labeled by REP-1, but not REP-2 (Fig. 1, compare lanes M and N), while the 23 kDa band appears equally intense when using either REP. This pattern of labeling is characteristic of all CHM extracts (Fig. 1, lanes M-T). As a control, we subjected extracts derived from patients with Usher Syndrome type II(5) , another disease leading to retinal degeneration, to the same procedure and observed that no proteins could be labeled, as observed for normal cells (data not shown). The 27 kDa band therefore meets the criteria for a protein that is preferentially processed by REP-1 both in vitro and in vivo. The 27-kDa protein is the only major protein observed upon two-dimensional SDS-PAGE (data not shown). The 23-kDa band is not a single band; it represents the sum of many faint bands, as could be demonstrated upon subsequent purification. They might represent a small fraction of other Rabs that accumulate unprenylated in CHM cells due to slower prenylation kinetics when REP-1 is missing.
We devised a
purification scheme in order to isolate and identify the 27-kDa
unprenylated protein (p27) (Fig. 2). All fractions were
subjected to in vitro prenylation with
[H]GG pyrophosphate to detect p27. We obtained a
cytosolic soluble fraction (S-100) from CHM-1 cells, discarding the
membranes that contain the majority of prenylated Rabs, and subjected
the S-100 fraction to ammonium sulfate fractionation. The 40-60%
ammonium sulfate fraction was incubated with an excess of recombinant
Rab GDI prior to gel filtration chromatography. Since only prenylated
Rabs form an 80-kDa complex with Rab GDI(8) , the rationale for
this step was to separate unprenylated p27 from other prenylated Rabs
present in the ammonium sulfate fraction. As predicted, p27 eluted from
the column at approximately 30 kDa, the behavior of a monomer (data not
shown). The fractions containing p27 were subjected to Triton X-114
partitioning where p27 partitioned into the aqueous phase as predicted
for an unprenylated hydrophilic protein. We then subjected the Triton
X-114 aqueous phase to ion-exchange chromatography on a Mono Q column (Fig. 3). p27 eluted at about 200 mM NaCl. When this
fraction was prenylated in vitro, p27 was the only protein
that was labeled (Fig. 3, inset).
Figure 2: Purification scheme for p27. Details are described under ``Experimental Procedures'' and ``Results.''
Figure 3:
Purification of p27 by Mono Q
chromatography. (A), the aqueous phase from Triton X-114
partitioning was processed as described under ``Experimental
Procedures'' and applied onto a Mono Q 5/5 column equilibrated in
buffer 3 (fractions 1-5). The column was washed with 10
ml in the same buffer (fractions 6-10), and proteins
were eluted with two sequential linear gradients, a 20-ml gradient from
0 to 250 mM NaCl (fractions 11-20), followed by
5 ml of a gradient from 250 mM to 1 M NaCl (fractions 21-23) in the same buffer, and then washed
for 5 ml with 1 M NaCl in the same buffer (fractions
24-26). The amount of protein present in each fraction was
determined by the method of Bradford (). An aliquot (45 µl)
of each fraction was assayed for incorporation of
[
H]geranylgeranyl into proteins (
) by in vitro prenylation, as described under ``Experimental
Procedures''. Assayed fractions were separated on 12.5%
SDS-polyacrylamide gel, transferred to nitrocellulose, and exposed for
15 h using a Fuji PhosphorImager. The units obtained from the digital
imager are arbitrary and plotted relative to the maximum value in
fraction 17 (assigned a value of 1.0). (B), the inset shows the actual image obtained to deduce these values. Only
fractions 16-22 are shown. The gel was calibrated with the
indicated
C-methylated protein
standards.
We then took
advantage of the fact that we could render p27 both radiolabeled and
hydrophobic by prenylating it in vitro with
[H]GG pyrophosphate. Therefore, we performed a
bulk prenylation of the p27-containing fractions from the Mono Q
column. The [
H]GG-p27 was again subjected to
Triton X-114 partitioning and the majority of
[
H]GG-p27 now partitioned into the detergent
phase as predicted (data not shown). This step resulted in a very
significant purification, since most proteins remained in the aqueous
phase. The fraction contained only about 15-20 proteins of
different molecular weights as visualized by gold staining of a
nitrocellulose filter, following SDS-polyacrylamide gel
electrophoresis, and transfer.
In order to identify the 27-kDa
protein, we digested the protein in vitro with proteases, and
then we isolated and sequenced the
[H]GG-radiolabeled peptide. This prenylated
peptide is considerably more hydrophobic than non-modified peptides and
was eluted after all other peptides upon C-18 reverse phase
chromatography. Fig. 4shows the profile obtained upon C-18
reverse phase chromatography of a sample digested with either
endoproteinase Lys-C (panel A) or endoproteinase Arg-C (panel B). The peak fractions in experiment A eluted between
5.75 and 5.95 ml. They contained 360 pmol of peptide which accounted
for 75% of radiolabel incorporated into starting material (fractions
23-24). In experiment B, the peak fractions eluted between 4.85
and 5.25 ml and contained 250 pmol of peptide or 41% of the starting
material (fractions 14-17). Peptide sequences were obtained (Table 1, peptides 1 and 2) and compared with sequences in a
protein data bank (Swiss-Prot, September 1994). We observed a close
match to the predicted sequence of a rat protein designated
Ram(26) . Ram is a member of the Rab family of low molecular
weight GTPases, and for the remainder of the text we will refer to it
as Ram/Rab27 (see below ``Discussion''). In later
experiments, when larger amounts of p27 were purified, we confirmed its
identity as Ram/Rab27 by proteolytic digestion of the protein and
isolation of two internal peptides using standard methods(30) .
The sequenced peptides showed a 100% match with rat Ram over 22 amino
acids (Table 1, peptides 3 and 4).
We isolated a cDNA encoding
Ram/Rab27 from rat spleen cDNA by polymerase chain reaction and
prepared a hexahistidine-tagged version of recombinant Ram/Rab27 by
expression in E. coli and purification by nickel-Sepharose
chromatography. Kinetic analysis of recombinant Ram/Rab27 in
prenylation reactions using REP-1 or REP-2 revealed that the maximal
reaction velocity was approximately the same when either REP was used
in the reaction, but there was a 3-fold higher apparent K with REP-2 as compared with REP-1 (Fig. 5). In other experiments, we compared directly the
prenylation kinetics of Rab1a, Rab3a, and Ram/Rab27 (Table 2). As
previously reported by Cremers et al.(25) , we
observed similar K
and V
values for REP-1 and REP-2 when Rab1a was the substrate, and a
similar K
but 3-fold lower V
for REP-2 when Rab3a was the substrate. In the same experiments,
we obtained a similar V
for REP-1 and REP-2 but
2-3-fold higher K
for REP-2 when Ram/Rab27
was the substrate, comparable to the values obtained in Fig. 5.
We observed the same result in three independent experiments using
different batches of REP-1 and REP-2.
Figure 5:
Geranylgeranylation of Ram/Rab27 by REP-1
or REP-2. Each reaction mixture contained, in a final volume of 50
µl, 5.5 µM [H]GG pyrophosphate,
50 ng of Rab GGTase, 50 ng of either REP-1 (
) or REP-2 (
),
and the indicated concentrations of Ram/Rab27. After incubation for 15
min at 37 °C, the amount of [
H]GG transfered
to Ram/Rab27 was measured by ethanol/HCl precipitation. The value
obtained in the absence of Ram/Rab27 was 0.11 pmol. Each value
represents the average of duplicate reactions. The apparent K
values obtained using k
cat
software are indicated. The standard errors for the mean were ±
0.4 for REP-1 and ± 0.8 for REP-2.
We obtained two different polyclonal antibodies against rat Ram/Rab27, one directed against a COOH-terminal peptide and the other against the entire recombinant protein. The antibodies were highly specific and reacted against the rat protein with high affinity. They also detected the human counterpart, albeit with lower affinity. Despite the presence of some nonspecific bands, we were able to study the distribution of Ram/Rab27 in human lymphoblasts from the same subjects shown in Fig. 1. In all of the normal cell lines, Ram/Rab27 was present exclusively in the membrane fraction (Fig. 6, lanes A-J). A different result was obtained in all four CHM cell lines studied. In these cells, Ram/Rab27 was significantly shifted to the cytosol and only 20-30% remained in the membrane fraction (Fig. 6, lanes K-R). Similar results were obtained with both anti-Ram/Rab27 antibodies. There were only slight changes, if any, in distribution between normal and CHM cells for other Rabs, such as Rab1 or Rab7 (Fig. 6). These data confirm that the prenylation defect in CHM cells affects specifically Ram/Rab27.
Figure 6: Subcellular distribution of Rabs in control and choroideremia lymphoblasts. Cytosolic (C) and membrane (M) fractions were prepared as described under ``Experimental Procedures'' from human normal lymphoblasts (controls, lanes A-J) or choroideremia lymphoblasts (lanes K-R). Extracts (25 µg each) were subjected to SDS-PAGE on 12.5% acrylamide gels, the proteins transferred to nitrocellulose filters, and Rabs were detected by immunoblot analysis, as described under ``Experimental Procedures.'' Anti-Ram/Rab27 affinity-purified N103 antibody was used at 1 µg/ml. Arrows at right denote the position of migration of Ram/Rab27, Rab7, and Rab1, as indicated. Arrowheads at right denote cross-reacting bands revealed by the anti-Ram/Rab27 antibody. Gels were calibrated with the indicated molecular weight standards.
To determine whether Ram/Rab27 is expressed in the retina, we used the anti-Ram/Rab27 antibody produced against the recombinant protein to probe membrane extracts derived from various rat tissues. Ram/Rab27 was present at high levels in eye, intestine, lung, pancreas, and spleen, and low or absent in brain, liver, heart, kidney, and skeletal muscle (Fig. 7). The high affinity and the specificity of this antibody allowed more detailed studies of the rat retina by immunohistochemistry. As shown in Fig. 8A, the anti-Ram/Rab27 antibody stained strongly the retinal pigment epithelium (RPE) and the choriocapillaris. This staining was competed with Ram/Rab27 protein (Fig. 8B) but not with Rab1a protein (Fig. 8C), suggesting that it is specific. Another antibody directed against the COOH terminus of Ram/Rab27 gave identical results (data not shown). Specific staining of other retinal layers was obtained when we used an anti-Rab7 antibody that stains the RPE and plexiform layers but not the choriocapillaris (Fig. 8D), an anti-cellular retinaldehyde-binding protein that stains the RPE (Fig. 8E) and an anti-opsin antibody that stains the rod outer segments (Fig. 8F). In each case, the staining pattern was different from that of Ram/Rab27.
Figure 7: Tissue distribution of rat Ram/Rab27 as determined by immunoblot analysis. Membrane fractions of the indicated rat tissues were prepared as described under ``Experimental Procedures,'' and subjected (50 µg each) to SDS-PAGE on 12.5% acrylamide gels. The proteins were transferred to nitrocellulose filters and stained by Ponceau S to verify that equal amounts of proteins were loaded in each lane. Ram/Rab 27 was detected by immunoblot analysis using 0.1 µg/ml affinity-purified N103 antibody as described under ``Experimental Procedures.'' Arrow at right denotes the position of migration of Ram/Rab27. Gels were calibrated with the indicated molecular weight standards.
Figure 8:
Immunohistochemical localization of
Ram/Rab27 in the rat retina. Longitudinal frozen rat retina sections
(10 µm) were prepared and immunostained with 0.5 µg/ml of
affinity-purified anti-Ram/Rab27 N103 antibody (A), 0.5
µg/ml of affinity-purified anti-Ram/Rab27 N103 antibody in the
presence of 10 molar excess of His-Ram/Rab27 (B) or
His-Rab1A (C), anti-Rab7 immune sera (D),
anti-cellular retinaldehyde-binding protein antibody immune sera (E), and anti-opsin immune sera (F), as described
under ``Experimental Procedures.'' The abbreviations used
are: sc, sclera; ch, choroid; rpe, retinal
pigment epithelium; ros, rod photoreceptor outer segments; onl, outer nuclear layer; inl, inner nuclear
layer.
We previously proposed that in CHM cells where REP-1 is missing, REP-2 can assist in the prenylation of most Rabs thereby compensating for the loss of REP-1 and preventing overt cellular dysfunction(5, 25) . The question remained: why does the retina degenerate? The current data provide a potential explanation. We have found that CHM lymphoblasts efficiently prenylate the majority of its Rab proteins, except for one protein that accumulates unprenylated. This protein, Ram/Rab27, is expressed at high levels in the retinal pigment epithelium and choriocapillaris, the two sites of earliest degeneration in CHM.
We demonstrate that CHM lymphoblasts contain one protein that requires preferentially REP-1 for prenylation both in vivo and in vitro. We isolated the REP-1-dependent substrate and identified it by peptide sequencing as the human homolog of a rat protein previously cloned by Nagata et al.(26) and designated Ram. These investigators studied GTP-binding proteins present in human platelets and purified a protein designated c25KG (32) . Using peptide sequences obtained from c25KG, they cloned a related but not identical gene from a rat megakaryocyte library, Ram (26) . Ram is a member of the Ras superfamily of GTPases. It shows less than 30% identity with members of the Ras, Rho/Rac, Arf, and Ran families and more than 30% identity with members of the Rab family (26) . Ram contains a carboxyl-terminal prenylation motif, CysGlyCys and an effector domain characteristic of Rab proteins. For these reasons we propose to rename it Rab27, in accordance with widely accepted nomenclature rules(33) . Ram/Rab27 is more closely related to Rab3 (40% identical) than to any other subfamily of Rabs, but the level of identity suggests that Ram/Rab27 represents a distinct subfamily (which may include c25KG). No clues exist to date as to the physiological role of Ram/Rab27, except to speculate that it is regulating one step of intracellular vesicular transport, as proposed for other better studied Rabs.
We raised specific antibodies directed against Ram/Rab27 and demonstrate that it has a different subcellular distribution in normal versus CHM lymphoblasts. In normal lymphoblasts the protein is mostly membrane bound, but in CHM cells Ram/Rab27 is predominantly cytosolic. Two other Rabs studied show similar distribution in both normal and CHM lymphoblasts. These findings support the conclusion that the correct protein was identified insofar as it has been extensively documented that Rabs require prenyl groups for membrane attachment and that they become cytosolic and inactive in their absence (see Introduction).
When we analyzed the kinetics of in vitro prenylation of Ram/Rab27, we observed a 2-3-fold higher
apparent K in the reaction when REP-2 was present
as compared with REP-1. This finding suggests that there is lower
affinity for the REP-2-Ram/Rab27 interaction, as compared to
REP-1-Ram/Rab27. We think that this change in apparent K
is significant and suggests a mechanism for its deficient
prenylation in CHM cells. We previously analyzed different recombinant
Rabs (Rab1a, Rab3a, Rab3b, Rab3d, Rab5a, and Rab6) and were never able
to demonstrate a change in the apparent K
of the
reaction, either between different Rabs or the same Rab toward REP-1
and REP-2(25) . Since all other Rabs tested show higher
affinity for REP-2 than Ram/Rab27, it is possible that Ram/Rab27 cannot
compete efficiently with other Rabs for prenylation in CHM cells where
REP-1 is missing. By this formulation, the deficient prenylation of
Ram/Rab27 in CHM cells would result in its inactivation and subcellular
redistribution from the membrane into the cytosol and is entirely
consistent with the data presented here.
We demonstrate here that Ram/Rab27 is present in the retina, namely in the two cell layers believed to degenerate primarily in CHM, the RPE and the choriocapillaris. The photoreceptors did not show significant expression of Ram/Rab27. Labeling of the RPE and the choriocapillaris layers was intense, reproduced with two different antibodies, and competed by soluble Ram/Rab27 protein but not by the related protein Rab1a, which suggest that the staining was specific.
Taken together, these results suggest a mechanism for the retinal degeneration in CHM. Ram/Rab27 is postulated to play an important and unique role in the regulation of a specific vesicular transport step in RPE and/or choriocapillaris cells. The inability to efficiently prenylate Ram/Rab27 in these cells leads to a functional defect that ultimately results in cellular death. The dysfunction of the RPE/choriocapillaris results in the secondary degeneration of photoreceptors and visual impairment. The slow progression of the disease may be explained by the fact that not all Ram/Rab27 is unprenylated and that a fraction is membrane bound and presumably fully functional. With age, these cells may become more sensitive to the functional loss of Ram/Rab27 and degenerate.
Why is CHM confined to the retina if Ram/Rab27 is expressed in other tissues? It is possible that in other tissues, Ram/Rab27 function is redundant and compensated by a related Rab (such as c25KG); alternatively, enough function may be provided by the remainder of active Ram/Rab27, or the dysfunctional transport step may not be so crucial to the viability of those tissues. Another possibility is that in the retina there is a more severe defect in geranylgeranylation of Ram/Rab27, either because there is less REP-2 present or because there are more competitor Rabs to be prenylated.
We have not yet ruled out that the crucial Rab that is not prenylated in CHM retinas is a protein related to Ram/Rab27 and not Ram/Rab27 itself. As discussed above, there is at least one related protein, c25KG and others may exist. Also, it remains to be shown that human retinas express Ram/Rab27 in the same cell types and that the findings in the rat are relevant to the human disease. These and other issues may be addressed with future studies detailing the function of Ram/Rab27 and related proteins in the RPE, the choriocapillaris, and other tissues.