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Vol. 14, Issue 8, 3400-3413, August 2003
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* Department of Biochemistry and Molecular Biology, University of British
Columbia, Vancouver, British Columbia, Canada V6T 1Z3;
Department of Ophthalmology, University of British Columbia, Vancouver,
British Columbia, Canada V6T 1Z3; and
Department of Neuroscience, University of Connecticut Health Center,
Farmington, Connecticut 06032-3705
Submitted February 10, 2003;
Revised March 19, 2003;
Accepted April 9, 2003
Monitoring Editor: Benjamin Glick
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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).
Peripherin-2 (also known as peripherin/rds) is a membrane protein localized
to the rims and incisures of photoreceptor disks
(Molday et al., 1987;
Arikawa et al., 1992).
It plays a crucial role in outer segment morphogenesis because rds
mice homozygous for the disrupted peripherin-2 gene fail to develop outer
segments and heterozygous mice produce highly disorganized disk structures
(Sanyal and Jansen, 1981;
Hawkins et al., 1984;
Travis et al., 1989,
1992;
Connell et al., 1991).
The importance of peripherin-2 in photoreceptor viability is further
highlighted by the finding that >40 different mutations in peripherin-2
have been linked to human retinopathies, including autosomal dominant
retinitis pigmentosa (ADRP), a retinal degenerative disease characterized by
night blindness, progressive loss of vision, and photoreceptor cell death
(Farrar et al., 1991;
Kajiwara et al.,
1991; Saga et al.,
1993; Weleber et al.,
1993).
Peripherin-2 is a member of the tetraspanin family of membrane proteins
characterized by four transmembrane segments and a large intradiskal (EC-2)
domain between the third and fourth membrane-spanning segments (see
Figure 1;
Connell and Molday, 1990;
Travis et al., 1991;
Hemler, 2001;
Seigneuret et al.,
2001). The EC-2 domain contains one N-linked glycosylation site
and seven conserved cysteine residues that participate in intramolecular and
intermolecular disulfide bonds important for protein folding and subunit
assembly (Goldberg et al.,
1998; Loewen and Molday,
2000).
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In mammalian photoreceptors, peripherin-2 associates with itself and with
rom-1, a related tetraspanin protein, to form a mixture of core homo- and
heterotetramers (Goldberg and Molday,
1996b; Loewen and Molday,
2000; Loewen et al.,
2001). These tetramers further link together through
intermolecular disulfide bonds to form octamers and higher order oligomers
believed to be crucial for disk rim formation
(Loewen and Molday, 2000;
Wrigley et al.,
2000). In contrast to peripherin-2, rom-1 plays a relatively minor
role in disk morphogenesis and structure because rom-1 knockout mice develop
outer segments that are only modestly altered in appearance
(Clarke et al., 2000).
Furthermore, rom-1 is absent in lower vertebrates including amphibians
(Kedzierski et al.,
1996).
Several recent studies have examined the effect of disease-causing missense
mutations on the biochemical properties of peripherin-2 and the structure of
ROS. The C214S peripherin-2 mutation linked to a monogenic form of ADRP is
misfolded and defective in its ability to form core tetramers
(Goldberg and Molday, 1996a;
Goldberg et al.,
1998; Loewen et al.,
2001). The L185P peripherin-2 mutation associated with a digenic
form of ADRP assembles with wild-type (WT) peripherin-2 and rom-1 to form
functional heterotetramers, but is unable to self-associate into tetramers.
Transgenic mice harboring disease-linked mutations generally show disorganized
outer segments that correlate with photoreceptor degeneration (Kedzierski
et al., 1997,
2001). However, because the
mutant protein could not be distinguished from endogenous WT peripherin-2 in
these mice, the effect of disease-linked mutations on peripherin-2 targeting
to outer segment disks could not be determined. Hence, it is unclear if
defective targeting of mutant peripherin-2 to outer segment disk membranes
contributes to the cellular mechanism underlying ADRP.
In the present study we have generated transgenic X. laevis tadpoles expressing WT, C214S, P216L, L185P, and C150S peripherin-2 green fluorescent fusion protein (GFP) to identify determinants responsible for the targeting of peripherin-2 to ROS disks and to define cellular and molecular mechanisms responsible for ADRP. Here, we show that peripherin-2 core tetramer formation is required for proper targeting and incorporation of peripherin-2 into newly formed disks membranes. We also provide evidence that ADRP caused by tetramerization-defective peripherin-2 mutations (C214S and L185P) occurs through a mechanism involving a deficiency in WT peripherin-2, whereas ADRP associated with the tetramer-competent P216L peripherin-2 mutation takes place through a dominant negative mechanism.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). The sequence differed slightly from the published Xrds-38
sequence (Kedzierski et al.,
1996). Five amino acid differences (A78, A92, D187, F188, and
S189) were found that are conserved in mouse, rat, cat, dog, bovine, and human
orthologues, suggesting that these amino acids are bona fide residues of
Xenopus peripherin-2 (Figure
1A). The GenBank accession number is AY062004
[GenBank]
.
The X. laevis peripherin-2-GFP construct for COS-1 cell expression
was created by PCR, removing the stop codon and cloning in frame into the
XhoI and EcoRI sites of peGFP-N2 (CLONTECH Laboratories,
Inc., Palo Alto, CA). For transgenic X. laevis expression, the
XhoI-NotI fragment of Xenopus peripherin-2-GFP
(including GFP) was subcloned into the XhoI and NotI sites
of XOP1.3-eGFP-N1. This plasmid was derived from peGFP-N1 by replacing the CMV
promoter with a portion of the X. laevis opsin promoter
(Batni et al., 2000;
Tam et al., 2000).
Quickchange PCR-based mutagenesis (Stratagene, La Jolla, CA) was used to
introduce the L185P, C214S, P216L, and C150S mutations. Bovine
WT-peripherin-2-GFP and C214S-peripherin-2-GFP fusion constructs were created
in a similar manner. All PCR products and constructs were verified by
sequencing. Transgenic expression constructs were linearized by SfoI
digestion.
mAb Production
Oligonucleotides coding for the Xenopus peripherin-2 C-terminal
amino acid sequence KDTIKSSWELVKSMGKLNKVE were synthesized with appropriate
restriction sites and cloned into the pGEX vector. The GST fusion protein,
expressed in Escherichia coli, was purified on a
glutathione-Sepharose affinity column and used to immunize Swiss Webster mice
for generation of the Xper5A11 mAb as previously described
(MacKenzie and Molday,
1982).
Production of Transgenic X. laevis
Transgenic frogs were generated using a modified protocol
(Moritz et al., 1999)
based on the method of Kroll and Amaya
(1996). X. laevis
sperm nuclei were incubated with 0.3x high-speed egg extract, 0.05 U
restriction enzyme, and 100–200 ng linearized plasmid DNA. The reaction
mixture was then diluted to 0.3 nuclei/nl and 10 nl was injected per egg. The
resulting embryos were kept at 18°C in 0.1x Marc's modified Ringer,
6% Ficoll solution for 48 h and then switched to 0.1x Gerhart's Ringer
solution. At 5–6 d postfertilization (dpf) roughly corresponding to
stages 40–42, tadpoles were screened for GFP expression using a Leica
MZ8 dissecting microscope (Leica Microsystems, Wetzlar, Germany) equipped with
epifluorescence optics and a GFP filter set. Animals were immobilized in glass
Pasteur pipettes and tadpoles expressing GFP were identified by the green
fluorescence emitted from their eyes. At 14 dpf, the transgenic tadpoles were
placed in tanks with 0.1x Gerhart's Ringer solution and reared at
18°C on a 12/12 h light/dark cycle. Adult X. laevis were obtained
from Nasco or Xenopus Express (Plant City, FL).
Immunocytochemistry
For X. laevis immunofluorescence studies, tadpoles were sacrificed
between 14 and 28 dpf (stage 48–62). After immobilizing the tadpoles in
0.02% Tricaine, their eyes were excised and fixed in 4% paraformaldehyde,
sodium phosphate buffer, pH 7.5, overnight. Fixed eyes were embedded in OCT
tissue embedding medium (Tissue-Tek) and frozen in a dry ice/isopentane bath.
Cryostat sections (14 µm) were blocked (10% BSA, 0.1% Triton X-100 in PBS)
and labeled overnight with 0.1 mg/ml Texas Red–conjugated wheat germ
agglutinin (TR-WGA; Molecular Probes, Eugene, OR) and 0.01 mg/ml Hoescht 33342
stain (Sigma-Aldrich, Oakville, ON) to label photoreceptor membranes and
nuclei, respectively. Sections were also labeled with anti-Xenopus
peripherin-2 mAb Xper5A11. Labeling was done in the presence of 1 mM
CaCl2, 1 mM MgCl2, 1% BSA, and 0.1% Triton X-100 in PBS
for visualization under a Zeiss 510 confocal laser scanning microscope
(Thornwood, NY). At least three transgenic animals were examined for each
construct.
For immunoelectron microscopy, transgenic tadpoles were killed at 14 and 28 dpf (stage 48–62) and fixed overnight in 4% paraformaldehyde, 0.1 M sodium phosphate buffer, pH 7. The eyes were embedded in LR White resin, incubated overnight with a rabbit anti-GFP polyclonal antibody (Clontech Laboratories, Inc.) diluted 1:100 in 1% bovine serum albumin (BSA), 0.1 M Tris, pH 7.4, and labeled for 1 h with anti-rabbit Ig-gold (10 nm; British BioCell International, Cardiff, United Kingdom) diluted 1:5 in 0.1 M Tris, pH 7.4, 1% BSA. A minimum of two transgenic animals was examined for each construct.
COS-1 Cell Expression and Analysis
COS-1 cells (ca. 6 x 105 cells/100-mm dish) were
transfected with a total of 30 µg of pcDNAI/amp containing the appropriate
peripherin-2 construct as previously described
(Goldberg et al.,
1995). For immunofluorescence studies, the cells transfected with
GFP constructs were fixed and labeled with an anticalnexin antibody
(Stressgene, Victoria, BC) as an ER marker for analysis by confocal scanning
microscopy. For biochemical studies, transfected COS-1 cells were washed and
incubated in PBS in the presence (reduced) or absence (nonreduced) of 20 mM
dithiothreitol (DTT) for 90 min at 25°C. The cells were solubilized with
an equal volume of PBS (pH 7.4) containing 2% Triton X-100, 80 mM
N-ethyl maleimide, and phenyl methyl sulfonyl fluoride for 10 min on
ice, and the detergent extract was centrifuged at 90,000 x g
for 30 min at 4°C to remove any aggregated material and analyzed by SDS
gels and Western blotting. Bovine peripherin-2 was purified on a
Per2B6-Sepharose matrix as previously described
(Loewen and Molday, 2000).
For velocity sedimentation experiments the solubilized extract was applied
to 5–20% (wt/wt) sucrose gradients in PBS containing 0.1% Triton X-100
(Loewen and Molday, 2000).
After centrifugation for 16 h at 50,000 rpm in a Beckman TLS-55 rotor
(Beckman, Mississauga, ON) at 4°C, the bottom of the centrifuge tube was
punctured and fractions were analyzed on Western blots.
Protein cross-linking was performed on untreated or DTT-treated
detergent-solubilized cell extracts with 0.005% glutaraldehyde for 15 min at
37°C. Samples in the absence or presence of 5% 
-mercaptoethanol were
fractionated on 6% or 8% SDS-polyacrylamide gels. Western blots were labeled
with Per 2B6 mAb specific for bovine peripherin-2
(Molday et al., 1987)
and a GFP polyclonal antibody (Clontech Laboratories, Inc.) specific for the
GFP fusion protein for detection by ECL. PNGase F deglycosylation of
peripherin-2 was carried out as recommended by the manufacturer (New England
BioLabs, Mississauga, ON).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Loewen and Molday,
2000). A significant fraction of these tetramers link together
through C150 mediated disulfide bonds to form higher-order oligomers
(Loewen and Molday, 2000). As
a result, peripherin-2 migrates as a 35-kDa monomer on SDS gel under disulfide
reducing conditions and a mixture of monomers and disulfide-linked dimers
under nonreducing conditions.
We have expressed Xenopus and bovine peripherin-2-GFP fusion
proteins in COS-1 cells in order to determine the effect of the C-terminal GFP
on its oligomeric properties. Like the endogenous protein, Xenopus
and bovine peripherin-2-GFP fusion proteins migrated on SDS gels as monomers
under reducing conditions (Figure
1B, lane a) and a mixture of monomers and dimers under nonreducing
conditions (Figure 1B, lane d).
Replacement of cysteine at position 150 with serine (C150S) in
Xenopus as well as bovine peripherin-2-GFP abolished dimer formation
(Figure 1B, lane e) consistent
with the role of C150 residues in intermolecular disulfide bonding.
Glutaldehyde cross-linking of Xenopus peripherin-2-GFP resulted in a
mixture of monomers and dimers under reducing conditions
(Figure 1B, lane b) and dimers
and higher molecular weight species under nonreducing conditions
(Figure 1B, lane c) as
previously reported for bovine peripherin-2
(Loewen and Molday, 2000).
When coexpressed in COS-1 cells, Xenopus peripherin-2-GFP
coprecipitated with bovine peripherin-2 on a Per2B6 immunoaffinity matrix
specific for bovine peripherin-2, indicating that Xenopus and bovine
peripherin-2 assembled into multisubunit complexes
(Figure 1B, lane f). Together,
these results indicate that GFP fused to the C termini of peripherin-2 does
not affect the subunit assembly of peripherin-2.
Immunolocalization of Endogenous Peripherin-2 in Xenopus
laevis
Confocal scanning microscopy was used to visualize the distribution of
peripherin-2 in a Xenopus retina labeled with a peripherin-2 mAb.
Figure 2, A and B, shows that
labeling was confined to rod and cone outer segments. The vertical striations
observed in ROS and the restricted labeling to one edge of cone outer segments
are indicative of peripherin-2 localization to the rims and incisures of disk
membranes (Molday et al.,
1987; Arikawa et al.,
1992; Kedzierski et
al., 1996).
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Expression of Xenopus and Bovine WT Peripherin-2-GFP in Xenopus
Rods
tk;2Xenopus peripherin-2-GFP under the control of the
Xenopus rhodopsin promoter was found only in the outer segments of
rod photoreceptors (Figure 2, C and
D). The contiguous row of photoreceptor nuclei and long uniform
appearance of the ROS indicated that peripherin-2-GFP expression did not
affect outer segment structure or induce retinal degeneration. Furthermore,
the peripherin-2-GFP was observed in phagosomes within the retinal pigment
epithelial cells, indicating that normal disk shedding and phagocytosis had
occurred.
Peripherin-2-GFP expression varied along the length of a ROS, between rod
photoreceptors within the retina and from animal to animal
(Figure 2, C and E). A similar
variation in protein expression has been observed previously in transgenic
X. laevis tadpoles expressing rhodopsin-GFP
(Tam et al., 2000;
Moritz et al.,
2001b). This variable rod expression has been attributed to random
transgene silencing, with a probability dependent on the chromosomal location
of transgene integration (position-effect variegation). Together with the
unique mechanisms of ROS renewal, transient silencing results in bands of
varying fusion protein concentration
(Karpen, 1994;
Moritz et al.,
2001b). This variation permitted us to examine the effects of
different expression levels within the same retina, and even within the same
ROS.
In regions of moderate expression, peripherin-2-GFP fluorescence showed vertical striations in longitudinal sections and a scalloped appearance in transverse sections (Figure 2E), similar to endogeneous peripherin-2 and characteristic of protein targeting to the disk rims and incisures. In regions of high expression, GFP fluorescence saturated the ROS and the distinctive striated pattern of fluorescence was lost.
The distribution of peripherin-2-GFP was mapped more precisely by electron microscopy using an anti-GFP antibody with postembedding immunogold labeling. In longitudinal and transverse sections, peripherin-2-GFP was confined to the disk rims and incisures of ROS at moderate expression levels (Figure 2, F and G). In regions of high expression, peripherin-2-GFP was also observed in the disk lamellae (Figure 2H). Intense immunogold labeling correlated with a reduction in the diameter of the ROS and the presence of smaller, more disorganized disks.
Bovine peripherin-2-GFP was also expressed in X. laevis in order to determine if the requirements for targeting of peripherin-2 to ROS are conserved between mammalian and amphibian species. Like Xenopus peripherin-2-GFP, the bovine fusion protein targeted to the outer segments and showed partial colocalization with endogenous Xenopus peripherin-2 as shown in Figure 3D.
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Transgenic Expression of C214S Peripherin-2-GFP
The disease-linked C214S peripherin-2 mutation prevents tetramer formation,
but does not affect dimerization of peripherin-2
(Goldberg et al.,
1998). To determine the effect of this mutation on the subcellular
localization of peripherin-2, Xenopus C214S peripherin-2-GFP was
expressed in Xenopus rod photoreceptors.
Figure 3, A and B, shows that
this mutant is retained in the inner segment and cell body of all rods.
However, in some cells expressing high levels of the transgene, fusion protein
was also detected at the base of their ROS. Photoreceptor degeneration was not
evident in animals up to 4 weeks old.
Immunoelectron microscopy confirmed the retention of Xenopus C214S peripherin-2-GFP within the inner segment (Figure 3C). Furthermore, a significant amount of the mutant protein was observed to accumulate near the base of the connecting cilium indicating that a portion of the mutant protein passed through the quality control of the ER. The ultrastructure of the ROS appeared normal.
The distribution of bovine C214S peripherin-2-GFP expressed in X. laevis retina was also examined. Like Xenopus C214S peripherin-2-GFP, the bovine mutant was localized throughout the rod inner segments and cell body, but absent in the ROS (Figure 3E). The targeting of endogenous Xenopus peripherin-2 to ROS was unaffected by the presence of bovine C214S peripherin-2-GFP.
Transgenic Expression of P216L Peripherin-2-GFP
The effect of the P216L peripherin-2 mutation, associated with another
monogenic form of ADRP, was also examined
(Kajiwara et al.,
1991). Figure 4A
shows the expression pattern of Xenopus P216L-peripherin-2-GFP in a
tadpole retina. Peripheral rods were long and uniform in appearance, with the
fusion protein specifically localized to the outer segments
(Figure 4C). By electron
microscopy, the ultrastructure of peripheral ROS appeared normal with the
P216L peripherin-2-GFP protein specifically targeted to the disk rims and
incisures (Figure 4F). In
contrast, the central rods were shorter and highly disorganized with an
apparent decrease in cell number indicative of photoreceptor degeneration
(Figure 4, D and E).
Immunoelectron microscopy further showed the presence of the P216L fusion
protein in whorls of outer segment disk membranes
(Figure 4, G and H).
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Transgenic Expression of L185P Peripherin-2-GFP
A L185P mutation in peripherin-2 together with either a null allele or a
G113E mutation in rom-1 is responsible for a digenic form of ADRP
(Kajiwara et al.,
1994). Biochemical studies have shown that this mutant is unable
to self-associate into homotetramers, but a significant fraction can interact
with WT peripherin-2 and WT rom-1 of mammalian photoreceptors
(Goldberg and Molday, 1996a;
Loewen et al., 2001).
To evaluate the effect of the L185P mutation on the subcellular localization
of peripherin-2, we expressed Xenopus L185P-peripherin-2-GFP in rods.
The mutant GFP fusion protein targeted to both the inner and outer segments of
rod cells (Figure 5, A and B).
Photoreceptor degeneration was not observed.
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Expression of C150S peripherin-2-GFP
The C150S peripherin-2 mutant assembles into noncovalent tetramers, but
these core complexes are incapable of forming higher order disulfide-linked
oligomers (Loewen and Molday,
2000). To assess the role of disulfide-mediated oligomerization of
peripherin-2 in protein targeting and retinal degeneration, we expressed
Xenopus C150S peripherin-2-GFP in rod cells.
Figure 5, C and D, show that
the C150S mutant targeted specifically to ROS. Vertical striations
characteristic of fusion protein localization to disk incisures, however, were
not readily visible with the C150S mutant. Instead, a more mottled labeling
pattern was observed, often with the presence of a thick central column of
fluorescence. By electron microscopy, C150S peripherin-2 localized to the disk
lamellae as well as the rims indicating that some missorting of the protein
within the disks had occurred. There was no apparent photoreceptor
degeneration in these transgenic tadpoles.
Localization and Biochemical Characterization of P216L and C214S
Peripherin-2 Expressed in COS Cells
The subcellular localization of WT and mutant X. laevis
peripherin-2-GFP expressed in COS-1 cells was examined by confocal microscopy
(Figure 6A). WT
peripherin-2-GFP exhibited a punctuate appearance indicative of localization
to intracellular vesicles that did not label with the ER marker calnexin. In
contrast the C214S mutant localized both to ER and intracellular vesicles
suggesting that a portion of the mutants passed through the quality control of
the ER and accumulated in intracellular vesicles. The P216L mutant was also
present both in the ER and in intracellular vesicles.
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The biochemical properties of these proteins were also examined on SDS gels under reducing conditions. Figure 6B shows that P216L peripherin-2-GFP migrated more slowly than either the WT or C214S fusion proteins. After PNGase F treatment to remove N-linked oligosaccharides, all proteins migrated at the same rate, slightly ahead of the untreated WT and C214S peripherin-2-GFP. These studies indicate that WT and the C214S mutant are glycosylated and the P216L mutant is hyperglycosylated.
Velocity Sedimentation Analysis of P216L and C214S Peripherin-2
Previously, the oligomeric states of bovine WT, C150S, and L185P
peripherin-2 mutants were determined by velocity sedimentation analysis
(Loewen and Molday, 2000;
Loewen et al., 2001).
We have used this technique to examine the effect of the P216L and C214S
mutations on the oligomeric structure of peripherin-2
(Figure 7, A and B). Like WT
peripherin-2, the P216L mutant sedimented as a mixture of core tetramers
(species a), consisting of noncovalently associated monomeric subunits, and
higher order oligomers (species b), comprised of disulfide-linked dimers
(Loewen and Molday, 2000).
This suggests that the bovine P216L mutation does not adversely affect the
assembly of peripherin-2 into core tetramers or higher order disulfide-linked
oligomers. The C214S mutant sedimented with a distinctly different profile.
Under nonreducing conditions, it sedimented as a mixture of noncovalent dimers
(species c), tetramers composed of two disulfide-linked dimers (species d),
and aggregated, disulfide-linked species not found for WT or P216L
peripherin-2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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WT Peripherin-2-GFP Exhibits Normal Biochemical Properties and
Targeting to Disk Rims and Incisures
To validate the use of peripherin-2 containing a C-terminal GFP tag in
transgenic studies, we first examined the biochemical properties and
subcellular distribution of WT peripherin-2-GFP in COS-1 cells. Both bovine
and Xenopus peripherin-2-GFP exhibited properties similar to WT
peripherin-2 without GFP when analyzed by covalent cross-linking,
immunoprecipitation, migration behavior on reducing and nonreducing SDS gels,
and fluorescence microscopy. On this basis, we conclude that GFP fused to the
C termini of peripherin-2 does not affect noncovalent tetramer formation or
disulfide-linked oligomerization. This is consistent with earlier studies
showing that the EC-2 domain, and not the C termini, of peripherin-2, is
important in tetramerization and disulfide-linked oligomerization
(Goldberg and Molday, 1996a;
Goldberg et al.,
1998; Loewen and Molday,
2000; Loewen et al.,
2001). The finding that bovine and Xenopus peripherin-2
associate with one another when coexpressed in COS-1 cells further indicates
that the conformation of the EC-2 domain of these orthologues is similar. This
is consistent with the high degree of sequence identity between bovine and
Xenopus peripherin-2 (72% overall identity and 83% identity within
the EC-2 domain). Finally, peripherin-2-GFP is found in vesicles within COS-1
cells, indicating that, like peripherin-2, the fusion protein passes through
the quality control system of the ER.
Like endogenous peripherin-2, bovine and Xenopus peripherin-2-GFP
fusion proteins targeted specifically to the rims and incisures of ROS disks
at moderate expression levels without affecting ROS structure or inducing
photoreceptor degeneration. This indicates that the GFP tag does not affect
peripherin-2 trafficking to the disk rims and incisures or ROS organization.
At high expression levels, however, a portion of the peripherin-2-GFP is
observed in the lamellar region of disks. This abnormal localization is likely
due to the inability of the rims and incisures to accommodate excessive
amounts of peripherin-2-GFP. A constriction in the diameter of the outer
segment and shortening and disorganization of the disks also correlates with
high peripherin-2-GFP expression. In contrast, enlarged disks have been
observed when peripherin-2 (or rom-1) expression is low or absent
(Hawkins et al.,
1984; Kedzierski et
al., 1997; Clarke et
al., 2000). Therefore, peripherin-2 expression levels appear
to play a central role in establishing the size of the disks, possibly by
controlling disk closure during morphogenesis. In such a case, excess
peripherin-2 would increase the rate of closure resulting in smaller disks,
whereas low levels would decrease the rate of closure leading to larger
disks.
Tetramerization Is Required for Peripherin-2 Targeting to ROS
Disks
Our studies indicate that there is a direct correlation between the
assembly of peripherin-2 into core noncovalent tetramers and the ability of
this complex to target to outer segment disk membranes. WT and P216L
peripherin-2, which assemble into core noncovalent tetramers and
disulfide-linked oligomers as measured by velocity sedimentation in this and
previous studies (Goldberg and Molday,
1996b; Loewen and Molday,
2000), targeted normally to the rims and incisures of ROS disk
membranes. The C150S mutant, which forms tetramers, but not disulfide-linked
oligomers (Loewen and Molday,
2000), also targeted to disks, indicating that disulfide-linked
oligomerization is not required for the incorporation of peripherin-2 into
disks.
In contrast tetramerization-defective mutants were not targeted to ROS. The
C214S mutant, which forms homodimers but fails to form tetramers even with WT
peripherin-2, is retained in the cell body, and inner segment of the rod
cells. The L185P mutant, which exists as a mixture of homodimers and
heterotetramers with WT peripherin-2
(Loewen et al.,
2001), localized to both the rod outer and inner segments. The
fraction of L185P peripherin-2 found in ROS disks most likely corresponds to
L185P peripherin-2-GFP that associates with endogenous Xenopus
peripherin-2 to form tetramers, whereas the fraction that is retained in the
inner segments and cell body represents L185P peripherin-2 that exists as
homodimers.
On the basis of these results and COS-1 expression studies, we suggest that
rod photoreceptors have two quality control mechanisms to assure that only
peripherin-2 tetramers traffick to the outer segment and get incorporated into
disk membranes. First, photoreceptors, like other cells, have a quality
control mechanism to retain grossly misfolded proteins in the ER or inclusion
bodies known as aggresomes for eventual degradation
(Sung et al., 1991;
Illing et al., 2002;
Saliba et al., 2002).
However, a portion of poorly assembled peripherin-2 mutants (C214S and L185P
mutants) exits the ER as shown by their presence in intracellular vesicles in
COS-1 cells and at the apical region of the rod inner segment near the base of
the cilium, an area in the rod cell that is rich in post-Golgi vesicles, but
devoid of ER and Golgi (Papermaster et
al., 1985; Deretic and
Papermaster, 1991; Moritz
et al., 2001a). Another checkpoint in the vicinity of the
connecting cilium prevents these tetramerization-defective proteins from
becoming incorporated into nascent disk membranes of the ROS. As a result,
these proteins remain in the inner segment where they do not affect outer
segment morphogenesis or structure.
By analogy with rhodopsin (Marszalek
et al., 2000), a model for the transport of peripherin-2
from the inner to the outer segment can be envisioned
(Figure 9). WT peripherin-2
together with a fraction of the tetramerization-defective variants are
processed through the ER and Golgi and exit in post-Golgi vesicles, distinct
from rhodopsin-containing vesicles (Fariss
et al., 1997). Tetramerization-competent peripherin-2 is
transported through the cilium by a kinesin-dependent mechanism
(Marszalek et al.,
2000) and incorporated into nascent disks, whereas mutant dimers
are retained in the inner segment. The mechanism that prevents incompletely
assembled peripherin-2 from targeting to the outer segment is not known, but
it may involve the inability of dimeric peripherin-2 to associate with
accessory proteins required for trafficking through the cilium. Mechanisms
that link subunit assembly to cell membrane targeting have been reported for
other multisubunit transmembrane proteins, including K channels and GABA
receptors (Zerangue et al.,
1999; Manganas and Trimmer,
2000; Margeta-Mitrovic et
al., 2000).
|
ADRP Associated with the C214S and L185P Peripherin-2 Mutations
Results from a Deficiency in WT Peripherin-2
The C214S mutation in peripherin-2 is responsible for photoreceptor cell
death in individuals with this monogenic form of ADRP
(Saga et al., 1993).
Studies carried out here, however, indicate that expression of the C214S
peripherin-2 on a WT peripherin-2 background does not, itself, affect ROS
structure or induce photoreceptor degeneration. This suggests that
photoreceptor degeneration in individuals with the C214S mutation results
primarily from a deficiency in WT peripherin-2 similar to photoreceptor
degeneration displayed in heterozygous rds mice
(Hawkins et al.,
1984; Cheng et al.,
1997) and individuals with a peripherin-2 null allele
(Jacobson et al.,
1996). The possibility that long-term accumulation of misfolded
C214S peripherin-2 in rod inner segments and cilium further contributes to
photoreceptor degeneration in these individuals, however, cannot be ruled
out.
Digenic ADRP linked to the L185P peripherin-2 mutation also appears to
occur through a deficiency in WT peripherin-2 because L185P peripherin-2-GFP
expression does not affect ROS structure or induce photoreceptor degeneration
in our system. In this instance, individuals who inherit the L185P
peripherin-2 mutation along with a rom-1 null allele
(Kajiwara et al.,
1994) will have levels of functional peripherin-2 containing
tetramers below the threshold required to sustain disk morphogenesis and
photoreceptor viability and as a result will be affected with ADRP. In
contrast individuals who inherit only the L185P peripherin-2 mutation will be
essentially normal since L185P peripherin-2 can associate with rom-1 and WT
peripherin-2 to generate sufficient levels of functional tetramers to support
ROS disk morphogenesis (Goldberg and
Molday, 1996a; Loewen et
al., 2001). In transgenic X. laevis, there is
sufficient WT peripherin-2 to support disk morphogenesis and as a result
photoreceptor degeneration is not observed in these transgenic animals.
Recently, Kedzierski et al.
(2001) have also suggested
that a deficiency in peripherin-2 underlies digenic ADRP on the basis of the
phenotypes of mice harboring the L185P and/or rom-1 null mutations, although
the molecular basis or targeting aspects were not addressed in this study.
ADRP Associated with the P216L Peripherin-2 Mutation Occurs through a
Dominant Negative Mechanism
The P216L peripherin-2-GFP mutant exhibits distinct properties. It targets
specifically to the rims and incisures of Xenopus ROS disks similar
to WT peripherin-2, suggesting that the mutant protein, perhaps in association
with endogeneous WT peripherin-2, is properly folded so as to pass through the
quality control system of the rod cell. In COS-1 cells, however, only a
portion of the Xenopus P216L GFP fusion protein exits the ER into
intracellular vesicles, indicating that in this cell environment not all the
mutant is properly folded. Evidently coassembly of the P216L mutant with
endogenous WT peripherin-2 in rod cells and/or the presence of photoreceptor
specific chaperone proteins facilitates the proper folding of the mutant and
the effective translocation of the peripherin-2 complex from the ER to the ROS
disks. However, unlike the C214S and L185P mutants, P216L peripherin-2-GFP has
a profound effect on photoreceptors. Peripheral rod cells of 4-week-old X.
laevis tadpoles expressing the P216L mutant appear normal with proper
targeting of the P216L peripherin-2-GFP to the disk rims and incisures. In
contrast, central rod cells have short, highly disorganized outer segments
displaying whorls of disk membrane with clear evidence of photoreceptor
degeneration. Photoreceptors of the central retina of X. laevis are
older than peripheral photoreceptors
(Hollyfield, 1971). The
differential appearance and degeneration of central and peripheral
photoreceptors expressing this mutant may arise from the age difference of
these cells. Similar differential degeneration has been observed in
Xenopus rods expressing rab8 mutants
(Moritz et al.,
2001a). Analysis of transgenic animals at later stages is required
to define the time course of peripheral and central rod degeneration.
The question arises "What is the molecular basis for the dominant
negative effect of the P216L mutant on ROS structure and photoreceptor
degeneration?" Sequence analysis suggests the possible involvement of
N-linked glycosylation. The proline at position 216, which is part of the
sequence 215N-P-S217, prevents glycosylation from
occurring at asparagine 215 in WT peripherin-2. Substitution of proline with
leucine, however, creates a new N-linked glycosylation site
215N-L-S217 in peripherin-2. Indeed, Xenopus
P216L peripherin-2 migrates more slowly than WT peripherin-2 on SDS gels in
the absence of PNGase treatment, but not after deglycosylation, suggesting
that this site is glycosylated (Figure
6). During our studies, Wrigley et al.
(2002) have also demonstrated
that in vitro expression of P216L peripherin-2 mutant is hyperglycosylated. It
is possible that introduction of a bulky oligosaccharide chain at
N215 of the P216L mutant may lead to ROS disk instability and
progressive photoreceptor degeneration.
In summary, our studies indicate peripherin-2 tetramerization is essential for the normal targeting of peripherin-2 to ROS disk membranes. A checkpoint in the vicinity of the cilium prevents poorly assembled peripherin-2 mutants from being incorporated into nascent disks. Individuals with tetramerization-defective mutations (C214S, C167Y, L185P) succumb to ADRP by a mechanism primarily involving a deficiency of WT peripherin-2, similar to heterozygous rds mice, whereas individuals with tetramerization-competent ADRP mutations (P216L) are affected through a dominant negative effect of the mutant on disk morphogenesis and structure. These mechanisms have implications in the design of gene therapy approaches. Individuals with C214S and L185P tetramerization-defective mutations will likely benefit from genetic approaches that simply increase the expression of WT peripherin-2 before photoreceptor degeneration, whereas individuals with a dominant negative mutation such as P216L will require disruption of the mutant gene in addition to increased expression of the WT gene.
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
Footnotes |
|---|
Abbreviations used: GFP, green fluorescent protein; ROS, rod outer segments; ADRP, autosomal dominant retinitis pigmentosa; WT, wild-type; DTT, dithiothreitol.
Corresponding author. E-mail address:
molday{at}interchange.ubc.ca.
|
REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
Batni, S., Mani, S.S., Schlueter, C., Ji, M., and Knox, B.E. (2000). Xenopus rod photoreceptor: model for expression of retinal genes. Methods Enzymol. 316, 50–64.[Medline]
Bok, D. (1985). Retinal photoreceptor-pigment
epithelium interactions. Friedenwald lecture. Invest Ophthalmol. Vis.
Sci. 26,
1659–1694.
Cheng, T., Peachey, N.S., Li, S., Goto, Y., Cao, Y., and Naash,
M.I. (1997). The effect of peripherin/rds haploinsufficiency on
rod and cone photoreceptors. J. Neurosci.
17,
8118–8128.
Clarke, G. et al. (2000). Rom-1 is required for rod photoreceptor viability and the regulation of disk morphogenesis. Nat. Genet. 25, 67–73.[CrossRef][Medline]
Connell, G., Bascom, R., Molday, L., Reid, D., McInnes, R.R., and
Molday, R.S. (1991). Photoreceptor peripherin is the normal
product of the gene responsible for retinal degeneration in the rds mouse.
Proc. Natl. Acad. Sci. USA 88,
723–726.
Connell, G.J., and Molday, R.S. (1990). Molecular cloning, primary structure, and orientation of the vertebrate photoreceptor cell protein peripherin in the rod outer segment disk membrane. Biochemistry 29, 4691–4698.[CrossRef][Medline]
Deretic, D., and Papermaster, D.S. (1991). Polarized
sorting of rhodopsin on post-Golgi membranes in frog retinal photoreceptor
cells. J. Cell Biol. 113,
1281–1293.
Fariss, R.N., Molday, R.S., Fisher, S.K., and Matsumoto, B. (1997). Evidence from normal and degenerating photoreceptors that two outer segment integral membrane proteins have separate transport pathways. J. Comp. Neurol. 387, 148–156.[CrossRef][Medline]
Farrar, G.J., Kenna, P., Jordan, S.A., Kumar-Singh, R., Humphries, M.M., Sharp, E.M., Sheils, D.M., and Humphries, P. (1991). A three-base-pair deletion in the peripherin-RDS gene in one form of retinitis pigmentosa. Nature 354, 478–480.[CrossRef][Medline]
Goldberg, A.F., Loewen, C.J., and Molday, R.S. (1998). Cysteine residues of photoreceptor peripherin/rds: role in subunit assembly and autosomal dominant retinitis pigmentosa. Biochemistry 37, 680–685.[CrossRef][Medline]
Goldberg, A.F., and Molday, R.S. (1996a). Defective
subunit assembly underlies a digenic form of retinitis pigmentosa linked to
mutations in peripherin/rds and rom-1. Proc. Natl. Acad. Sci.
USA 93,
13726–13730.
Goldberg, A.F., and Molday, R.S. (1996b). Subunit composition of the peripherin/rds-rom-1 disk rim complex from rod photoreceptors: hydrodynamic evidence for a tetrameric quaternary structure. Biochemistry 35, 6144–6149.[CrossRef][Medline]
Goldberg, A.F., Moritz, O.L., and Molday, R.S. (1995). Heterologous expression of photoreceptor peripherin/rds and Rom-1 in COS-1 cells: assembly, interactions, and localization of multisubunit complexes. Biochemistry 34, 14213–14219.[CrossRef][Medline]
Hawkins, R.K., Jansen, H.G., and Sanyal, S. (1984). Development and degeneration of retina in rds mutant mice: photoreceptor abnormalities in the heterozygotes. Exp. Eye Res. 41, 701–720.
Hemler, M.E. (2001). Specific tetraspanin functions.
J. Cell Biol. 155,
1103–1107.
Hollyfield, J.G. (1971). Differential growth of the neural retina in Xenopus laevis larvae. Dev. Biol. 24, 264–286.[CrossRef][Medline]
Illing, M.E., Rajan, R.S., Bence, N.F., and Kopito, R.R.
(2002). A rhodopsin mutant linked to autosomal dominant retinitis
pigmentosa is prone to aggregate and interacts with the ubiquitin proteasome
system. J. Biol. Chem. 277,
34150–34160.
Jacobson, S.G., Cideciyan, A.V., Kemp, C.M., Sheffield, V.C., and
Stone, E.M. (1996). Photoreceptor function in heterozygotes with
insertion or deletion mutations in the RDS gene. Invest. Ophthalmol.
Vis. Sci. 37,
1662–1674.
Kajiwara, K., Berson, E.L., and Dryja, T.P. (1994).
Digenic retinitis pigmentosa due to mutations at the unlinked peripherin/RDS
and ROM1 loci. Science 264,
1604–1608.
Kajiwara, K., Hahn, L.B., Mukai, S., Travis, G.H., Berson, E.L., and Dryja, T.P. (1991). Mutations in the human retinal degeneration slow gene in autosomal dominant retinitis pigmentosa. Nature 354, 480–483.[CrossRef][Medline]
Karpen, G.H. (1994). Position-effect variegation and the new biology of heterochromatin. Curr. Opin. Genet. Dev. 4, 281–291.[CrossRef][Medline]
Kedzierski, W., Lloyd, M., Birch, D.G., Bok, D., and Travis, G.H.
(1997). Generation and analysis of transgenic mice expressing
P216L-substituted rds/peripherin in rod photoreceptors. Invest.
Ophthalmol. Vis. Sci. 38,
498–509.
Kedzierski, W., Moghrabi, W.N., Allen, A.C., Jablonski-Stiemke, M.M., Azarian, S.M., Bok, D., and Travis, G.H. (1996). Three homologs of rds/peripherin in Xenopus laevis photoreceptors that exhibit covalent and non-covalent interactions. J. Cell Sci. 109, 2551–2560.[Abstract]
Kedzierski, W., Nusinowitz, S., Birch, D., Clarke, G., McInnes,
R.R., Bok, D., and Travis, G.H. (2001). Deficiency of
rds/peripherin causes photoreceptor death in mouse models of digenic and
dominant retinitis pigmentosa. Proc. Natl. Acad. Sci. USA
98,
7718–7723.
Kroll, K.L., and Amaya, E. (1996). Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development 122, 3173–3183.[Abstract]
Loewen, C.J., and Molday, R.S. (2000).
Disulfide-mediated oligomerization of Peripherin/Rds and Rom-1 in
photoreceptor disk membranes. Implications for photoreceptor outer segment
morphogenesis and degeneration. J. Biol. Chem.
275,
5370–5378.
Loewen, C.J., Moritz, O.L., and Molday, R.S. (2001).
Molecular characterization of peripherin-2 and rom-1 mutants responsible for
digenic retinitis pigmentosa. J. Biol. Chem.
276,
22388–22396.
MacKenzie, D., and Molday, R.S. (1982). Organization
of rhodopsin and a high molecular weight glycoprotein in rod photoreceptor
disc membranes using monoclonal antibodies. J. Biol. Chem.
257,
7100–7105.
Manganas, L.N., and Trimmer, J.S. (2000). Subunit
composition determines Kv1 potassium channel surface expression. J.
Biol. Chem. 275,
29685–29693.
Margeta-Mitrovic, M., Jan, Y.N., and Jan, L.Y. (2000). A trafficking checkpoint controls GABA(B) receptor heterodimerization. Neuron 27, 97–106.[CrossRef][Medline]
Marszalek, J.R., Liu, X., Roberts, E.A., Chui, D., Marth, J.D., Williams, D.S., and Goldstein, L.S. (2000). Genetic evidence for selective transport of opsin and arrestin by kinesin-II in mammalian photoreceptors. Cell 102, 175–187.[CrossRef][Medline]
Molday, R.S., Hicks, D., and Molday, L. (1987).
Peripherin. A rim-specific membrane protein of rod outer segment discs.
Invest. Ophthalmol. Vis. Sci.
28,
50–61.
Moritz, O.L., Tam, B.M., Hurd, L.L., Peranen, J., Deretic, D., and
Papermaster, D.S. (2001a). Mutant rab8 impairs docking and fusion
of rhodopsin-bearing post-Golgi membranes and causes cell death of transgenic
Xenopus rods. Mol. Biol. Cell
12,
2341–2351.
Moritz, O.L., Tam, B.M., Knox, B.E., and Papermaster, D.S.
(1999). Fluorescent photoreceptors of transgenic Xenopus laevis
imaged in vivo by two microscopy techniques. Invest. Ophthalmol. Vis.
Sci. 40,
3276–3280.
Moritz, O.L., Tam, B.M., Papermaster, D.S., and Nakayama, T.
(2001b). A functional rhodopsin-green fluorescent protein fusion
protein localizes correctly in transgenic Xenopus laevis retinal rods
and is expressed in a time-dependent pattern. J. Biol. Chem.
276,
28242–28251.
Papermaster, D.S., Schneider, B.G., and Besharse, J.C.
(1985). Vesicular transport of newly synthesized opsin from the
Golgi apparatus toward the rod outer segment. Ultrastructural
immunocytochemical and autoradiographic evidence in Xenopus retinas.
Invest. Ophthalmol. Vis. Sci.
26,
1386–1404.
Poetsch, A., Molday, L.L., and Molday, R.S. (2001).
The cGMP-gated channel and related glutamic acid-rich proteins interact with
peripherin-2 at the rim region of rod photoreceptor disc membranes. J.
Biol. Chem. 276,
48009–48016.
Saga, M., Mashima, Y., Akeo, K., Oguchi, Y., Kudoh, J., and Shimizu, N. (1993). A novel Cys-214-Ser mutation in the peripherin/RDS gene in a Japanese family with autosomal dominant retinitis pigmentosa. Hum. Genet. 92, 519–521.[CrossRef][Medline]
Saliba, R.S., Munro, P.M., Luthert, P.J., and Cheetham, M.E.
(2002). The cellular fate of mutant rhodopsin: quality control,
degradation and aggresome formation. J. Cell Sci.
115,
2907–2918.
Sanyal, S., and Jansen., H.G. (1981). Absence of receptor outer segments in the retina of rds mutant mice. Neurosci. Lett. 21, 23–26.[CrossRef][Medline]
Seigneuret, M., Delaguillaumie, A., Lagaudriere-Gesbert, C., and
Conjeaud, H. (2001). Structure of the tetraspanin main
extracellular domain. A partially conserved fold with a structurally variable
domain insertion. J. Biol. Chem.
276,
40055–40064.
Sung, C.H., Schneider, B.G., Agarwal, N., Papermaster, D.S., and
Nathans, J. (1991). Functional heterogeneity of mutant rhodopsins
responsible for autosomal dominant retinitis pigmentosa. Proc. Natl.
Acad. Sci. USA 88,
8840–8844.
Tam, B.M., Moritz, O.L., Hurd, L.B., and Papermaster, D.S.
(2000). Identification of an outer segment targeting signal in
the COOH terminus of rhodopsin using transgenic Xenopus laevis. J. Cell
Biol. 151,
1369–1380.
Travis, G.H., Brennan, M.B., Danielson, P.E., Kozak, C.A., and Sutcliffe, J.G. (1989). Identification of a photoreceptor-specific mRNA encoded by the gene responsible for retinal degeneration slow (rds). Nature 338, 70–73.[CrossRef][Medline]
Travis, G.H., Groshan, K.R., Lloyd, M., and Bok, D. (1992). Complete rescue of photoreceptor dysplasia and degeneration in transgenic retinal degeneration slow (rds) mice. Neuron 9, 113–119.[CrossRef]
