A Rhodopsin Mutant Linked to Autosomal Dominant RetinitisPigmentosa Is Prone to Aggregate and Interacts with theUbiquitin Proteasome System*

Received for publication, May 20, 2002, and in revised form, June 26, 2002Published, JBC Papers in Press, June 28, 2002, DOI 10.1074/jbc.M204955200

Michelle E. Illing‡§¶, Rahul S. Rajan§�, Neil F. Bence‡**, and Ron R. Kopito‡ ‡‡

From the Departments of ‡Biological Sciences and �Chemistry, Stanford University, Stanford, California 94305

The inherited retinal degenerations are typified byretinitis pigmentosa (RP), a heterogeneous group of in-herited disorders that causes the destruction of photo-receptor cells, the retinal pigmented epithelium, andchoroid. This group of blinding conditions affects over1.5 million persons worldwide. Approximately 30–40% ofhuman autosomal dominant (AD) RP is caused by dom-inantly inherited missense mutations in the rhodopsingene. Here we show that P23H, the most frequent RPmutation in American patients, renders rhodopsin ex-tremely prone to form high molecular weight oligomericspecies in the cytoplasm of transfected cells. AggregatedP23H accumulates in aggresomes, which are pericent-riolar inclusion bodies that require an intact microtu-bule cytoskeleton to form. Using fluorescence resonanceenergy transfer (FRET), we observe that P23H aggre-gates in the cytoplasm even at extremely low expressionlevels. Our data show that the P23H mutation destabi-lizes the protein and targets it for degradation by theubiquitin proteasome system. P23H is stabilized by pro-teasome inhibitors and by co-expression of a dominantnegative form of ubiquitin. We show that expression ofP23H, but not wild-type rhodopsin, results in a general-ized impairment of the ubiquitin proteasome system,suggesting a mechanism for photoreceptor degenera-tion that links RP to a broad class of neurodegenerativediseases.

Retinitis pigmentosa (RP)1 is a heterogeneous group of in-herited diseases that cause blindness due to progressive degen-

eration of rod and cone photoreceptors in the human retina(reviewed in Ref. 1). Approximately 43% of RP is inherited asautosomal dominant (AD) alleles of genes that are expressedpredominantly or exclusively in rod photoreceptors. The reces-sive forms of RP are primarily the result of null mutationseliminating enzymes critical to photoreceptor function. Morethan 100 distinct mutations in the rhodopsin gene have beendocumented, accounting for 30–40% of ADRP. The most com-mon abnormality found to cause RP (2) is a single-base substi-tution in codon 23 (P23H) of the rhodopsin gene, accounting for�7% of all cases of dominant retinitis pigmentosa in the UnitedStates (3, 4).

Although the signal transduction cascades leading to caspaseactivation in RP are unknown (reviewed in Ref. 5), studies inrats and mice expressing rhodopsin transgenes with mutationsthat are equivalent to those known to cause ADRP in humanssuggest that photoreceptor loss is due to apoptotic cell death(6–8). Humans (9) and mice (10, 11) with a single null rhodop-sin allele exhibit minimal photoreceptor degeneration, suggest-ing that ADRP is not the result of haploinsufficiency at therhodopsin locus.

Studies of mutant rhodopsin molecules expressed heterolo-gously in mammalian cells in culture have suggested thatADRP-linked rhodopsin mutations fall into two classes basedon the differing intracellular fates of the mutant proteins.Class I mutants are expressed at wild-type levels in HEK293 orCOS cells, where they traffic to the plasma membrane andproduce functional photopigment when reconstituted with 11-cis-retinal, suggesting that they are correctly folded (12–14).These mutants, the majority of which are strikingly clusteredwithin the carboxyl-terminal cytoplasmic domain of rhodopsin,appear to be defective in their ability to traffic correctly to rodouter segments (15, 16), possibly because of defective interac-tion with the retrograde microtubule motor cytoplasmic dynein(17). By contrast, the vast majority of ADRP-causing rhodopsinmutations are class II mutants, which are defective in theirability to fold (18, 19). How rhodopsin misfolding causes ADRPis unknown, although misfolding has recently been suggestedto be closely linked to the gain-of-function that underlies dis-ease pathogenesis (19).

When expressed in HEK293 or COS cells, class II mutantsfail to acquire complex oligosaccharides indicative of transitthrough the Golgi apparatus and are severely defective orunable to produce functional photopigment on reconstitutionwith 11-cis-retinal (12, 14). These findings strongly support theconclusion that class II mutants are unable to fold correctlywithin the endoplasmic reticulum. This conclusion is also sup-ported by the observation that class II mutants fail to accumu-late to high levels in transfected cells (as wild-type rhodopsin),suggesting that they are subject to enhanced intracellular deg-

* This work was supported in part by Grants 1R01-DK43994 and1R01-DK52795 from the National Institutes of Health (to R. R. K.). Thecosts of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “adver-tisement” in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

§ Both authors contributed equally to this work.¶ Supported by a postdoctoral fellowship from the Canadian Insti-

tutes of Health Research. Present address: Ingenuity Systems, 2160Gold St., Alviso, CA 95002.

** Supported by a predoctoral training grant from National Institutesof Health.

‡‡ To whom correspondence should be addressed. Tel.: 650-723-7581;Fax: 650-723-8475; E-mail: kopito@stanford.edu.

1 The abbreviations used are: RP, retinitis pigmentosa; ADRP, auto-somal dominant RP; Ub, ubiquitin; FRET, fluorescence resonance en-ergy transfer; GFP, green fluorescent protein; YFP, yellow fluorescentprotein; CFP, cyan fluorescent protein; PBS, phosphate-buffered saline;PNGase, protein:N-glycanase; ALLN, N-acetyl-Leu-Leu-norleucinal;DAPI, 4�,6-diamidino-2-phenyl-indole; FITC, fluorescein isothiocya-nate; UPS, ubiquitin-proteasome system; ER, endoplasmic reticulum;CFTR, cystic fibrosis transmembrane conductance regulator; FALS,familial amyotrophic lateral sclerosis; SOD, superoxide dismutase;mAb, monoclonal antibody.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 37, Issue of September 13, pp. 34150–34160, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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radation (12). The mechanism by which class II mutants aredegraded, however, is unknown.

Despite an extensive body of data, the mechanisms by whichrhodopsin mutations initiate the signaling events leading tophotoreceptor death and retinal degeneration remain a mys-tery. The dominant inheritance pattern, together with the lackof a haploinsufficient phenotype, imply that that rhodopsin-linked ADRP results from a toxic gain of function at the rho-dopsin locus. However, the only phenotypic consequence to beattributed to class II mutations in rhodopsin, an inability tofold correctly, is a loss of function.

The discovery that most class II mutants are folding-defec-tive places ADRP within a family of conformational diseasesthat includes most neurodegenerative disorders (20, 21). LikeADRP, the familial forms of these diseases are usually inher-ited as delayed onset, highly penetrant, dominant traits. Insome cases, pathogenesis has been linked to enhanced suscep-tibility of the mutant gene product to degradation by the ubiq-uitin proteasome system (22, 23). Degenerating neurons inthese diseases accumulate high molecular weight forms of mu-tant gene product that are usually segregated within intracel-lular inclusion bodies that are often heavily modified withubiquitin (Ub) (24–26). By contrast, inclusion bodies and Ubimmunoreactivity have not been reported to be prominent his-topathological features of degenerating photoreceptors intransgenic animals or in retinas from human RP patients inearly stages of the disease (27). Thus, although RP and othercentral nervous system neurodegenerations appear to share acommon etiology, insofar as they are all linked to the produc-tion of abundantly expressed, folding-defective polypeptides,they differ in that overt inclusion bodies have not been reportedin RP. A simple explanation for this difference might be thatthe retina may be more effective at eliminating misfolded pro-teins or preventing the accumulation of aggregated proteinproducts than other central nervous system tissues.

In this study, we have investigated the role of the ubiquitin-proteasome system in the disposal of RP-linked mutant rho-dopsin. Our data reveal that the class II rhodopsin mutant,P23H, is a substrate for Ub-dependent degradation by theproteasome. We find that high molecular weight, ubiquitiny-lated forms of P23H accumulate when expressed in culturedcells. We have used fluorescence resonance energy transfer(FRET) to demonstrate that the aggregation of P23H is intra-cellular, and furthermore, occurs even at very low expressionlevels, unlike wild-type rhodopsin and the class I mutant,V345M. Finally, our data indicate that aggregation of P23H incells leads to impairment of the function of the ubiquitin pro-teasome system, as has recently been demonstrated for otheraggregated proteins, including the mutant form of huntingtinlinked to Huntington’s disease (28). Therefore, rhodopsin ag-gregation may represent a toxic gain of function associatedwith class II mutations in the pathogenesis of ADRP, raisingthe possibility that retinal degeneration may share a commonpathogenic mechanism with other late-onset neurodegenera-tive diseases of the central nervous system.

EXPERIMENTAL PROCEDURES

Antibodies and Plasmids—The following antibodies were used in thisstudy: Rho1D4 (29) (gift from R. Molday, University of British Colum-bia), B6–30 (gift from P. Hargrave, University of Florida), anti-His6

(MMS-156P, BAbCO/Covance Research Products, Denver, PA), mono-clonal �-tubulin (GTU-88, Sigma Chemical Co., St. Louis, MO). Thec-myc-Ub plasmids have been described previously (30). Wild-type andP23H rhodopsin in pRK were obtained from J. Nathans (Johns HopkinsUniversity). V345M was made by site-directed mutagenesis usingQuikChange (Stratagene, La Jolla, CA). YFP and CFP rhodopsin andP23H plasmids have been previously described (31).

Cell Culture—Human embryonic kidney 293 (HEK) cells were main-tained in Dulbecco’s modified Eagle’s medium and transfected as de-

scribed previously (30). Transient transfections were carried out byadding plasmid DNA as a calcium phosphate precipitate (32).

Biochemical Characterization of Rhodopsin Aggregates—Cell pelletsfrom transfected and washed HEK cells were lysed in 250 �l of ice-coldbuffer A (PBS, pH 7.5, 5 mM EDTA, 1% TX-100) plus protease inhibitormixture (Roche Molecular Biochemicals) for 30 min on ice. Insolublematerial was recovered by centrifugation at 13,000 � g for 15 min andsolubilized in 50 �l (PBS, 1% SDS) for 10 min at room temperature.After addition of 200 �l of buffer A, samples were sonicated for 20 s witha microtip sonicator.

For immunopurification of the Ub-rhodopsin complexes, 150 �l of thedetergent-soluble fraction from His6-myc-Ub-rhodopsin co-transfectedcells was incubated with 5 �l of 1D4 Sepharose beads (a kind gift of D.Oprian, Brandeis University) at 4 °C for 1 h. The beads were washedextensively with cold buffer A, and protein was eluted into 20 �l of SDSsample buffer �-mercaptoethanol).

Endoglycosidase H and PNGase (Roche Molecular Biochemicals,Mannheim, Germany) digestion was performed on detergent-solubleand insoluble fractions for 1 h at 37 °C in a 10:1 dilution with the buffersupplied by the manufacturer.

For immunoblotting, cell fractions were separated on Ready Gelprecast 4–20% gradient SDS-PAGE (Bio-Rad, Hercules, CA) and elec-troblotted to Immobilon (Millipore, Bedford, MA) membranes. Chemi-luminescence detection was carried out with the ECL detection kit(Amersham Biosciences, Piscataway, NJ). Band intensities were quan-tified from non-saturated exposures using IMAGE software (NationalInstitutes of Health).

Fluorescence Microscopy—Cells were seeded onto #1 coverslips. Fordrug treatments, ALLN (5–10 mg/ml, Calbiochem, La Jolla, CA) andnocodazole (10 mg/ml, Sigma Chemical Co.) in Me2SO were added to theculture medium 12 h before fixation. Cells were fixed in �20 °C meth-anol or 4% paraformaldehyde (20 min). After fixation, cells werewashed extensively in PBS and blocked with 10% bovine serum albu-min for 10 min and then incubated with primary antibodies for 60 minat room temperature. Cells were washed 5� for 5 min each in PBS andincubated with fluorophore-conjugated secondary antibodies at a 10�g/ml final concentration. Cells were washed again (5� 5 min each) andthen incubated for 3 min in PBS plus 10 �g/ml bisbenzimide (SigmaChemical Co.) to stain DNA. Cells were washed a final time, mountedonto slides in Fluoromount-G (Electron Microscopy Sciences, FortWashington, PA). For C/YFP fusion proteins, the cells were fixed in 4%paraformaldehyde. Epifluorescence micrographs were obtained on aZeiss Axiovert microscope with a 63� oil objective (numerical aperture,1.4). Digital (12-bit) images were acquired with a cooled charge-coupleddevice (Princeton Instruments, Trenton, NJ) and processed by usingMetamorph software (Universal Imaging, Media, PA). The excitationfilters used were: 355DF20 (DAPI), 440 DF20 (CFP), 490DF10 (YFPand FITC), and 570DF10 (Texas red). Emission filters were: 460DF20(DAPI), 475 DF20 (CFP), 535DF25 (FITC and YFP), and 630 DF20(Texas red). The dichroic measurements were: 420 DCLP (DAPI),445DCLP (CFP), 505 DCLP (FITC and YFP), and 595DCLP (Texasred).

FRET Measurements—FRET was determined as previously de-scribed (31). Fluorescence spectra were recorded on suspended cells(�106 cells/ml) in a Spex Fluorolog fluorometer with a Spex 1620 dualgrating emission monochromator (Spex Industries, Metuchen, NJ).FRET measurements were made by exciting the donor (CFP) at 425 nmand monitoring the emission spectrum between 450 and 600 nm. AllFRET spectra were corrected for background YFP fluorescence using anon-FRET pair consisting of identical amounts of YFP fusion constructand unlabeled rhodopsin (wild-type or mutant). To quantify FRET, theratio of fluorescence at 525 nm (YFP) to the fluorescence at 476 nm(CFP) was measured. For P23H-CFP alone, this ratio was found to be0.42 � 0.013 (S.E., n � 5). This value was subtracted from the ratioobtained for any given sample to yield the “FRET value.” Intrinsic YFPfluorescence was recorded by excitation at 490 nm and emission be-tween 505 and 600 nm. Slit widths were 2 mm for all experiments.

Ubiquitin Proteasome System Activity—GFPu-1 cells (28) were tran-siently transfected using the calcium phosphate method. Cells wereharvested by trypsinization 48 h post-transfection and fixed in 4%p-formaldehyde in PBS for 20 min at room temperature. Cells werepermeabilized in a solution of PBS, 0.1% Triton X-100, and 2% bovineserum albumin and stained in suspension using the indicated antibodyat room temperature for 1 h followed by phycoerythrin-conjugatedsecondary antibody (1:500) for 1 h at room temperature. GFP andphycoerythrin intensities were simultaneously measured on 50,000cells for each sample using an XLII analyzer (Coulter). Histogramswere generated using FlowJo software (Tree Star, Inc.).

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RESULTS

P23H Rhodopsin Is Prone to Aggregate—Aggregation of wild-type rhodopsin and the RP-linked mutants P23H and V345Mwas assessed by SDS-PAGE immunoblot analysis of detergent-soluble and detergent-insoluble extracts from HEK293 cellstransiently expressing these constructs at varying levels (Fig.1A). At low expression levels (�0.5 �g), wild-type rhodopsinmigrated predominantly as a detergent-soluble, diffuse band at

a molecular mass of �40–43 kDa. This species corresponds tomonomeric, mature rhodopsin containing complex N-linkedglycans, as evidenced by its mobility and by its sensitivity tocleavage by protein:N-glycanase (PNGase) but not by endogly-cosidase H (Fig. 1B). Endoglycosidase H is specific for highmannose N-linked oligosaccharide structures typical of pro-teins that have not matured beyond the endoplasmic reticu-lum, whereas PNGase cleaves all N-linked glycans. At higher

FIG. 1. High molecular weight complexes and intracellular processing of ADRP-linked rhodopsin mutants in HEK293 cells. A,formation of high molecular weight oligomers is a function of expression and genotype. Cells were transiently transfected with the indicatedamount of plasmid-encoding wild-type (WT) rhodopsin or the ADRP mutants P23H (class II) or V345M (class I), separated into detergent soluble(upper panels) or insoluble fractions (lower panels), and subjected to immunoblot analysis with a rhodopsin (1D4) mAb. Control cells (lane v) weretransfected with vector alone. Some cells were incubated in the presence of the proteasome inhibitor ALLN as indicated. B, glycosylation statusof mutant and wild-type rhodopsin species. Detergent-soluble and -insoluble fractions of lysates from HEK293 cells transfected with 1 �g ofplasmid were subjected to digestion with endoglycosidases H or PNGase, as indicated, prior to immunoblot analysis. The data in this analysis arerepresentative of at least three independent experiments.

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expression levels of wild-type rhodopsin (Fig. 1A), additionalhigh molecular weight species, suggestive of SDS-resistantmultimers, were also detected. All of the monomeric rhodopsin

partitioned into the detergent-soluble fraction, whereas theslower migrating forms partitioned into both detergent-solubleand -insoluble fractions. These data, together with the results

FIG. 2. Aggregated rhodopsin accumulates in aggresomes. A, intracellular distribution of wild-type, V345M, and P23H in HEK293 cells.Low levels of each plasmid (0.5 �g) were transfected into cells, and immunostained for rhodopsin with monoclonal antibody B6–30 (red) and forDNA with bis-benzimide (blue). B, effect of proteasome inhibition and microtubule disruption on localization of P23H rhodopsin. Cells weretransfected with low levels (0.5 �g) of rhodopsin plasmid and treated with the drugs indicated and immunolocalized for rhodopsin (red pseudocolor)and DNA (blue). C, P23H aggresomes are pericentriolar. Cells were transfected with 8 �g of P23H in the absence of drugs and immunostained withantibodies against rhodopsin (red) and �-tubulin (green). Bar, 10 �m. D, microtubules are not required for rhodopsin aggregation. Cells weretransfected and treated overnight with ALLN and nocodazole as indicated and subjected to detergent solubilization and immunoblotting withanti-rhodopsin mAb (1D4).

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FIG. 3. Rhodopsin expression and spontaneous inclusion body formation in the absence of proteasome inhibitors. A–C, fusion ofYFP to rhodopsin does not alter its trafficking. HEK cells were transfected with 0.5 �g of wild-type-YFP (A), V345M-YFP (B), or P23H-YFP (C)and fixed 48 h later, and visualized for YFP (green). DNA was stained (blue) with bis-benzimide. The arrow in C indicates a spontaneous P23H

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from immunofluorescence studies (Figs. 2 and 3), are in agree-ment with previous studies (12), which concluded that themajority of rhodopsin, expressed in HEK293 cells, folds into anative conformation. At high levels of expression, a minorfraction of wild-type rhodopsin forms high molecular weightcomplexes that are insoluble in SDS and non-ionic detergent,suggesting that the capacity of these cells to fold rhodopsin islimited.

P23H differed markedly from wild-type rhodopsin both interms of mobility and detergent solubility. The small amountsof monomeric P23H detected migrated with a slightly fastermobility than that of monomeric wild-type rhodopsin and weresensitive to cleavage by endoglycosidase H (Fig. 1B), indicatingthat they were retained within the ER. The majority of P23H,however, migrated as dimers and higher molecular weightoligomers. These oligomers are evidence of a non-native con-formation, because rhodopsin is monomeric in its native mem-brane (33). A substantial fraction of P23H partitioned into thedetergent-insoluble fraction, suggesting that P23H is moreprone to aggregate than wild-type rhodopsin (Fig. 1). In con-trast to P23H, both the mobility and the detergent solubilitybehavior of the class I mutant, V345M, were indistinguishablefrom those of wild-type rhodopsin (Fig. 1, A and B).

Exposure of cells expressing low levels of wild-type or V345Mrhodopsin to the proteasome inhibitor (ALLN) resulted in asubstantial increase in the abundance of all electrophoreticrhodopsin species (Fig. 1A), suggesting that some folding-com-petent rhodopsin molecules are degraded by the proteasome inHEK cells. Indistinguishable results were obtained in cellsexposed to the more specific inhibitors MG132 (34) and lacta-cystin (35) (data not shown). In contrast, exposure of P23H-expressing cells to ALLN increased the proportion of oligomericP23H species but failed to promote the formation of maturemonomer, suggesting that proteasomes also participate in thedegradation of this folding-incompetent class II mutantrhodopsin.

Together, these data confirm that, in HEK cells, wild-typerhodopsin and a class I mutant (V345M) are able to fold andmature beyond the ER, whereas the class II mutant P23H isunable to fold productively. Moreover, our data suggest thatP23H is considerably more prone to forming non-native oli-gomers than is wild-type or V345M.

Misfolded Rhodopsin Accumulates in Aggresomes—We usedimmunofluorescence microscopy to determine the intracellularlocalization of wild-type and mutant rhodopsins. As shown inFig. 2A, wild-type and V345M were present in a predominantlyplasma membrane distribution, consistent with previous re-ports and with our observation (Fig. 1) that wild-type andV345M rhodopsin are detergent soluble and modified by com-plex oligosaccharides. In contrast, under the same conditions,P23H was present in a predominantly ER distribution (Fig.2A), consistent with its designation as a class II mutant andwith data indicating that it remains core-glycosylated (Fig. 1B).Exposure to ALLN (or other proteasome inhibitors, not shown)led to the accumulation of wild-type and mutant rhodopsins injuxtanuclear inclusion bodies resembling aggresomes (Fig. 2B).

Aggresomes are pericentriolar inclusion bodies into whichaggregated proteins are sequestered by active retrogradetransport on microtubules (26, 36). To test whether P23H-containing inclusion bodies are aggresomes, we compared the

distribution of P23H foci to that of �-tubulin, a centrosomalprotein (Fig. 2C). As observed for other aggresome substrates,P23H inclusions were localized around �-tubulin-immunoreac-tive sites. Rhodopsin inclusions were also associated with peri-centriolar depositions of rearranged vimentin intermediate fil-aments and were distinct from markers for Golgi apparatusand ER (data not shown).

To assess the role of microtubules in the formation of P23Hinclusion bodies, cells expressing low levels of P23H weretreated simultaneously with ALLN to induce accumulation ofaggregates and the microtubule-destabilizing agent, nocoda-zole (Fig. 2B, right-hand panel). In contrast to cells treatedwith ALLN alone (Fig. 2B, left panel), cells treated with ALLNand nocodazole together had numerous intense, small foci ofP23H that were diffusely distributed throughout the cyto-plasm. However, the extent of rhodopsin aggregation, as as-sessed by detergent solubility and electrophoretic mobility (Fig.2D) and by fluorescence resonance energy transfer (31), wasunaffected by nocodazole treatment. These data demonstratethat protein aggregation and inclusion body formation are dis-tinct, separable processes and that delivery of misfolded mu-tant or wild-type rhodopsin to cytoplasmic inclusion bodiesrequires an intact microtubule cytoskeleton. A direct inferencefrom this observation is that the absence of rhodopsin-contain-ing inclusion bodies, as in retinas from human ARDP andanimal models thereof, does not necessarily rule out a role forrhodopsin aggregation in pathogenesis.

Aggregation Is a Gain of Function Linked to the P23H Mu-tation—To elucidate the relationship between protein expres-sion and aggregation, we examined the cellular distributionand steady-state levels of P23H, V345M, and wild-type rhodop-sin in HEK cells transfected with varying levels of plasmid(Figs. 3 and 4). To study the intrinsic aggregation properties ofthese proteins, these studies were conducted in the absence ofany added proteasome inhibitors. We used fusion proteins be-tween the C terminus of wild-type, V345M, or P23H rhodopsinwith GFP or the spectrally shifted variants cyan (CFP) oryellow (YFP) fluorescent protein (31). As shown in Fig. 3 (A–C),the appended fluorescent proteins did not influence the intra-cellular distribution of the rhodopsins studied. Wild-type rho-dopsin and V345M were predominantly localized at the plasmamembrane, whereas the majority of P23H exhibited a diffuse,ER-like distribution. Cells expressing wild-type or V345M rho-dopsin fusions were 4- to 5-fold brighter than those expressingP23H, consistent with the suggestion (12–14) that the P23Hmutation destabilizes rhodopsin (Fig. 3E). At low expressionlevels (0.5 �g), inclusion bodies were found only in rare (�1%)cells expressing wild-type rhodopsin-YFP or V345M-YFP, butmore frequently (�3%) in cells transfected with equal amountsof P23H-YFP plasmid (Fig. 3D, left). At higher transfectionlevels, the fraction of cells with inclusion bodies increased forboth wild-type and P23H rhodopsin fusions (Fig. 3D, right).This reveals that rhodopsin inclusion bodies can form in theabsence of proteasome inhibitor. However, the increased tend-ency of P23H-YFP to aggregate (compared with wild-type) isdramatically illustrated when the fraction of cells with inclu-sion bodies is normalized to the total rhodopsin fluorescence.Total rhodopsin expression was quantified by monitoring theintrinsic CFP or YFP fluorescence, because the absolute fluo-rescence is not significantly influenced by subcellular localiza-

inclusion body. Exposure times have not been normalized. Bar, 15 �m. D, percentage of cells, transfected as indicated, with a microscopicallyobservable inclusion body. Each bar represents a mean of three independent transfections (n � 400 for each count). E, dose-response curves for thethree fusions. HEK cells were transfected with increasing amounts of plasmid DNA corresponding to wild-type YFP (open squares), V345M-YFP(open circles), or P23H-YFP (closed squares) and harvested 42–48 h later. YFP fluorescence was quantified by spectrofluorometry. F, inclusion bodyindex for the same set of transfections as in D, as determined by dividing the percentage of cells with inclusion bodies by the total rhodopsin(wild-type or mutant) level as assessed by YFP fluorescence.

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tion or non-linearity in the detection system. Total wild-typerhodopsin-YFP accumulation increased much more steeply as afunction of transfected DNA than did P23H-YFP (Fig. 3E). Incontrast, the steady-state levels of V345M-YFP and wild-type-YFP were indistinguishable. When the fraction of cells withinclusion bodies was normalized to total rhodopsin levels (“in-clusion body index” � percentage of cells with inclusion bodies/total rhodopsin) (Fig. 3F), it is apparent that P23H is far moreaggregation-prone than is wild-type rhodopsin.

We have recently reported that FRET is a highly sensitivemethod to monitor, in vivo, the aggregation of misfolded pro-teins (31). FRET results from the transfer of energy from afluorescent donor (CFP) in its excited state to another excitablemoiety, the acceptor (YFP), via non-radiative dipole-dipole in-teractions. To establish that P23H aggregation occurs intracel-lularly and is not an artifact of cellular lysis or proteasomalinhibition, we used FRET between rhodopsin C/YFP fusions tomonitor rhodopsin aggregation in living cells, in the absence ofdrugs. The fluorescence emission spectrum obtained from amixture of HEK cells singly transfected with either P23H-CFPor P23H-YFP revealed distinct peaks at 476 and 505 nm, cor-responding to CFP emission (Fig. 4A, solid line). In contrast,the spectrum of cells co-transfected with both plasmids re-vealed a reduction in the CFP emission peaks and an appear-ance of a shoulder at 525 nm, corresponding to the sensitizedemission from YFP as a result of FRET. We then monitoredFRET (using a FRET scale based on the ratio of fluorescenceemission at 525–476 nm (31)) in cells expressing wild-type,V345M, and P23H as a function of total rhodopsin expressionlevel. Total rhodopsin levels were quantified either by the

intrinsic CFP (Fig. 4B) or YFP (Fig. 4C) fluorescence emission.At low expression levels, FRET was undetectable in cells ex-pressing wild-type rhodopsin-C/YFP, despite a robust fluores-cence signal (Fig. 4B). The FRET value increased with increas-ing wild-type rhodopsin expression, reaching a plateau at alevel of �0.4. In contrast, in cells expressing P23H, FRET wasessentially maximal at the same plateau as the wild-type pro-tein, even at the lowest levels of expression. The aggregationbehavior of V345M, assessed using FRET (Fig. 4C), is similar tothat of wild-type rhodopsin.

Remarkably, the aggregation behavior of wild-type andP23H rhodopsin, as assessed by FRET, correlates extremelywell with the extent of association of these molecules intoSDS-insoluble non-native oligomers (Figs. 1A and 4D). To-gether these results confirm that P23H is more aggregation-prone than wild-type rhodopsin and further demonstrate thatthe propensity to aggregate is a gain of function attributable tothe P23H mutation.

Misfolded Rhodopsin Is Degraded by the Ubiquitin-Protea-some System—The increased steady-state level of mutant andwild-type rhodopsin induced by exposure of cells to proteasomeinhibitors (Fig. 1A) suggests a role for proteasome-dependentproteolysis in rhodopsin turnover. Because degradation of mis-folded integral membrane proteins from the ER requires afunctional Ub pathway (30, 37), we used two different ap-proaches to evaluate the role of Ub in the degradation of mu-tant and wild-type rhodopsin expressed in HEK cells (Fig. 5).In the first approach, we used immunopurification to show thatP23H is ubiquitinylated to a far greater extent than is wild-type rhodopsin. Cells were transfected with rhodopsin or P23H

FIG. 4. Aggregation is a gain offunction for P23H. A, fluorescence res-onance energy transfer was used to studyrhodopsin aggregation in the absence ofproteasome inhibitor. The solid line rep-resents cells singly transfected withP23H-CFP mixed with cells singly trans-fected with P23H-YFP. The dashed linerepresents cells that were co-transfectedwith P23H-CFP and P23H-YFP andmixed with mock transfected cells. Thespectra were corrected for cellularautofluorescence and background YFPfluorescence. B, relationship between ex-tent of wild-type (open squares) or P23H(closed squares) rhodopsin aggregation(determined by FRET) and total rhodop-sin expression (assessed by CFP fluores-cence). C, a comparison of FRET in wild-type, P23H, and V345M at low and highprotein expression levels (mean of n � 5observations). Protein expression wasquantified by intrinsic YFP fluorescenceand divided into two regimes of low (�5 �105 counts per second) and high (�106

counts per second) YFP fluorescence. Allexperiments had equal amounts of CFPand YFP plasmids. D, relationship be-tween the fraction of oligomeric wild-type(open squares) or P23H (closed squares)rhodopsin (determined from the immuno-blot in Fig. 1A) and DNA used fortransfection.

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plasmids, together with either a control plasmid (empty vector)or a plasmid encoding His6-myc-tagged Ub. Immunopurifiedrhodopsin, harvested from cell lysates using anti-rhodopsinantibody, was subjected to immunoblotting with anti-His6 an-tibody (Fig. 5A). The amount of His6-immunoreactive materialpresent in anti-rhodopsin immunoprecipitates from cells ex-pressing wild-type rhodopsin was not significantly higher inHis6-myc-Ub expressing than in vector-transfected controls(compare lanes 1 and 2). This indicates that, at the expressionlevel used, wild-type rhodopsin expressed in HEK cells is notdetectably modified with Ub. In sharp contrast, high molecularweight P23H Ub conjugates were readily detected even in theabsence of proteasome inhibitor, indicating the presence ofsteady-state P23H-Ub conjugates (lanes 3 versus 4). Followingtreatment of the co-transfected cells with proteasome inhibitor,we observed an increase in steady-state ubiquitinylation ofboth rhodopsin and P23H, consistent with our observation thatproteasome inhibitors also increase the steady-state level ofboth forms of rhodopsin, and supporting the conclusion thatturnover of both forms of rhodopsin is mediated by the Ub-proteasome system.

In the second approach, we tested the effect of dominantnegative Ub on the steady-state level of wild-type and mutant

rhodopsin. Protein degradation by the proteasome is stronglyenhanced by attachment of multiple Ub moieties in a polymerformed by isopeptide linkages between the carboxyl terminusof one Ub and the �-amino group of Lys-48 of another Ub(38–40). Mutations at Lys-48 can function as chain-terminat-ing dominant negative modulators of proteolysis (38). To con-firm a role for Ub in rhodopsin and P23H turnover, cells wereco-transfected with the respective rhodopsin plasmid togetherwith excess wild-type or K48R-Ub plasmid (or empty vectorcontrol). The steady-state levels of both wild-type and P23Hwere substantially increased by co-expression of K48R-Ubwhen compared with vector co-transfection. This strongly sup-ports the conclusion that the turnover of both forms is Ub-de-pendent (Fig. 5B). As with the proteasome inhibitors, K48R-Ubco-expression increased rhodopsin levels in both detergent-soluble and -insoluble fractions, indicating that undegradedrhodopsin molecules aggregate. Interestingly, co-expression ofwild-type Ub led to a reproducible and significant decrease insteady-state levels of the rhodopsin proteins, suggesting that,under these experimental conditions, the levels of free Ub maybe rate-limiting for degradation.

P23H Aggregation Leads to Impairment of the Ubiquitin-Proteasome Pathway—Recently we reported that cytoplasmicaggregation of a mutant form of cystic fibrosis transmembraneconductance regulator (F508-CFTR) and a mutant form ofhuntingtin (Q103) causes a decrease in the function of theubiquitin-proteasome system (UPS) (28). We developed a novelreporter (GFPu) of UPS activity consisting of GFP conjugated toa Ub-specific degron (28). Because GFPu is a rapidly degradedUPS substrate, impaired UPS function leads to increasedsteady-state GFPu concentration, which can be monitored as achange in mean fluorescence. To investigate the cellular con-sequences of P23H aggregation, we measured the fluorescenceof GFPu-1, an HEK293 line harboring stable expression ofGFPu, following transient expression of wild-type or mutantrhodopsin. As controls, GFPu-1 cells were transfected with highlevels of a fragment of wild-type huntingtin Q25, a solubleprotein that is very resistant to aggregation, or with F508CFTR, an integral membrane protein, which we have previ-ously shown to be extremely aggregation-prone (36). Trans-fected cells were immunostained for rhodopsin (or Q25 andCFTR), and the levels of the transfected protein and GFPu wereanalyzed simultaneously by two-color flow cytometry. GFPu

levels in the cells expressing low or high levels of transfectedprotein are represented as normalized population histograms(Fig. 6). The control experiments (Fig. 6, A and B) confirmedour previous finding that GFPu fluorescence was increased byexpression of F508 CFTR but not by expression of Q25. Sim-ilarly, there was no detectable increase in GFPu fluorescence incells expressing wild-type rhodopsin (Fig. 6C) even at levels upto 4 �g. However, expression of P23H resulted in a significantand reproducible increase in GFPu fluorescence that was evi-dent even at expression levels below those used for wild-type(Fig. 6D). Together, these data indicate that P23H, like otheraggregation-prone proteins, leads to a generalized impairmentof the ubiquitin proteasome system and suggests a mechanismby which mutations affecting the folding of rhodopsin might belinked to the biochemical events leading to photoreceptordeath.

DISCUSSION

Accumulation of aggregated, multiubiquitinylated proteinswithin intracellular inclusion bodies in affected neurons haslong been recognized as a hallmark of most neurodegenerativediseases, suggesting that these diverse disorders may be re-lated through a common pathogenic mechanism linked to pro-tein misfolding and aggregation. The presence of highly ele-

FIG. 5. Rhodopsin is a substrate of the ubiquitin proteasomesystem. A, undegraded P23H is ubiquitinylated. Cells were co-trans-fected with wild-type or mutant rhodopsin (4 �g) together with His6-myc-tagged Ub (4 �g) and treated overnight with Me2SO (control) orALLN as indicated. Cell lysates were immunoprecipitated with anti-body to rhodopsin and subjected to immunoblotting with anti-His6antibody. B, dominant negative Ub inhibits rhodopsin degradation.Cells were co-transfected with wild-type or mutant rhodopsin (4 �g)together with plasmid encoding His6-mycUb or His6-mycK48R-Ub orcontrol vector (4 �g) as indicated. Lysates were separated into deter-gent-soluble and -insoluble fractions and probed with anti-rhodopsinmAb.

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vated levels of multiubiquitinylated protein within inclusionbodies in affected neurons in most sporadic and inherited neu-rodegenerative diseases (24–26), together with recent biochem-ical (28) and genetic studies (41–45), suggests that dysfunctionof the ubiquitin proteasome pathway (UPS) is also intimatelytied to the underlying cellular pathogenesis. The data pre-sented in this study reveal that a mutation linked to ADRP, aleading cause of adult onset blindness, results in the produc-

tion of a misfolded and highly aggregation-prone form of rho-dopsin that in cultured cells is both a substrate and an inhib-itor of the UPS.

These data suggest that UPS impairment may contribute toADRP pathogenesis and, therefore, that retinal degenerationmay share hitherto unsuspected common pathogenic featureswith other adult onset degenerative diseases of the centralnervous system. Class II mutants like P23H fail to acquire

FIG. 6. Aggregation of P23H rhodopsin impairs the function of the Ub-proteasome system. GFPu-1 cells (harboring the GFPu UPSreporter) were transfected with 4 �g of plasmid encoding huntingtin Q25-myc (A), F508 CFTR (B), wild-type rhodopsin (C), or P23H (D).Transfected cells were fixed, immunostained with c-myc antibody (A), CFTR antibody (B), or rhodopsin (1D4) mAb (C and D) and analyzedsimultaneously by flow cytometry for expression of the respective antigens and GFPu fluorescence. Traces shown are histograms (normalized tototal cell number) of GFPu fluorescence in cells containing high (dashed line) or low (solid line) levels of each transgene.

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complex oligosaccharides and are not able to bind 11-cis-retinalto form a rhodopsin chromophore, suggesting that class IIADRP, like many other dominantly inherited neurodegenera-tive diseases, is associated with defective protein folding (12–14). Our finding that P23H is ubiquitinylated and is a sub-strate for the UPS, reveals a second feature common to ADRPand other neurodegenerative diseases such as familial amyo-trophic lateral sclerosis (FALS), in which disease-causing mu-tations enhance the susceptibility of the affected protein (cop-per and zinc superoxide dismutase (SOD)) to degradation bythe UPS (23, 46, 47). Finally, the data in the present studyshow that the P23H mutation renders rhodopsin highly proneto aggregation, revealing a third feature common betweenADRP and nearly all other neurodegenerative diseases.

In this study, three lines of evidence, formation of highmolecular weight SDS-resistant oligomers, enhanced seques-tration of P23H in aggresomes and FRET, all support theconclusion that P23H is significantly more prone to aggrega-tion than is wild-type rhodopsin. Strikingly, unlike otheraggregation-prone proteins, like SOD, CFTR, and mutant hun-tingtin, in which we find a mixture of aggregated and non-aggregated forms at steady-state, P23H is remarkable in thataggregates are the predominant form detectable in cells at allexpression levels. Whether this property results from the ex-tremely hydrophobic nature of rhodopsin or an increased pro-pensity to form �-sheet structures, which underlie the aggre-gation of many other proteins (48), will require furtherinvestigation.

It is essential to distinguish between aggregates, which canbe defined as non-native protein oligomers, and inclusion bod-ies, which are microscopically distinct cellular regions intowhich aggregated proteins are sequestered (26). For example,formation of inclusion bodies composed of aggregated mutantSOD is a relatively late event in the progression of spinal motorneuron disease in a transgenic mouse model of FALS, occurringseveral months after the onset of detectable cellular pathology(46, 49). In contrast to inclusion bodies, formation of biochem-ically distinct, detergent-insoluble, SOD oligomers and highmolecular weight aggregates precedes by several months theappearance of inclusion bodies and coincides with the earliestmanifestations of cellular pathology (23). These findings sug-gest that it is the adoption of a non-native oligomeric confor-mation, i.e. aggregation, and not inclusion body formation, thatunderlies FALS and perhaps other aggregation-linked neuro-degenerative diseases. The oligomeric forms of rhodopsin,much like those of SOD, are non-native structures, given thefact that the wild-type is monomeric (33). Thus, they fit ourdefinition of aggregates, being “non-native oligomers.”

One possible explanation for the apparent absence of inclu-sion bodies in photoreceptors from degenerating retina is thatphotoreceptors may be more susceptible to the toxicity of pro-tein aggregates than are other central nervous system neuronsor transfected HEK cells. Microtubules in photoreceptors areorganized around the basal bodies within the specialized non-motile connecting cilium at the junction between the lightsensitive outer segment and the metabolically and biosyntheti-cally active inner segment (50, 51). Perhaps this highly special-ized microtubule array organization limits the capacity ofphotoreceptors to clear protein aggregates by the type ofdynein-mediated processes, which operate in non-ciliated cellsto concentrate aggregated proteins in pericentriolar inclusionbodies (26). Lacking such a mechanism to sequester toxic P23Haggregates into inclusion bodies could lower the threshold foraggregate toxicity and cause photoreceptors to die without theformation of readily detectable foci.

It is also possible that the absence of detectable inclusion

bodies within photoreceptors in ADRP reflects an increasedcapacity of the retina to eliminate damaged cells comparedwith other central nervous system regions. Lysis of the photo-receptor inner segment is a prominent and early feature ofADRP pathogenesis, suggesting that cells with the highestaggregate burden sufficient to produce detectable inclusionbodies may be eliminated preferentially. Because of the lami-nar organization of the retina and the close proximity of theretinal pigmented epithelium, a layer of highly phagocytic cellscontacting the photoreceptor outer segments, it is likely thatremoval of moribund neurons or fragments thereof is moreefficient in retina than in other regions of the brain. In Dro-sophila, mutations affecting rhodopsin folding also cause pho-toreceptor degeneration (52). However, in flies, which lack theequivalent of a retinal pigmented epithelium, expression offolding-defective rhodopsin mutants does lead to the formationof cytoplasmic inclusion bodies and a generalized disruption ofinternal membranes (53). These observations confirm that ag-gregation into cytoplasmic inclusion bodies is a property intrin-sic to folding-defective mutant forms of rhodopsin and suggestthat the mammalian retina may possess specialized mecha-nisms to prevent their accumulation. Our observation thatP23H-expressing cells that have negligible inclusion bodies(�3%) still have maximal FRET ties in very well with the lackof observable inclusion bodies in ADRP. It suggests that aggre-gates, and their potential toxicity, can persist in the absence ofobservable inclusion bodies.

Although it is formally possible that the high molecularweight rhodopsin and P23H species observed in this study arepost-lysis artifacts generated during sample preparation, threelines of evidence argue strongly that these species are gener-ated intracellularly. First, the observation of aggresomes incells expressing P23H strongly indicates that P23H aggrega-tion occurs in vivo. Aggresome formation by P23H does notrequire proteasome inhibitors, because they are observed incells in the absence of any drug treatment. Second, becauseFRET is efficient only for C/YFP fluorophores that are within100 Å of each other (54), our data suggest that the P23Hmutation endows the rhodopsin molecule, normally a monomereven at the high concentrations present within the rod outersegments (33), with the ability to self-associate into compactstructures. Finally, we observe identical patterns of electro-phoretic motilities of P23H rhodopsin when the cells are lysedin the presence of alkylating agents such as iodoacetamide,which prevent the formation of non-native disulfide-linked spe-cies in the highly oxidizing environment of SDS-PAGE (55).

The data presented here demonstrate that an increased pro-pensity to self-associate into high molecular weight aggregatesis a robust gain of function, which is directly linked to patho-genesis caused by a class II ADRP mutant rhodopsin. One wayin which this property might be cytotoxic is by interfering withthe function of the UPS, a proteolytic system that plays acrucial role as a cellular “master regulator” in all eukaryoticcells (56). Whether or not rhodopsin aggregation and UPSimpairment are general features of other class II mutants mustawait future studies. Nevertheless, our data suggest a novellink between ADRP and other hereditary neuropathies andsuggest that the consequences of protein aggregation may havea more general role in neurodegeneration than hithertoanticipated.

Acknowledgments—We thank J. Flannery and E. Lee for usefuldiscussions and R. Molday, J. Nathans, D. Oprian, and P. Hargrave forreagents. We are also grateful to J. Johnston and members of the Kopitolaboratory for helpful discussions.

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Michelle E. Illing, Rahul S. Rajan, Neil F. Bence and Ron R. Kopitoto Aggregate and Interacts with the Ubiquitin Proteasome System

A Rhodopsin Mutant Linked to Autosomal Dominant Retinitis Pigmentosa Is Prone

doi: 10.1074/jbc.M204955200 originally published online June 28, 20022002, 277:34150-34160.J. Biol. Chem. 

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