Molecular Vision 2005; 11:665-676 <>
Received 15 June 2005 | Accepted 19 July 2005 | Published 30 August 2005

Stargardt-like macular dystrophy protein ELOVL4 exerts a dominant negative effect by recruiting wild-type protein into aggresomes

Vidyullatha Vasireddy,1 Camasamudram Vijayasarathy,2 Jibiao Huang,1 Xiaofei F. Wang,3 Monica M. Jablonski,3 Howard R. Petty,1 Paul A. Sieving,2,4 Radha Ayyagari1

1Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, MI; 2National Eye Institute and 4National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD; 3University of Tennessee Health Science Center, Hamilton Eye Institute, Memphis, TN

Correspondence to: Radha Ayyagari, PhD, Department of Ophthalmology and Visual Sciences, W. K. Kellogg Eye Center, The University of Michigan, 1000 Wall Street, Room number 325, Ann Arbor, MI, 48105; Phone: (734) 647-6345; FAX: (734) 936-7321; email:


Purpose: Mutations in the gene Elongation of very long-chain fatty acids-4 (ELOVL4) have been shown to be associated with autosomal dominant Stargardt-like macular dystrophy (STGD3). ELOVL4 is expressed in photoreceptors and encodes a putative transmembrane protein of 314 amino acids with an endoplasmic reticulum (ER) retention signal. A 5 bp deletion in exon 6 of ELOVL4 observed in some STGD3 patients results in the truncation of the protein and loss of the ER retention signal. To understand the disease mechanism underlying STGD3 we studied the intracellular trafficking of the wild-type and a 5 bp deletion mutant of ELOVL4.

Methods: Wild-type and mutant ELOVL4 proteins with the N-terminal GFP/V5 tags were expressed in COS-7 cells. Expression and the intracellular localization of the wild-type and mutant proteins were characterized by immunocytochemistry and western blot analysis using tag- and organelle-specific antibodies. Interaction between the wild-type and mutant proteins was studied by two-dimensional gel electrophoresis and fluorescence resonance energy transfer (FRET) analysis.

Results: The mutant ELOVL4 protein exerted a dominant negative effect when the wild-type and 5 bp deletion mutant ELOVL4 proteins were co-expressed in COS-7 cells. Immunocytochemical analysis, two-dimensional gel electrophoresis and FRET revealed that the mutant ELOVL4 interacts with the wild-type protein, forming higher molecular mass complexes that accumulate in aggresomes.

Conclusions: In the presence of mutant ELOVL4 protein, the wild-type protein was recruited into perinuclear cytoplasmic inclusions that resemble aggresomes. The interaction between the wild-type and mutant forms of ELOVL4 and the resultant alteration in the trafficking of the wild-type ELOVL4 protein suggest a mechanism for the pathogenicity observed in patients with autosomal dominant STGD3.


Stargardt-like autosomal dominant macular dystrophy is characterized by progressive macular degeneration leading to central macular and peripapillary retinal pigment epithelium (RPE) atrophy and accumulation of flecks. The onset of the disease is during the teenage years [1,2]. For these macular degenerations, two loci, one on chromosome 6q (STGD3) and the other on chromosome 13 (STGD2), have been mapped. Mutations in the elongation of very long chain fatty acids-4 gene (ELOVL4) have been identified in families linked to the locus on the 6q [3,4]. The ELOVL4 protein has sequence and structural similarities with the ELO family of proteins described in yeast [5]. ELOVL4 encodes a protein with 314 aminoacids with a dilysine motif (KXKXX) at the C-terminus that is thought to signal ER retention. Three different mutations have been identified in the ELOVL4 gene associated with Stargardt-like macular degeneration: (1) a 5 bp deletion mutation in exon 6 of ELOVL4, which causes a frameshift leading to the loss of 51 amino acids; (2) a two-base-deletion, which also results in the loss of 51 terminal amino acids; and (3) a nonsense mutation at codon 270. All three mutations result in premature termination of protein translation and loss of the ER retention signal [3,4,6]. We previously demonstrated that the dilysine ER-retention signal at the C-terminal end of the ELOVL4 protein is functional, and its loss results in misrouting of the protein [7]. However, the mechanism underlying the degeneration process due to the mutations in the ELOVL4 gene is not known. Mutations present in the heterozygous state could cause disease either due to haploinsufficiency or a dominant negative effect exerted by the mutant protein. Expression of the ELOVL4 mutant protein in COS-7 cells indicated that the mutant protein is localized to cytosolic juxtanuclear compartments [7]. Several neurodegenerative diseases, including retinitis pigmentosa due to mutations in the rhodopsin gene, are found to result from the dominant negative effect of the misfolded mutant proteins [8-10]. In order to evaluate whether the mutant ELOVL4 exerts similar dominant negative effect, we studied the cellular fate of the wild-type and 5 bp deletion mutant ELOVL4 proteins following their co-expression in COS-7 cells. Our studies suggest that the mutant protein interacted with the wild-type protein to form higher molecular weight aggregates. These aggregates were localized to perinuclear inclusions that have the characteristic features of aggresomes. Interaction of the wild-type and mutant proteins, and the alteration in their cellular trafficking may explain the molecular pathology underlying the degeneration process due to mutations in the ELOVL4 gene.


Reagents and antibodies

The following antibodies and chemicals were used in this study. Antibodies: mouse monoclonal anti-vimentin (Sigma-Aldrich, Inc., St. Louis, MO, dilution 1:100), mouse monoclonal anti-γ-tubulin (Sigma-Aldrich, dilution 1:500), rabbit polyclonal anti-V5 (Sigma-Aldrich, dilution 2 μg/ml), mouse monoclonal anti-V5 (Invitrogen Corporation, Carlsbad, CA, dilution 1:500), mouse monoclonal anti-GFP (Covance, Berkeley, CA, dilution 1:10,000), mouse monoclonal anti-golgin-97 (Molecular Probes, Eugene, OR, concentration 1 μg/ml), mouse monoclonal anti-trans Golgi network-38 (TGN-38, Abcam, MA, dilution 1:1000).

Secondary antibodies: Alexa fluor-555 conjugated goat anti-mouse IgG (Molecular probes, Eugene, OR, concentration 8 μg/ml), mouse monoclonal anti-rabbit IgG (Pierce Biotechnology, Rockford, IL, dilution 1:7500), goat monolclonal anti-mouse IgG (Pierce Biotechnology, IL, dilution 1:7500). Chemicals: proteasome inhibitor MG-132 and nocodozole were purchased from Calbiochem, San Diego, CA. Fetal bovine serum was obtained from ATCC (Manassas, VA) and the other chemicals were obtained form Sigma-Aldrich, Inc.

Cloning of ELOVL4 into GFP tagged and V5 tagged expression vectors

Preparation of EGFP-ELOVL4 wild-type (GFP-Ewt) and EGFP-ELOVL4 5 bp deletion mutant (GFP-Emut) constructs was reported earlier [7]. The V5-tagged ELOVL4 wild-type (V5-Ewt) and V5 tagged ELOVL4 mutant (V5-Emut) constructs were prepared by standard two-step method using Gateway cloning technology (Invitrogen Corp., Carlsbad, CA). Gateway is a universal cloning technology that takes advantage of the site specific recombination properties of bacteriophage lambda to provide a rapid and highly efficient method to move the gene of interest into multiple vector systems.

The complete cDNA sequence for ELOVL4 was amplified from human brain cDNA library, using gene specific primers carrying BamHI cohesive ends using the forward (5'-CTC GGA TCC GCG ATG GGG CTC CTG GAC-3') and reverse (5'-GCG GAT CCC AGT TCA ATT TAA TCT CCT TTT GCT TTT CC-3') primers and Pfx DNA polymerase. wild-type and mutant ELOVL4 cDNAs were first cloned into the pENTER-directional-TOPO entry vectors. These TOPO entry clones were used for recombination reactions and thereby directional cloning into the Gateway pcDNA 3.1/nV5 DEST mammalian expression vector carrying a V5 tag. Sequencing confirmed that the resulting clone, pcDNA 3.1/nV5 DEST-ELOVL4, contained the complete ELOVL4 wild-type or the mutant sequence in frame and the N-terminal V5 extension.

Cell culture

COS-7 cells were cultured in DMEM with glutamine and 10% fetal bovine serum with an atmosphere of 10% CO2 at 37 °C. The cells were seeded and grown to 80-90% of confluence. After 24 h, the cells were transfected with expression constructs or control vectors using Lipofectamine Plus (Invitrogen) according to the manufacturer's instructions. The effects of proteasomal inhibition and Golgi disruption were studied by treating the cells 48 h after transfection with 10 μM MG-132 for 12 h and 10 μg/ml nocodazole for 1 h, respectively, at 37 °C and then washed with PBS and immunolabeled.

Immunofluorescence analysis of transfected COS-7 cells by confocal microscopy

Cells were washed with cold PBS 48 h after transfection and fixed in absolute methanol at -10 °C for 15 min. Fixed cells were blocked for 1 h with goat serum in PBS, incubated for 1 h with primary antibody followed by washing with PBS, and further incubation with secondary antibody. Subsequently, washed cells were mounted with Vectashield anti-fade mounting medium containing DAPI. Immunofluorescence was visualized with a Zeiss LSM 510 laser scanning confocal microscope.

Preparation of cell extracts and western blotting

Cells transfected with the EGFP or V5 tagged ELOVL4 constructs were lysed with buffer containing 25 mM Tris-HCl (pH 7.5), 1% (w/v) SDS, 1 mM DTT, glycerol, and protease inhibitor cocktail (Sigma-Aldrich). Cell lysates were separated by 10% SDS-PAGE and blotted onto nitrocellulose membrane for western analysis. The blot was developed by the chemiluminescent method using ECL reagents according to the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ).

Colorless native polyacrylamide gel electrophoresis (CN-PAGE)

CN-PAGE was employed to determine whether ELOVL4 exists as a higher molecular mass oligomeric complex and was carried out essentially as described earlier with slight modifications [8-10]. CN-PAGE is identical to Blue-Native PAGE (BN-PAGE) but Coomassie dye is omitted from sample and cathode buffer and sodium taurodeoxycholate is used to induce the charge shift, which facilitates the migration of protein complexes according to their molecular mass on a nondenaturing gel. The whole cell lysates derived from cells transfected with GFP and/or V5 tagged ELOVL4 constructs, containing about 25 μg of protein were loaded onto the 6-13% native gel. The samples were electrophoresed at 80 V for 1 h and 200 V (current not exceeding 15 mA) for 2 h at 4 °C. The relative migration rates of standards on native gel were visualized by staining the first dimension native gel with Simply Blue safe stain (Invitrogen). The oligomeric complexes separated initially by CN-PAGE were further resolved into individual polypeptide components in second dimension on a 10% SDS-polyacrylamide gel, and the protein components were identified by immunoblot analysis using epitope-specific (anti-GFP or anti-V5) antibodies according to standard protocols [9].

FRET analysis to study the interaction between Ewt and Emut proteins

The interaction of wild-type and mutant ELOVL4 proteins was explored by fluorescence resonance energy transfer (FRET), in which molecular proximity is determined by the energy transfer from a fluorescent donor to an acceptor. COS-7 cells were transfected with GFP-Ewt and GFP-Emut, V5-Ewt and GFP-Emut, GFP-Ewt and V5-Ewt, or GFP-Emut, and V5-Emut constructs. After 24 h of transfection, cells were fixed with 2% paraformaldehyde, permeabilized with 1% Brij-58, and fixed again with 2% paraformaldehyde for 20 min at room temperature [11]. These cells were labeled with Alexa fluor 555-conjugated anti-V5 antibodies, and were washed for 1 h with Hank's Balanced Salt solution with constant shaking at room temperature.

Emission spectrophotometry

Energy transfer was examined by means of a microscope spectrophotometer apparatus [12,13]. Fluorescence emission spectra were collected from single cells by a Peltier-cooled IMAX camera with a liquid nitrogen cooled intensifier (Princeton Instruments, Princeton, NJ) attached to a modified Zeiss Axiovert fluorescence microscope. Microspectrophotometry used a 485/22-nm narrow band pass discriminating filter for excitation, a 510 nm longpass dichroic mirror, and a 520 nm long-pass emission filter. The emission spectrum was monitored between 400-800 nm. Winspec software (Princeton Instruments) was used to analyze spectrophotometric data. The excitation and emission wavelength pairs used for EGFP and Alexa fluor labeled anti-V5-antibody are 490 nm, 509 nm, and 555 nm, 565 nm, respectively.


Analysis of wild-type and mutant ELOVL4 expressed in COS-7 cells

To express wild-type and mutant ELOVL4 proteins in COS-7 cells, mammalian expression constructs carrying EGFP-ELOVL4 wild-type and mutant, and V5-tagged ELOVL4 wild-type and mutant constructs were generated as described earlier [7]. COS-7 cells were transfected or co-transfected with the following constructs: (1) GFP-Ewt, (2) GFP-Emut, (3) GFP-Ewt and GFP-Emut, (4) V5-E wt, (5) V5-E mut, (6) GFP-Ewt and V5-E mut, (7) V5-E wt and GFP-Emut, (8) GFP vector, and (9) mock transfection with transfection reagents only. Cell lysates were analyzed on SDS-PAGE followed by western blot analysis with appropriate antibodies to determine the expression of ELOVL4 fusion proteins. The GFP antibody detected a prominent band migrating at about 64 kDa in the lysates (Figure 1) of cells expressing the GFP-Ewt protein (lane 2), and GFP-Emut protein (lane 3). Cells co-expressing GFP-Ewt and GFP-Emut protein (lane 4), GFP-Ewt and V5-Emut protein (lane 7), and V5-Ewt and GFP-Emut protein (lane 8) also showed a signal at about 64 kDa. The band size was consistent with the expected molecular mass of 67 kDa (ELOVL4 contributing 37 kDa and GFP contributing 30 kDa) for wild-type protein and 63 kDa for mutant protein (ELOVL4 contributing a mass of 33 kDa and GFP contributing a mass of 30 kDa). A 30 kDa band was detected in control GFP vector transfected cell lysate (lane 9). No specific signal was detected in lysates of cells transfected with V5-Ewt (lane 5), V5-Emut (lane 6) or mock-transfected cells (lane 10).

When these cell lysates were analyzed with the V5 antibody, a specific signal at a molecular mass of about 41 kDa (ELOVL4 contributes a mass of 37 kDa and V5 a mass of 4 kDa, summing to an expected mass about 41 kDa, Figure 2) was detected in cells expressing V5-Ewt protein (lane 5). A polypeptide of about 37 kDa band was detected in lysates of cells expressing V5-Emut (lane 6, ELOVL4 contributing 33 and V5 contributing 4 kDa) and in lysates of cells co-expressing GFP-Ewt and V5-Emut (lane 7), V5-Ewt and GFP-Emut (lane 8). There was no specific band in mock transfected cell lysates (lane 9), or cells transfected with GFP-Ewt protein (lane 2), GFP-Emut protein (lane 3), or GFP-Ewt and GFP-Emut protein (lane 4). In summary, these results suggest that the COS-7 cells transfected with wild-type and/or mutant ELOVL4 constructs carrying a GFP or V5 tag expressed ELOVL4 fusion proteins of the expected sizes (Figure 2, Figure 3, Table 1).

Intracellular misrouting of Ewt in presence of Emut protein

To determine the subcellular localization of the wild-type and mutant ELOVL4 proteins in cells expressing both these proteins, COS-7 cells were transfected with constructs encoding ELOVL4 in four combinations: (1)GFP-Ewt and GFP-Emut, (2) V5-E wt and V5-E mut, (3) V5-E wt and GFP-Emut, and (4) GFP-Ewt and V5-E mut. In addition, COS-7 cells were transfected separately and individually with the GFP-Ewt, GFP-Emut, V5-E wt, or V5-E mut construct. Mock transfections were performed with transfection reagents only. Transfected cells were labeled with Alexa flour 555 conjugated anti-V5 antibody, then observed using laser confocal microscopy (Figure 3B,E). In cells expressing the wild-type ELOVL4 fusion protein, the ER localization of ELOVL4 is consistent with its In vivo localization in photoreceptors [7]. The localization of mutant protein to the juxtanuclear region was consistent with our previous report (data not shown) [7]. However, in the cells co-transfected with both wild-type and mutant ELOVL4, both proteins were localized to cytoplasmic aggregates in the perinuclear region (Figure 3). In these cells, localization of the wild-type ELOVL4 protein was indistinguishable from the distribution of mutant protein by confocal microscopy (Figure 3C,F). Furthermore, the wild-type protein did not co-localize with ER-specific marker protein disulfide isomerase (PDI; data not shown). This suggests that in the presence of mutant protein, the wild-type protein also was misrouted to cytoplasmic juxtanuclear aggregates along with the mutant protein.

Mutant ELOVL4 oligomerizes with wild-type ELOVL4

A combination of CN-PAGE and SDS-PAGE in a two-dimensional approach was used to determine the interaction between wild-type and mutant ELOVL4 proteins when they are co-expressed. Immunoblotting of the second dimension gel either with anti-GFP or anti-V5 identified the migration pattern of ELOVL4 fusion proteins (Figure 4). Under the conditions employed, the monomeric forms of proteins migrate towards the extreme right end of the second dimension gel while high molecular mass proteins or complexes remain on the left side of the gel. The GFP (Figure 4A), GFP-Ewt (Figure 4B), and V5-Ewt (Figure 4G) proteins appeared toward the right side of the second dimension blot and migrated in accordance with their expected monomeric molecular weight indicating that these proteins exist as monomers. Lysates of cells co-transfected with wild-type and mutant forms of ELOVL4 constructs showed the presence of ELOVL4 protein on the left side of the gel (Figure 4D-F,H), suggesting that these proteins are components of higher molecular mass oligomeric complexes. The mutant ELOVL4 in lysates from cells transfected with GFP-Emut construct also formed similar high molecular mass complexes on the left side of the gel (Figure 4C). When co-expressed, both the wild-type and mutant forms of ELOVL4 showed a much broader distribution in molecular weight of ELOVL4 protein (about 60-200 kDa) in both horizontal and vertical directions (Figure 4D-F,H). This is indicative of the protein being present both as a monomeric species and as a higher molecular mass oligomeric complex (Figure 4D-F). Thus, these results suggested that when expressed together, the wild-type ELOVL4 and its mutant form tend to aggregate and form higher molecular mass oligomeric complexes.

Interaction of wild-type and mutant ELOVL4 proteins

Physical interaction of wild-type and mutant ELOVL4 proteins was determined by FRET. This energy transfer between the donor and acceptor takes place through a nonradiative, dipole-dipole interaction. The transfer of energy depends upon the distance and the orientation between the two fluorophores, occurring over a donor to acceptor separation range of about 10-100 Å. Energy transfer in the nanometer scale makes the FRET an ideal technique to study the wild-type and mutant ELOVL4 protein interaction in intact cells. In our studies we used EGFP as the donor and Alexa 555 as the acceptor. When the two fluorophores are within a range of about 10-100 Å, the energy of the donor (EGFP) will transfer to the acceptor that leads to two peaks in the emission spectrum with one peak originating from EGFP at 485 nm and a second peak from Alexa 555 at 565 nm. We assessed the proximity of GFP-Ewt and V5-Emut (Figure 5A-C) in cells co-expressing wild-type and mutant proteins, and the emission spectra of the two molecules are shown in Figure 5A,B, respectively. FRET between these molecules was detected (Figure 5C). Similar results were observed in cells co-expressing V5-Ewt and GFP-Emut (Figure 5D-F), and FRET was also detected between V5-Ewt and GFP-Emut (Figure 5F). As a negative control, GFP-Ewt and V5-Ewt co-expressing cells were labeled with Alexa 555-conjugated anti-V5 antibodies, and emission spectra are shown in Figure 5G-I. There was no FRET between the two wild-type proteins (Figure 5I). However, as a positive control, FRET (Figure 5L) was observed on GFP-Emut and V5-Emut co-expressing cells (Figure 5J-L). Thus, these results provide evidence that the wild-type and mutant ELOVL4 are in close proximity in cells co-expressing both of them. The close proximity, as indicated by FRET signals, suggested that a mutant and wild-type polypeptide must be immediately adjacent to each other, implying that the two types of polypeptides are together in a complex or aggregate.

ELOVL4 aggresomes are associated with the microtubule organizing center (MTOC)

COS-7 cells expressing either the mutant ELOVL4 alone or co-expressing the wild-type and mutant ELOVL4 proteins appeared to form intracellular, perinuclear inclusions that have characteristics of aggresomes. Aggresomes are pericentriolar membrane-free, cytoplasmic ubiquitinated inclusion bodies formed at the microtubule organizing center (MTOC), and ensheathed in a cage of intermediate filaments [14,15]. It is well established that misfolded and aggregated proteins are sequestered into aggresomes [14,15]. To test whether the wild-type and mutant ELOVL4 complex is associated with aggresomes [14,15], localization of the mutant and wild-type ELOVL4 was compared with that of γ-tubulin, a centrosomal protein (Figure 6). The wild-type and mutant ELOVL4 protein complex was found to be co-localized with γ-tubulin (Figure 6G-I). Similar co-localization of ELOVL4 with γ-tubulin was also observed in cells expressing the mutant ELOVL4 protein alone (Figure 6D-F), whereas the wild-type ELOVL4 protein (Figure 6A-C) did not co-localize with γ-tubulin when it was expressed independently (Figure 6A-C). The γ-tubulin distribution was found to be similar in cells transfected with ELOVL4 fusion constructs or mock transfected cells (Figure 6J-L).

Reorganization of vimentin distribution in cells with ELOVL4 aggresomes

We have tested the localization and nature of intermediate filament network protein, vimentin, in relation to the distribution of the perinuclear inclusions that contained GFP-Ewt and GFP-Emut (Figure 7). Staining the cells with antibody specific to vimentin showed a typical dispersed distribution of vimentin as a long cytoplasmic filamentous network, in wild-type ELOVL4 expressed cells and mock transfected cells (Figure 7B,K). In contrast to its normal reticular distribution throughout the cell, vimentin showed juxtanuclear localization in cells co-expressing wild-type and mutant ELOVL4 proteins (Figure 7H). This altered distribution pattern of vimentin was reported to be associated with the formation of aggresomes [16]. Similar altered distribution of vimentin was observed in cells expressing the mutant ELOVL4 protein alone (Figure 7D-F), but the distribution of vimentin did not change in cells expressing the wild-type ELOVL4 protein alone (Figure 7G-I).

To further evaluate the association of co-expressed mutant and wild-type ELOVL4 proteins with aggresomes, cells transfected with the GFP-Ewt and GFP-Emut constructs were treated with MG-132, a low molecular weight proteasome inhibitor [17]. The localization of ELOVL4 proteins was examined in comparison to the localization of vimentin in these cells (data not shown). In cells co-expressing the wild-type and mutant ELOVL4 proteins, vimentin was found to be completely redistributed from its normal reticular pattern to a juxtanuclear region (data not shown). Though redistribution of vimentin was observed in cells expressing the wild-type ELOVL4 protein, the extent of vimentin redistribution was significantly different from the pattern of vimentin distribution observed in cells expressing mutant and mutant and wild-type ELOVL4 proteins (data not shown). This suggests a possible impairment of proteasomal degradative pathway in cells co-expressing wild-type and mutant ELOVL4 proteins. However additional analysis is needed to establish these observations.

ELOVL4 aggregates are distinct from Golgi

The juxtanuclear localization of aggregates formed in cells expressing mutant ELOVL4 protein alone or co-expressing GFP-Ewt and GFP-Emut proteins resembled the localization of Golgi. To determine their specific localization, COS-7 cells expressing mutant ELOVL4 protein alone and cells co-expressing both wild-type and mutant proteins were labeled for the Golgi markers, golgin-97 and TGN-38. Confocal microscopy revealed a clear distinction between the localization of co-expressed Ewt and Emut proteins and the distribution of golgin-97 (Figure 8D-F) and TGN-38 (Figure 8J-L). A similar difference in distribution was observed in the cells expressing the mutant protein alone (Figure 8A-C). golgin-97 signal in these cells was not completely co-localized with the GFP fluorescence instead there was a partial co-localization of golgin-97 signal with mutant ELOVL4 (Figure 8A-C).

To further evaluate the localization of ELOVL4 cytoplasmic juxtanuclear inclusions in comparison to Golgi, cells expressing either the wild-type or mutant ELOVL4 or co-expressing wild-type and mutant ELOVL4 proteins were incubated with nocodazole and immunostained with antibody to golgin-97 or TGN-38 (Figure 9). As nocodazole is a microtubule disrupting agent, and intact microtubules are required to form and maintain aggresomes and the Golgi apparatus, treatment with nocodazole disrupts both the aggresome and the Golgi [14]. Immunolabeling of nocodazole-treated cells with Golgi specific antibodies will distinguish disrupted Golgi from aggresomes. As reported previously [7], following nocodazole treatment, the GFP fluorescence of GFP-Emut did not co-localize with either golgin-97 (Figure 9A-F) or TGN-38 (Figure 9G-I), and appeared as spots at multiple places in the cytoplasm, instead of a large, juxtanuclear aggregate. This suggests that the perinuclear inclusions containing mutant ELOVL4 protein alone (Figure 8A-C) or co-expressed wild-type and mutant ELOVL4 proteins (Figure 8D-L) were associated with microtubules of the centrosome and were probably distinct from Golgi.


We have reported that the mutant ELOVL4 protein, which lacks an ER retention signal, is misrouted into the perinuclar region [7]. Previously, we speculated that the 5 bp deletion in ELOVL4 observed in families with autosomal dominant macular degeneration (STGD3) exerts a dominant negative effect operating at the protein level [7]. Here we demonstrate that the mutant ELOVL4 protein altered the trafficking of the wild-type protein by direct physical interactions using three independent approaches. Immunocytochemical analysis showed the co-localization of the wild-type ELOVL4 protein with the mutant ELOVL4 protein as cytoplasmic perinuclear aggregates. When co-expressed, wild-type and mutant ELOVL4 proteins were detected as high molecular weight aggregates as shown by a combination of CN-PAGE and SDS-PAGE in a two-dimensional electrophoretic approach. These findings and the results of FRET analysis suggested a direct physical interaction between the wild-type and mutant forms of ELOVL4 proteins. Presently, it is not known whether this interaction involves other proteins.

Although COS cells are distinct from highly specialized photoreceptor cells, they are useful tools in understanding the basic biochemical mechanisms that underlie the mutations linked to diseases such as retinitis pigmentosa. It is known that when overexpressed in heterologous systems, proteins tend to aggregate; however, in this study overexpression of wild-type ELOVL4 alone in COS-7 cells did not cause aggregation, a finding that validated the specificity of the observations. The juxtanuclear location of aggregates containing wild-type and mutant ELOVL4 proteins, the co-localization of centrosome components with these aggregates, the redistribution of intermediate filaments (IF), and the acceleration of ELOVL4 clustering in response to proteasome inhibition indicated that these aggregates were sequestered in aggresomes [18]. The altered trafficking of the wild-type ELOVL4 protein in the presence of mutant ELOVL4 and their accumulation in the perinuclear region suggested that this physical interaction underlies macular degeneration in patients with STGD3.

Formation and accumulation of aggresomes were shown to result in adverse biochemical events that involve the proteasomal system and apoptosis [19]. The degeneration of retina observed in patients with ELOVL4 mutations is progressive with onset in the second decade of life [1]. Recruitment of the wild-type protein by the mutant protein may result in the loss of functional ELOVL4. However, it is likely that a residual amount of wild-type ELOVL4 protein escapes the interaction with mutant ELOVL4, to reach its target site in the ER. This residual amount of functional ELOVL4 may be sufficient for the normal development and maintenance of retina in early stages of life. However, accumulation of the wild-type protein with the mutant protein in aggresomes may cause a cumulative effect leading to degeneration of ELOVL4 expressing photoreceptors over time, as observed in patients with STGD3. Alternatively, the degeneration observed in these patients could be due to the lack of sufficient functional ELOVL4 later in life, as normal adult mouse retinal tissue shows higher levels of ELOVL4 expression with maturation indicating greater demand for this protein in older age [20].

Hetero-oligomerization of mutant protein with wild-type protein, their misrouting and formation of aggresomes has been found to be a common feature observed in many neurodegenerative disorders including retinitis pigmentosa [21-23]. The possible mechanism of protein aggregation in these cells could be the inability of cells to process misfolded and aggregated proteins. The mechanism by which the ER retention signals present in the wild-type ELOVL4 of the aggregates are overridden, resulting in altered trafficking of the complexes, remains to be elucidated. This may provide important clues towards understanding the degenerative process and in developing therapeutic strategies to treat protein trafficking diseases. In this regard, further evaluation of aggresome formation in cells co-expressing mutant and wild-type ELOVL4 may serve as a model system for studying the cellular fate of mutant proteins and their disease mechanism.


We thank Bruce Donohoe (University of Michigan) for helping in the operation of the confocal microscope, Austra Liepa and Regina Belaney (University of Michigan) for assistance in manuscript preparation, and Swaroop Mahaveer Bhojani (University of Michigan) for helpful discussions. This work is supported by grants to RA from NIH (R01EY13198) and the Foundation Fighting Blindness, a research grant to RA and an unrestricted grant to the Department of Ophthalmology, UTHSC from Research to Prevent Blindness, Inc., and a NIH core grant to UTHSC (P30EY13080) and two core grants to the University of Michigan, Department of Ophthalmology (P30EY007003) and Vision Research (P30EY07060). This work was presented, in part, at the Association for Research in Vision and Ophthalmology (ARVO) meeting, May 2005, Fort Lauderdale, FL.


1. Griesinger IB, Sieving PA, Ayyagari R. Autosomal dominant macular atrophy at 6q14 excludes CORD7 and MCDR1/PBCRA loci. Invest Ophthalmol Vis Sci 2000; 41:248-55.

2. Stone EM, Nichols BE, Kimura AE, Weingeist TA, Drack A, Sheffield VC. Clinical features of a Stargardt-like dominant progressive macular dystrophy with genetic linkage to chromosome 6q. Arch Ophthalmol 1994; 112:765-72.

3. Bernstein PS, Tammur J, Singh N, Hutchinson A, Dixon M, Pappas CM, Zabriskie NA, Zhang K, Petrukhin K, Leppert M, Allikmets R. Diverse macular dystrophy phenotype caused by a novel complex mutation in the ELOVL4 gene. Invest Ophthalmol Vis Sci 2001; 42:3331-6.

4. Zhang K, Kniazeva M, Han M, Li W, Yu Z, Yang Z, Li Y, Metzker ML, Allikmets R, Zack DJ, Kakuk LE, Lagali PS, Wong PW, MacDonald IM, Sieving PA, Figueroa DJ, Austin CP, Gould RJ, Ayyagari R, Petrukhin K. A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy. Nat Genet 2001; 27:89-93.

5. Oh CS, Toke DA, Mandala S, Martin CE. ELO2 and ELO3, homologues of the Saccharomyces cerevisiae ELO1 gene, function in fatty acid elongation and are required for sphingolipid formation. J Biol Chem 1997; 272:17376-84.

6. Maugeri A, Meire F, Hoyng CB, Vink C, Van Regemorter N, Karan G, Yang Z, Cremers FP, Zhang K. A novel mutation in the ELOVL4 gene causes autosomal dominant Stargardt-like macular dystrophy. Invest Ophthalmol Vis Sci 2004; 45:4263-7.

7. Ambasudhan R, Wang X, Jablonski MM, Thompson DA, Lagali PS, Wong PW, Sieving PA, Ayyagari R. Atrophic macular degeneration mutations in ELOVL4 result in the intracellular misrouting of the protein. Genomics 2004; 83:615-25.

8. Camacho-Carvajal MM, Wollscheid B, Aebersold R, Steimle V, Schamel WW. Two-dimensional Blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates: a proteomics approach. Mol Cell Proteomics 2004; 3:176-82.

9. Schagger H, Cramer WA, von Jagow G. Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal Biochem 1994; 217:220-30.

10. Vijayasarathy C, Damle S, Lenka N, Avadhani NG. Tissue variant effects of heme inhibitors on the mouse cytochrome c oxidase gene expression and catalytic activity of the enzyme complex. Eur J Biochem 1999; 266:191-200.

11. Pedley KC, Jones GE, Magnani M, Rist RJ, Naftalin RJ. Direct observation of hexokinase translocation in stimulated macrophages. Biochem J 1993; 291:515-22.

12. Petty HR, Worth RG, Kindzelskii AL. Imaging sustained dissipative patterns in the metabolism of individual living cells. Phys Rev Lett 2000; 84:2754-7.

13. Kindzelskii AL, Yang Z, Nabel GJ, Todd RF 3rd, Petty HR. Ebola virus secretory glycoprotein (sGP) diminishes Fc gamma RIIIB-to-CR3 proximity on neutrophils. J Immunol 2000; 164:953-8.

14. Johnston JA, Ward CL, Kopito RR. Aggresomes: a cellular response to misfolded proteins. J Cell Biol 1998; 143:1883-98.

15. Riley NE, Bardag-Gorce F, Montgomery RO, Li J, Lungo W, Lue YH, French SW. Microtubules are required for cytokeratin aggresome (Mallory body) formation in hepatocytes: an in vitro study. Exp Mol Pathol 2003; 74:173-9.

16. Kopito RR, Sitia R. Aggresomes and Russell bodies. Symptoms of cellular indigestion? EMBO Rep 2000; 1:225-31.

17. Tawa NE Jr, Odessey R, Goldberg AL. Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscles. J Clin Invest 1997; 100:197-203.

18. Wigley WC, Fabunmi RP, Lee MG, Marino CR, Muallem S, DeMartino GN, Thomas PJ. Dynamic association of proteasomal machinery with the centrosome. J Cell Biol 1999; 145:481-90.

19. Bence NF, Sampat RM, Kopito RR. Impairment of the ubiquitin-proteasome system by protein aggregation. Science 2001; 292:1552-5.

20. Mandal MN, Ambasudhan R, Wong PW, Gage PJ, Sieving PA, Ayyagari R. Characterization of mouse orthologue of ELOVL4: genomic organization and spatial and temporal expression. Genomics 2004; 83:626-35.

21. Ross CA, Poirier MA. Protein aggregation and neurodegenerative disease. Nat Med 2004; 10 Suppl:S10-7.

22. Junn E, Lee SS, Suhr UT, Mouradian MM. Parkin accumulation in aggresomes due to proteasome impairment. J Biol Chem 2002; 277:47870-7.

23. Tanaka M, Kim YM, Lee G, Junn E, Iwatsubo T, Mouradian MM. Aggresomes formed by alpha-synuclein and synphilin-1 are cytoprotective. J Biol Chem 2004; 279:4625-31.

Vasireddy, Mol Vis 2005; 11:665-676 <>
©2005 Molecular Vision <>
ISSN 1090-0535