IOVS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

(Investigative Ophthalmology and Visual Science. 2004;45:863-871.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.03-0708
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fan, J.
Right arrow Articles by Chepelinsky, A. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fan, J.
Right arrow Articles by Chepelinsky, A. B.

{gamma}E-crystallin Recruitment to the Plasma Membrane by Specific Interaction between Lens MIP/Aquaporin-0 and {gamma}E-crystallin

Jianguo Fan,1 Anna K. Donovan,1 Dolena R. Ledee,1 Peggy S. Zelenka,1 Robert N. Fariss,2 and Ana B. Chepelinsky1

1From the Laboratory of Molecular and Developmental Biology and the 2Laboratory of Mechanisms of Ocular Diseases, National Eye Institute, National Institutes of Health, Bethesda, Maryland.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Major intrinsic protein (MIP), also called aquaporin-0, is essential for lens transparency and is specifically expressed in the lens fiber cell membranes. The goal of the current study was to identify and characterize proteins that interact with MIP and to elucidate the role of these interactions in MIP functions.

METHODS. The C-terminal 74-amino-acid fragment of MIP was used as bait to screen a rat lens cDNA yeast two-hybrid library. The full-length MIP was expressed as enhanced green fluorescent protein (EGFP)–tagged or myc-tagged proteins, and {gamma}E-crystallin was expressed as FLAG-tagged or red fluorescent protein (HcRed)-tagged proteins, respectively, in the RK13 rabbit kidney epithelial cell line. Protein–protein interactions were analyzed by coimmunoprecipitation assays and visualized by confocal fluorescence microscopy.

RESULTS. {gamma}E-Crystallin, a water-soluble protein that is specifically expressed in lens fibers, was identified as a binding protein to the MIP C-terminal peptide. Coimmunoprecipitation assays demonstrated that {gamma}E-crystallin interacts specifically with full-length MIP in mammalian cells. MIP did not interact with {gamma}D-crystallin, another member of the highly conserved {gamma}-crystallin gene family. Confocal fluorescence microscopy demonstrated that MIP interacted with {gamma}E-crystallin in individual mammalian cells and that this interaction resulted in the recruitment of {gamma}E-crystallin from the cytoplasm to the plasma membrane.

CONCLUSIONS. These experiments provide the first demonstration of MIP interaction with other lens proteins at the molecular level and raise the possibility of a structural role of MIP in the organization of {gamma}-crystallins in lens fibers.

Lens major intrinsic protein (MIP), also known as aquaporin-0, is the most abundant protein of the ocular lens fiber membranes. It belongs to an ancient family of membrane channel proteins. Mutations in the MIP gene have been linked to genetic cataracts in mice and humans, suggesting an important role for MIP in maintaining lens transparency. MIP may play a role in reducing the interfiber space, as tightly packed fibers are essential for lens transparency, and vacuolated fibers are observed in the mouse mutant lenses. Four mouse MIP mutations, including a point mutation at amino acid 51 (A51P; lop), replacement of the last 61 amino acids (amino acids 203-263) at the MIP C-terminus by a transposon sequence (Cat Fr), deletion of MIP amino acids 121-175 (Hfi), and deletion of amino acids 46-49 (CatTohm) result in autosomal dominant cataracts and MIP trapping in the endoplasmic reticulum without being inserted into the plasma membrane.1 2 3 4 Similarly, two different point mutations in MIP identified in two human cataract families (E134G, T138R) do not integrate into the plasma membrane when expressed in Xenopus oocytes.5 6

Water channel activity measurements of reconstituted lens membrane vesicles derived from Cat Fraser or MIP null mice, showed a marked decrease in water channel activity compared with those from the wild-type lens.7 8 However, the facts that some water channel activity remains, even in the absence of MIP, and severe changes in lens fiber structure with dominant phenotype are observed in MIP mutant mice suggest additional functions for MIP in the lens, such as maintenance of fiber structure and arrangement required for optimal focusing of the lens.8 9

MIP forms pH- and Ca2+-dependent water channels when expressed in Xenopus oocytes10 and pH- and voltage-dependent channels in mammalian and insect cells.11 MIP voltage-dependent channels in lipid bilayer vesicles are regulated by protein kinase-A–dependent phosphorylation.12 MIP may also function as an adhesion molecule and may play a role in gap junction formation.13 14 15 16 Both the N- and C-termini of MIP are located in the cytoplasmic side of the plasma membrane. The MIP C-terminal domain may play an important role in its physiological function. This domain is cleaved in cataractogenesis and aging,17 18 19 is serine phosphorylated,20 and may interact with calmodulin.21 22

To understand the role of the MIP C-terminus in MIP functions, we screened a lens yeast two-hybrid expression library with the MIP C-terminal domain as the bait to search for novel proteins expressed in the lens that may interact with this MIP domain. We identified {gamma}E-crystallin, a soluble protein that is also specifically expressed in the lens fibers, as a binding protein interacting with the MIP C-terminal domain. We provided biochemical evidence for the specific interaction between the full-length MIP and {gamma}E-crystallin in mammalian cells. We further demonstrated that this specific interaction results in the recruitment of {gamma}E-crystallin to the plasma membrane in mammalian cells expressing MIP.

{gamma}-Crystallins are lens-fiber–specific soluble proteins that also play a critical role in maintaining lens transparency.23 Various mutations in different members of the {gamma}-crystallin family have been linked to genetic cataracts in both mice and humans.24 25 26 27 28 29 30 31 32 However, the mechanisms by which {gamma}-crystallins are involved in cataract formation are largely unknown. Thus, this study may provide a functional link between two distinct classes of lens proteins that are involved in genetic cataract formation.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and Reagents
Tween-20, Triton X-100, protease inhibitor cocktail for mammalian cells, and X-gal were obtained from Sigma-Aldrich (St. Louis, MO). Media and sera for cell culture were purchased from Invitrogen (Carlsbad, CA). Agarose and DNA polymerase (Protein A/Protein G Plus Agarose and turbo Pfu polymerase) were obtained from Stratagene (La Jolla, CA). A polymerase chain reaction (PCR) purification kit and plasmid DNA purification were purchased from Qiagen (Valencia, CA). A sandwich membrane kit (Polyvinylidene Difluoride [PVDF] Membrane Sandwich Assembly Kit) was obtained from Invitrogen. A Western blot detection kit (Enhanced Chemiluminescence [ECL] Plus) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Nonfat dry milk and pre-stained molecular weight standards (Kaleidoscope) were obtained from Bio-Rad (Hercules, CA). All other chemicals were reagent grade and from standard commercial sources.

Antibodies
Antibodies against FLAG (M2) and anti-FLAG (M2) agarose conjugates were purchased from Sigma-Aldrich. A rabbit polyclonal anti-myc tag antibody was obtained from Cell Signaling Technology, Inc. (Beverly, MA). C-myc monoclonal antibody-agarose beads were obtained from Clontech (Palo Alto, CA). The anti-MIP rabbit polyclonal antibody MIP31 was prepared and affinity purified using a synthetic peptide derived from the mouse MIP amino acids 1-31. Rabbit polyclonal antibody against {gamma}-crystallins was generously provided by Samuel Zigler (the Laboratory of Mechanisms of Ocular Diseases, National Eye Institute).

Cell Lines
The RK13 (rabbit kidney) cell line was obtained from American Type Culture Collection (Manassas, VA) and maintained as monolayer cultures at 37°C in a 5% CO2/95% air incubator in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS).

Plasmids
The plasmids pEGFP-C2, pEGFP-N2, and pHcRed1-C1 were purchased from Clontech. Plasmid DNAs were propagated in Escherichia coli strain DH5{alpha} or DH10B and purified by ion exchange chromatography using plasmid kits (Plasmid Midi Endonuclease-free Maxi Kits) from Qiagen.

DNA Sequencing
Sequencing of plasmid DNA was performed with a commercial system (CEQ DTCS Quick Start Kit; Beckman-Coulter, Hialeah, FL) and an automated DNA analysis system (CEQ 2000XL; Beckman-Coulter), according to the manufacturer’s instructions.

Construction of MIP Bait Plasmids
The MIP C-terminal bait plasmid pBDGal4-MIP-C74, which contains a MIP cDNA fragment encoding the C-terminal 74 amino acids (amino acids 190-263), was constructed as follows. The MIP C-terminal cDNA fragment was amplified from a full-length mouse MIP cDNA plasmid by polymerase chain reaction (PCR) using turbo Pfu DNA polymerase (Stratagene) with the amplimers 5'-ATCGAATTCGCTCCTGCCATCCTCACCCGGAAC-3' (forward) and 5'-ATCGTCGACTCAGGGCCTGAGTCTTCAGTTCGAC-3' (reverse). The PCR product was digested with EcoRI and SalI restriction enzymes, purified with a PCR purification kit (Qiagen), and subsequently cloned into the parental bait plasmid pBDGal4cam (Stratagene) at EcoRI and SalI sites. The mouse MIP full-length bait plasmid pBDGal4-MIP-F was constructed in the same manner, except that the amplimers used for PCR amplification of the full-length MIP cDNA were 5'-ATCGAATTCATGTGGGAACTTCGGTCTGCCTCC-3' (forward) and 5'-ATCGTCGACTCAGGGCCTGAGTCTTCAGTTCGAC-3' (reverse). DNA sequences of the insert and the junction regions flanking the insert in these plasmids were verified by DNA sequencing.

Construction of MIP/EGFP, MIP/Myc, FLAG/ {gamma}-crystallin, or HcRed/{gamma}-crystallin Fusion Vectors
To construct an enhanced green fluorescent protein (EGFP) fusion plasmid of MIP, the full-length MIP cDNA (0.8 kb) was cut from the plasmid pBDGal4-MIP-F with EcoRI and SalI restriction enzymes and purified on 1.2% agarose gel. This fragment was subsequently cloned into either pEGFP-C2 or pEGFP-N2 (Clontech) at EcoRI and SalI sites, producing plasmid pEGFP-MIP (i.e., EGFP is fused to the N-terminal site of MIP) or pMIP-EGFP (i.e., EGFP is fused to the C-terminal site of MIP), respectively.

To construct the myc/MIP fusion vector pmyc-MIP, the entire MIP coding sequence was amplified from the plasmid pBDGal4-MIP-F by PCR with turbo Pfu DNA polymerase, using the following amplimers: 5'-ATATGGATCCGGTTCTGGATCAATGTGGGAACTTCGGTCTGC-3'(forward) and 5'-ATCAGAATTCTTACAGGGCCTGAGTC TTCAGTTC-3' (reverse). The PCR product, after digestion with BamHI and EcoRI restriction enzymes, was cloned into the BamHI and EcoRI sites of pCMVTag3B (Stratagene), producing the vector pmyc-MIP (i.e., myc tag at the N-terminal side of MIP). To construct the myc/MIP fusion vector pMIP-myc (i.e., myc tag at the C-terminal side of MIP), the full-length mouse MIP cDNA fragment, cut from the plasmid pBDGal4-MIP-F with restriction enzymes EcoRI and SalI and purified on 1.2% agarose gel, was cloned into the EcoRI and SalI of the plasmid pCMVTag5C (Stratagene).

To construct the FLAG/{gamma}E-crystallin fusion expression plasmid pFLAG-{gamma}E-cry (i.e., FLAG tagged at the N- terminus of {gamma}E-crystallin), the full-length rat {gamma}E-crystallin cDNA fragment (0.5 kb), cut from pADc12-1b (a target cDNA clone obtained from the yeast two-hybrid screening of rat lens cDNA library), with EcoRI and XhoI restriction enzymes, was purified on a 1.2% agarose gel and cloned into the same restriction sites in the plasmid pCMV-Tag2B (Stratagene).

The FLAG/{gamma}D-crystallin fusion expression plasmid pFLAG-{gamma}D-cry was constructed as follows. The full-length rat {gamma}D-crystallin cDNA fragment (0.5 kb) was isolated from pADB2B2 (a target cDNA clone obtained from the yeast two-hybrid screening of rat lens cDNA library) by digestion with EcoRI and XhoI restriction enzymes. After purification by agarose gel electrophoresis, the fragment was cloned into the plasmid pCMV-Tag2B at the EcoRI-XhoI sites, producing pFLAG-{gamma}D-cry.

To construct the HcRed/{gamma}-E-cry fusion plasmid pHcRed-{gamma}-E-cry, the full-length rat {gamma}E-crystallin cDNA was amplified, by using PCR with turbo Pfu DNA polymerase, from pADc12-1b with the following amplimers: (forward) 5'-ATCAGATCTCTCGAG GCCACCATG GGGAAGATCACCTTCTATGAG-3' and (reverse) 5'-TTAGGTCGACACATAGAAATCCATGATTCTCCT CAG-3'. After digestion with BglII and SalI restriction enzymes and purification using a PCR purification kit (Qiagen), the PCR product was cloned into pHcRed1-C1 (Clontech) at the BglII and SalI sites.

To construct the HcRed/{gamma}-D-cry fusion plasmid pHcRed-{gamma}-D-cry, the full-length {gamma}D-crystallin cDNA fragment (0.5 kb), cut from pAD-B2B2 pADc12-1b with restriction enzymes BamHI and SalI and purified on a 1.2% agarose gel, was cloned into the BglII and SalI restriction sites of pHcRed1-C1 (Clontech).

All expression vectors constructed are under the control of a CMV promoter. DNA sequences of the insert and the junction regions flanking the insert in these plasmids were verified by DNA sequencing.

Construction and Preparation of the Rat Lens cDNA Library for Yeast Two-Hybrid Screening
mRNAs were isolated and purified from 18-day rat embryo lenses and reverse transcribed into cDNAs with an oligo dT primer. After addition of adapter sequences with appropriate restriction sites, the double-stranded cDNAs were size fractionated and ligated into the HybriZAP-2.1 vector (Stratagene). The ligation products were packaged by using a packaging extract (Gigapack III Gold; Stratagene). The resultant {lambda}-phage library was subsequently amplified, excised into pADGal4 phagemids, and amplified again. The library plasmid DNA (pADGal4-cDNA) was propagated in the XLOLR host cells and purified with a plasmid purification kit (Plasmid Maxi Kit; Qiagen).

Yeast Transformation and Two-Hybrid Screening
Yeast strain YRG-2 was transformed with the MIP bait plasmid pBDGal4-MIP-C74, after selection on Trp-minus agar plates the presence of the insert was confirmed by PCR. A transformed clone was subsequently transformed with 100 µg of the rat lens cDNA library plasmid DNA (pADGal4-cDNA), and the double transformed yeast cells were selected on synthetic medium (SD)-agar plates lacking Leu, Trp, and His (KD Medical, Inc., Columbia, MD). After 7 to 14 days of growth at 30°C, surviving colonies were screened for ß-gal activity in an additional filter-lift assay. Colonies positive for ß-gal were replated and their ß-gal activity was verified again by a filter-lift assay. Positive colonies were subsequently propagated in liquid medium. The plasmid DNA was isolated, amplified by transformation into E. coli and sequenced with the DNA analysis system (model CEQ 2000XL; Beckman-Coulter).

Transient Transfection and Extraction of Proteins
RK13 cells were grown to approximately 30% to 70% confluence in 150-mm tissue culture dishes. A transfection mixture was prepared by thoroughly mixing a total of 10 µg plasmid DNA (i.e., 5 µg each expression plasmid) and 2.5 µg of protein expression-enhancing vector pAdVAntage (Promega, Madison, WI) in 750 µL of serum-free media with 150 µL transfection reagent (Superfect; Qiagen), incubating the resultant mixture for 20 minutes at room temperature, and addition of 5 mL growth medium containing 10% heated-inactivated FBS. The transfection mixture was subsequently added to each RK13 culture dish in which the medium had been removed, and the dish was incubated at 37°C in a CO2 incubator for 4 hours. The transfection mixture in each culture dish was removed, and 30 mL of growth medium containing 10% heated-inactivated FBS was added. After incubation at 37°C for another 48 hours, the cells were harvested by scraping into 5 mL per dish ice-cold phosphate-buffered saline (PBS; pH 7.4) followed by centrifugation in a clinical centrifuge. After one wash with PBS, the cell pellet was either stored at -80°C or used immediately for preparation of total cellular extracts with an extract buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% (vol/vol) Triton X-100, a 1:100 dilution of a protease inhibitor cocktail (cat. no. P8340; Sigma-Aldrich) and 1 mM phenylmethanesulfonyl fluoride (4°C for 1 hour). After protein quantification, the cell extracts were used immediately for immunoprecipitation (IP) experiments or stored at -80°C. Two separate culture dishes were used for each transfection experiment, and transfected cells from these two dishes were pooled.

Immunoprecipitation and Immunodetection
One milligram of proteins from total cellular extracts prepared from transfected cells were diluted in a total of 1 mL buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1% (vol/vol) Triton X-100. After addition of 20 µL (bed volume) of anti-FLAG M2 antibody-agarose (Sigma-Aldrich) or anti-myc antibody-agarose (Clontech) beads that were prewashed extensively with the same buffer, the mixture was incubated at 4°C on a rotator for 16 hours. The affinity agarose beads were recovered by centrifugation and washed 5 times with a washing buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% (vol/vol) Tween-20. The immunoprecipitated proteins were eluted from the beads by addition of sodium dodecyl sulfate (SDS)-sample buffer followed by boiling for 5 minutes in a water bath. The eluted immunoprecipitate was loaded and separated on a 4% to 20% gradient SDS polyacrylamide gel. After electrotransfer of proteins from the gel to PVDF membranes, the immunoprecipitated proteins were visualized by western blot with specific antibodies against various proteins or tags including MIP, {gamma}-crystallins, and the FLAG tag.

Western Blot Analysis
After electrophoresis, the proteins were electro-transferred to PVDF membranes and the latter were probed with either anti-myc or anti-MIP or anti-{gamma} crystallin antibody followed by a second antibody conjugated with peroxidase or with anti-FLAG antibody conjugated with peroxidase (no secondary antibody step). Specific proteins were then visualized by treatment of the membranes with the enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia Biotech) followed by exposure to x-ray films.

Confocal Fluorescence Microscopy of Transfected Mammalian Cells
RK13 cells were grown to approximately 30% to 50% confluence in 60-mm tissue culture dishes. A transfection mixture was prepared by thoroughly mixing 5 µg of each plasmid (EGFP or HcRed vectors) in 150 µL of serum-free medium with 60 µL of transfection reagent (Superfect; Qiagen), incubating the resulting mixture for 20 minutes at room temperature, and addition of 1 mL growth medium containing 10% heated-inactivated (FBS). The transfection mixture was subsequently added to each RK13 culture dish in which the medium had been removed, and the dish was incubated at 37°C in a CO2 incubator for 1.5 hours. Four milliliters of growth medium containing 10% heated-inactivated FBS was added to each dish and incubation at 37°C in a CO2 incubator continued for an additional 40 hours. After washing with PBS three times and complete removal of buffer from the culture dish, cells were fixed by addition of 2 mL of PBS (pH 7.4; Digene, Beltsville, MD) containing 4% paraformaldehyde and incubation for 20 minutes at room temperature. Cells were washed three times with PBS and counterstained with 2 mL immunohistochemistry (ICC) buffer containing 1x PBS, 0.5% bovine serum albumin, 0.2% Tween-20, 0.05% sodium azide, and 1 µg/mL 4',6'-diamidino-2-phenylindole dihydrochloride hydrate (DAPI; pH 7.3), for 30 minutes at room temperature. Cells were washed three times with PBS and mounted (Gel Mount; BioMeda Corp., Foster City, CA), an aqueous mounting medium containing a proprietary anti-photograph bleaching reagent. The cells were imaged using a confocal microscope (model TCS SP2) with a 40x 0.85-NA (HCX Plan Apo CS) and a 40x 0.75- to 1.25-NA (Oil HCX Plan Apo CS) objective lens (all from Leica, Deerfield, IL). EGFP fluorescence was imaged at the wavelengths of 458, 476, and 488 nm (excitation) and 510 to 540 nm (emission). HcRed fluorescence was imaged at the wavelengths of 568 nm (excitation) and 600 to 650 nm (emission), and DAPI fluorescence was imaged at the wavelengths of 351 and 364 nm (excitation) and 400 to 500 nm (emission). To minimize crosstalk between channels, fluorochromes were excited sequentially rather than simultaneously, using the sequential scan mode of the confocal microscope.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Identification of {gamma}E-crystallin as a Binding Protein to the MIP C-Terminal Domain
A cDNA fragment encoding 74 amino acids of the mouse MIP C-terminal domain (amino acids 190-263) was cloned into the parental bait plasmid (pBDGal4Cam) of the yeast two-hybrid system and subsequently used as bait to screen a yeast two-hybrid library containing cDNAs isolated from 18-day rat embryo lenses (see the Materials and Methods section for details). The bait plasmid (pBDGal4-MIP-C74) and the library DNA were introduced in a sequential manner into yeast strain YRG-2. After growth selection of cells in medium lacking tryptophan, leucine, and histidine, positive clones were identified by a filter-lift assay that measures ß-galactosidase activity (blue). Identity of the target cDNA present in the plasmid from these positive clones was obtained by DNA sequencing, followed by a BLAST search of DNA/protein databases. Two of the positive, in-frame clones obtained with the MIP C-terminal fragment as bait encode {gamma}E-crystallin (containing full-length cDNA). No other {gamma}-crystallin (i.e., {gamma}A, {gamma}B, {gamma}C, {gamma}D, or {gamma}F) was identified with this MIP C-terminal bait, although all these {gamma}-crystallins are expressed in the lens yeast two-hybrid library, as determined by cloning and sequencing. Interaction between MIP C-terminal and {gamma}E-crystallin in yeast was confirmed by the yeast two-hybrid assay, in which bait and target plasmids were cotransfected into yeast followed by verification of ß-galactosidase activity (blue) of transformed yeast cells. The results indicate that {gamma}E-crystallin may interact with MIP and that the interaction may involve the C-terminus of MIP.

Specific Interaction between Full-Length MIP and {gamma}E-crystallin in Mammalian Cells
To demonstrate the specific interaction between MIP and {gamma}E-crystallin in mammalian cells, mammalian expression vectors for expressing the full-length MIP as myc- and EGFP-tagged proteins and the full-length {gamma}E- or {gamma}D-crystallin as FLAG- and HcRed-tagged proteins were constructed as indicated in Materials and Methods (Fig. 1) . The EGFP tag was placed at either the C- or N-terminal end of MIP, generating the fusion proteins MIP-EGFP and EGFP-MIP, respectively (Figs. 1A 1B) . Similarly, the myc tag was placed at either the N- or C-terminal end of MIP, generating the fusion proteins myc-MIP and MIP-myc, respectively (Figs. 1C 1D) . The FLAG tag was placed at the N-terminal end of {gamma}E- or {gamma}D-crystallin, generating the fusion proteins FLAG-{gamma}E-cry and FLAG-{gamma}D-cry, respectively (Figs. 1E 1F) . The HcRed tag was fused to the N-terminal side of {gamma}E- or {gamma}D-crystallin, producing the fusion proteins HcRed-{gamma}E-cry and HcRed-{gamma}D-cry, respectively (Figs. 1G 1H) .



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 1. Construction of expression vectors for tagged MIP-, {gamma}E-, or {gamma}D-crystallin proteins. (A) MIP-EGFP was constructed by fusing the full-length MIP (263 amino acids) to the N-terminal end of EGFP (265 amino acids). (B) EGFP-MIP was constructed by fusing the full-length MIP to the C-terminal end of EGFP. (C) An myc tag (10 amino acids) was fused to the N-terminal end of MIP to produce myc-MIP fusion protein. (D) An myc tag was fused to the C-terminal end of MIP to produce MIP-myc fusion protein. (E) FLAG-{gamma}E crystallin was constructed by fusing a FLAG tag (nine amino acids) to the N-terminal end of {gamma}E-crystallin, (F) FLAG-{gamma}D crystallin by fusing a FLAG tag (nine amino acids) to the N-terminal end of {gamma}D-crystallin, (G) HcRed-{gamma}E crystallin by fusing the full-length {gamma}E-crystallin to the C-terminal end of HcRed, and (H) HcRed-{gamma}D crystallin by fusing the full-length {gamma}D-crystallin to the C-terminal end of HcRed. All expression vectors are under the control of a CMV promoter.

 
Coimmunoprecipitation assays were performed to provide the biochemical evidence for the specific interaction between full-length MIP and {gamma}E-crystallin in mammalian cells. To this end, myc-tagged MIP (either myc-MIP or MIP-myc) and FLAG-tagged {gamma}E-crystallin expression vectors were cotransfected into rabbit kidney epithelial (RK13) cells (Fig. 2 , lanes 1 and 2) along with various controls (lanes 3–5 and 7). Total cellular extracts from the transfected cells were immunoprecipitated with either anti-myc (Figs. 2A 2B) or anti-FLAG (Figs. 2C 2D) antibody-agarose conjugate. The immunoprecipitated proteins were resolved on a denaturing SDS-polyacrylamide gel. After transfer of proteins to a PVDF membrane, the immunoprecipitated proteins were visualized by Western blot analysis with either anti-myc or anti-FLAG antibody.



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 2. Interaction of MIP with {gamma}-crystallins in mammalian cells. RK13 cells were cotransfected with one of the following combinations of myc-tagged MIP expression plasmid and/or FLAG-tagged {gamma}E- or {gamma}D-crystallin expression plasmid: pmyc-MIP and pFLAG-{gamma}E cry (lane 1), pMIP-myc and pFLAG-{gamma}E cry (lane 2), pFLAG-{gamma}E cry (lane 3), pmyc-MIP (lane 4), pMIP-myc (lane 5), pmyc-MIP and pFLAG-{gamma}D cry (lane 6), and no-DNA control (lane 7). Seventy-two hours after transfection, total cellular protein extracts were prepared from the transfected cells and immunoprecipitated with anti-myc (A, B) or anti-FLAG (C, D) antibody-agarose conjugates. The immunoprecipitated proteins were separated on a 4% to 20% gradient SDS-polyacrylamide gel. After transfer of proteins to a PVDF membrane, the immunoprecipitated proteins were visualized by Western blot with one of the following antibodies in conjunction with ECL detection: an anti-myc antibody (A, D) and an anti-FLAG monoclonal antibody (B, C). Total cellular protein extracts, without IP, from each transfection (lanes 1–7) were subjected to SDS-PAGE, followed by transfer of proteins to PVDF membranes and Western blot with anti-myc antibody (E) and anti-FLAG antibody (F). IP, immunoprecipitation; WB, Western blot. One milligram of proteins from the total cellular extracts was used for each IP reaction (A–D) and 80 µg of proteins was analyzed for protein expression for each cotransfection (E, F). Input: 80/1000 = 8%.

 
Coimmunoprecipitation using anti-myc antibody was examined first. The efficacy of IP of myc-tagged proteins with anti-myc antibody agarose conjugate was tested with IP followed by Western blot with anti-myc antibody (Fig. 2A) . The protein levels of the immunoprecipitated myc-tagged MIP from cotransfected cells (Fig. 2A , lanes 1 and 2) were comparable to each other and to those from control cells into which a single expression vector was transfected (Fig. 2A , lanes 4 and 5). Myc-tagged MIP protein was not detected in cells that were transfected with FLAG-tagged {gamma}E-crystallin alone (Fig. 2A , lane 3) or in cells that were mock transfected (Fig. 2A , lane 7). The relative levels of the immunoprecipitated myc-tagged proteins (Fig. 2A , lanes 1–2, 4, 5) were comparable to those detected in total cellular extracts (Fig. 2E , lanes 1, 2, 4, 5). These results indicate that myc-tagged proteins were expressed at comparable levels and were immunoprecipitated with similar efficacy in cotransfected cells as well as in control cells. When the anti-myc immunoprecipitates were analyzed by Western blot using anti-FLAG antibody (Fig. 2B) , the FLAG-tagged {gamma}E-crystallin was coimmunoprecipitated with either myc-MIP (Fig. 2B , lane 1) or MIP-myc (Fig. 2B , lane 2). The specificity of the coimmunoprecipitation reaction was demonstrated by the absence of coimmunoprecipitation in control experiments in which cells were transfected with only a single expression vector (Fig. 2B , lanes 3–5) or when cells were mock transfected (Fig. 2B , lane 7).

Next, the reciprocal coimmunoprecipitation using the anti-FLAG antibody was performed in a similar manner. The efficacy of IP of FLAG-tagged proteins with anti-FLAG antibody agarose conjugate was tested with IP followed by western blotting with anti-FLAG antibody (Fig. 2C) . The protein levels of the immunoprecipitated FLAG-tagged {gamma}E-crystallin from cotransfected cells (Fig. 2C , lanes 1, 2) were similar to each other and to those from control cells into which a single expression vector was transfected (Fig. 2C , lane 3). FLAG-tagged {gamma}E-crystallin protein was not detected in cells that were transfected with myc-tagged MIP vectors alone (Fig. 2C , lanes 4, 5) or in cells that were mock transfected (Fig. 2C , lane 7). The relative levels of the immunoprecipitated FLAG-tagged proteins (Fig. 2C , lanes 1–3) were very similar to those detected in total cellular extracts (Fig. 2F , lanes 1–3). These results indicate that FLAG-tagged {gamma}E-crystallin was expressed at comparable levels and was immunoprecipitated with similar efficiency in cotransfected cells as well as in control cells. When the anti-FLAG immunoprecipitates were analyzed by Western blot with anti-myc antibody (Fig. 2D) , the myc-tagged MIP protein was coimmunoprecipitated with FLAG-tagged {gamma}E-crystallin (Fig. 2D , lanes 1, 2). The specificity of the coimmunoprecipitation reaction was demonstrated by the absence of coimmunoprecipitation in control experiments in which cells were transfected with only a single expression vector (Fig. 2D , lanes 3–5) or when cells were mock transfected (Fig. 2C , lane 7). The appearance of a double band for MIP-myc (Figs. 2A 2D 2E , lane 2) is probably due to the use of the commercially available expression vectors (Stratagene), which tend to express different C-terminally tagged proteins as a double band (Fan J, et al., unpublished data, 2003).

Thus, our data showed that MIP is coimmunoprecipitated with {gamma}E-crystallin through an anti-FLAG antibody and, vice versa, that {gamma}E-crystallin is coimmunoprecipitated with MIP through an anti-myc antibody. These results indicate that full-length MIP specifically interacts with {gamma}E-crystallin in mammalian cells.

Involvement of the MIP C-terminal Domain in Interaction between MIP and {gamma}E-crystallin
Interaction between MIP and {gamma}E-crystallin was further studied in coimmunoprecipitation experiments using EGFP-tagged MIP and FLAG-tagged {gamma}E-crystallin expression vectors. EGFP-tagged MIP (either EGFP-MIP or MIP-EGFP; Fig. 1 ) and FLAG-tagged {gamma}E-crystallin vectors were cotransfected into RK13 cells. Total cellular extracts from the transfected cells were prepared and immunoprecipitated with anti-FLAG antibody-agarose conjugate. The immunoprecipitated proteins were resolved on a denaturing SDS-polyacrylamide gel. After transfer of proteins to a PVDF membrane, the immunoprecipitated proteins were visualized by Western blot with various antibodies. The efficacy of IP of FLAG-tagged proteins with anti-FLAG agarose was verified with IP followed by Western blot with both an anti-FLAG antibody (Fig. 3A) . The protein level of the immunoprecipitated FLAG-tagged {gamma}E-crystallin was comparable in various transfection experiments (Fig. 3A , lanes 1–3), indicating that FLAG-tagged proteins were expressed at comparable levels and were immunoprecipitated with similar efficacy among different samples. Western blot of the anti-FLAG immunoprecipitates with an anti-{gamma} crystallin antibody (Fig. 3C) confirmed this result and, in addition, verified the identity of the FLAG-tagged {gamma}E-crystallin fusion protein. The expression levels of EGFP-tagged MIP (EGFP-MIP or MIP-EGFP) in cells transfected with these plasmids were also comparable, as verified by confocal fluorescence microscopy (Figs. 4B 4G) . Under these conditions, when the anti-FLAG immunoprecipitates were analyzed by Western blot with the anti-MIP antibody MIP31 (Fig. 3B) , an EGFP-MIP fusion protein (~50 kDa) was detected in the immunoprecipitated samples prepared from cells cotransfected with EGFP-MIP and FLAG-{gamma}E-crystallin expression vectors (Fig. 3B , lane 1). In contrast, no MIP fusion protein band was detected in immunoprecipitates from cells cotransfected with MIP-EGFP and FLAG-{gamma}E-crystallin expression vectors (Fig. 3B , lane 2). Negative results were also obtained when cells were cotransfected with the parental EGFP plasmid (i.e., no MIP fusion) and FLAG-{gamma}E-crystallin expression vectors (Fig. 3B , lane 3), further confirming the specificity of the coimmunoprecipitation reaction. These results indicate that EGFP-MIP but not MIP-EGFP interacts with FLAG-tagged {gamma}E-crystallin, suggesting that a bulky EGFP moiety at the C-terminus of MIP may interfere with the interaction between MIP and {gamma}E-crystallin. These data, along with the fact that the C-terminus of MIP was actually used as the bait molecule for the yeast two-hybrid screening for MIP-binding proteins, strongly support the notion that C-terminus of MIP may play a critical role in the interaction between MIP and {gamma}E-crystallin.



View larger version (44K):
[in this window]
[in a new window]
  		FIGURE 3. Effect of the bulky EFGP tag on interaction between MIP and {gamma}-crystallins. RK13 cells were transfected with the following combinations of expression vectors: pEGFP-MIP and pFLAG-{gamma}E cry (lane 1), pMIP-EGFP and pFLAG-{gamma}E cry (lane 2), pEGFP and pFLAG-{gamma}E cry (lane 3), pEGFP-MIP and pFLAG-{gamma}D cry (lane 4), and pMIP-EGFP and pFLAG-{gamma}D cry (lane 5). Forty-eight hours after transfection, total cell extracts were prepared from transfected cells and immunoprecipitated with anti-FLAG antibody-agarose conjugates. The immunoprecipitates were then subjected to 4% to 20% gradient SDS-PAGE, which was followed by transfer of proteins to PVDF membranes, Western blot using an anti-FLAG antibody (A), the anti-MIP antibody MIP31 (B), and an polyclonal anti-{gamma}-crystallin antibody (C). IP, immunoprecipitation; WB, Western blot.

 


View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 4. Confocal fluorescence microscopy of RK13 cells cotransfected with EGFP-tagged MIP and HcRed-tagged {gamma}-crystallin fusion expression vectors. RK13 cells were transfected with one of the following combinations of expression plasmids: pEGFP-MIP and pHcRed-{gamma}E-cry (A–E), pMIP-EGFP and pHcRed-{gamma}E-cry (F–J), pEGFP and pHcRed-{gamma}E-cry (K–O), or pEGFP-MIP and pHcRed-{gamma}D-cry (P–T). Forty-eight hours after transfection, the cells were fixed with a buffer containing 4% paraformaldehyde and counterstained with the nucleus-staining dye DAPI (1 µg/mL). The cells were visualized by confocal fluorescence microscopy. Images from three channels of fluorescence were obtained: DAPI (A, F, K, P), EGFP (B, G, L, Q), and HcRed (C, H, M, R). Superimposed images (EGFP+HcRed) were obtained by combining the EGFP and HcRed channels (D, I, N, S). Spatial quantification (E, J, O, T) was performed along a path across the plasma membrane, indicated by a white line in (D), (I), (N), and (S), respectively. EGFP fluorescence and HcRed fluorescence was quantified separately and plotted as a function of distance along the path. Blue dashed lines (E, J, T): approximate location of the plasma membrane. Photographs and spatial quantification graphs shown are representative (>90%) of those obtained from at least 12 different fields in at least three independent experiments. Bar, 10 µm.

 
Lack of Interaction between MIP and {gamma}D-crystallin
Because {gamma}E-crystallin is a member of the highly conserved {gamma}-crystallin gene family, we chose {gamma}D-crystallin to test whether MIP interacts with other members of the {gamma}-crystallin family. Possible interaction between MIP and {gamma}D-crystallin was investigated in a manner similar to that described earlier for interaction between MIP and {gamma}E-crystallin (Fig. 2) . RK13 cells were cotransfected with myc-MIP and FLAG-{gamma}D-crystallin, and cell extracts from cotransfected cells were immunoprecipitated with either anti-myc or anti-FLAG antibody, followed by Western blot with anti-myc or anti-FLAG antibodies. In the coimmunoprecipitation experiment using anti-myc antibody, FLAG-tagged {gamma}D-crystallin did not coimmunoprecipitate with myc-MIP (Fig. 2B , lane 6), whereas FLAG-tagged {gamma}E-crystallin coimmunoprecipitated with myc-MIP (Fig. 2B , lane 1), although the expression levels of FLAG-tagged {gamma}E-crystallin and {gamma}D-crystallin (Fig. 2F , compare lanes 6 and 1) and efficiency of IP for myc-MIP (Fig. 2A , compare lanes 6 and 1) were very similar. In the reciprocal coimmunoprecipitation experiment using anti-FLAG antibody, myc-MIP did not coimmunoprecipitate with FLAG-tagged {gamma}D-crystallin (Fig. 2D , lane 6), whereas myc-MIP coimmunoprecipitated with FLAG-tagged {gamma}E-crystallin (Fig. 2D , lane 1). Again, the expression levels of myc-MIP (Fig. 2E , compare lanes 6 and 1) and efficacy of IP for FLAG-tagged {gamma}E-crystallin and {gamma}D-crystallin (Fig. 2C , compare lanes 6 and 1) were very similar.

Furthermore, in a separate coimmunoprecipitation experiment involving EGFP-tagged MIP and either FLAG-tagged {gamma}E crystallin or FLAG-tagged {gamma}D-crystallin, the protein level of the immunoprecipitates and IP efficacy of FLAG-tagged {gamma}D-crystallin were comparable to those of FLAG-tagged {gamma}E-crystallin (Fig. 2E , compare lanes 4, 5 with lanes 1–3). Expression levels of EGFP-MIP or MIP-EGFP in cells transfected with these vectors were also comparable, as determined by confocal fluorescence microscopy (Figs. 4B 4G , described later). Under these conditions, FLAG-tagged {gamma}D-crystallin did not coimmunoprecipitate with either EGFP-MIP or MIP-EGFP (Fig. 3B , lanes 4, 5), whereas FLAG-tagged {gamma}E-crystallin coimmunoprecipitated with EGFP-MIP (Fig. 3B , lane 1). These results further confirmed that MIP interacts with {gamma}E-crystallin but does not interact with {gamma}D-crystallin, although they are highly homologous to each other. Indeed, the lack of interaction between MIP and {gamma}D-crystallin serves as a good physiological control for the specificity of the interaction between MIP and {gamma}E-crystallin.

Recruitment of {gamma}E-crystallin to the Plasma Membrane by MIP
To determine the cellular localization of interaction between MIP and {gamma}E-crystallin in mammalian cells, MIP and {gamma}E-crystallin were expressed as EGFP- and red fluorescent protein (HcRed)–tagged proteins, respectively, and the interaction between MIP and {gamma}E-crystallin was visualized in individual mammalian cells by confocal fluorescence microscopy.

RK13 cells were transiently cotransfected with the EGFP/MIP fusion vector (either pEGFP-MIP or pMIP-EGFP) and the expression vector pHcRed-{gamma}E-cry in which HcRed was fused at the N-terminal end of {gamma}E-crystallin. The transfected cells were examined by confocal fluorescence microscopy. When RK13 cells were cotransfected with pEGFP-MIP and pHcRed-{gamma}E-cry (Figs. 4A 4B 4C 4D 4E) , the EGFP fluorescence was concentrated to the plasma membrane (Fig. 4B) , although cytoplasmic and nuclear expression was also evident. The HcRed fluorescence was also localized to the plasma membrane (Fig. 4C) although low HcRed fluorescence in the cytoplasm and strong HcRed fluorescence in the nucleus were also observed. When the EGFP and HcRed fluorescence images of the same field were superimposed (Fig. 4D) , a yellow signal around the plasma membrane was observed, indicating that MIP and {gamma}E-crystallin colocalized to the plasma membrane. The colocalization of MIP and {gamma}E-crystallin was further demonstrated by spatial quantification of both EGFP and HcRed fluorescence along a path that goes across the plasma membrane (Fig. 4E) . The EGFP and HcRed fluorescence showed almost identical profiles along the path, both of which peaked at the same location (most likely the plasma membrane).

In contrast, cells cotransfected with MIP-EGFP and pHcRed-{gamma}E-cry showed different results (Figs. 4F 4G 4H 4I 4J) . EGFP fluorescence was localized to the plasma membrane, similar to cells cotransfected with EGFP-MIP and pHcRed-{gamma}E-cry (Fig. 4A) . Cytoplasmic and nuclear EGFP fluorescence was significantly less in MIP-EGFP–transfected cells than in EGFP-MIP–transfected cells. However, the HcRed fluorescence was completely diffused throughout the cells (Fig. 4H) . No plasma membrane localization of {gamma}E-crystallin was evident. The absence of yellow-membrane–associated fluorescence in these cells (Fig. 4I) indicates that {gamma}E-cry did not colocalize with MIP in cells cotransfected with MIP-EGFP and pHcRed-{gamma}E-cry. This result was confirmed by a spatial profile measurement (Fig. 4J) that showed that EGFP fluorescence did not overlap with HcRed fluorescence at the vicinity of the plasma membrane. These data indicate that the presence of a relatively bulky EGFP moiety at the C-terminus of MIP interfered with the interaction between MIP and {gamma}E-crystallin. This result was confirmed by a separate coimmunoprecipitation experiment that showed that EGFP-MIP, but not MIP-EGFP, coimmunoprecipitated with FLAG-tagged {gamma}E-crystallin in cotransfected RK13 cells (Fig. 3B) . In contrast, the presence of the relatively small myc tag at the C-terminus of MIP did not affect the interaction between MIP and {gamma}E-crystallin (Figs. 2B 2D , lanes 1 and 2). These data, together with the fact that the C-terminal peptide of MIP was actually used as the bait for yeast two-hybrid screening to identify MIP interacting proteins, suggest that the C-terminus of MIP may play a key role in its interaction with {gamma}E-crystallin.

In a control experiment, RK13 cells were cotransfected with pEGFP and pHcRed-{gamma}E-cry (Figs. 4K 4L 4M 4N 4O) . In the absence of the MIP moiety, EGFP showed a diffuse intracellular distribution typical of a soluble cytoplasmic protein (Fig. 4L) . HcRed fluorescence also displayed diffuse distribution under these conditions (Fig. 4M) . The superimposed image of EGFP and HcRed fluorescence (Fig. 4N) as well as the spatial profile measurement (Fig. 4O) showed that there is no localization of either EGFP or HcRed on the plasma membrane.

Lack of Recruitment of {gamma}D-crystallin to the Plasma Membrane by MIP
As shown earlier (Figs. 2 3) , MIP does not interact with {gamma}D-crystallin. This lack of interaction between MIP and {gamma}D-crystallin was further demonstrated using confocal fluorescence microscopy. RK13 cells were cotransfected with EGFP-MIP and HcRed-tagged {gamma}D-crystallin or {gamma}E-crystallin and then visualized by confocal fluorescence microscopy. As shown in Figure 4 , EGFP-MIP colocalized with {gamma}E-crystallin and recruited {gamma}E-crystallin to the plasma membrane (Figs. 4A 4B 4C 4D 4E) . In contrast, in cells coexpressing EGFP-MIP and HcRed-tagged {gamma}D-crystallin, only EGFP-MIP localized to the plasma membrane (Figs. 4Q 4S) , whereas HcRed-tagged {gamma}D-crystallin displayed a diffuse intracellular localization pattern without any membrane localization (Figs. 4R 4S) . This conclusion was confirmed by spatial quantification shown in Figure 4T . These data indicate that MIP does not interact with {gamma}D-crystallin in RK13 cells, and is thereby unable to recruit {gamma}D-crystallin to the plasma membrane.

In summary, the data presented in this article demonstrate that MIP interacts specifically with {gamma}E-crystallin in mammalian cells and this interaction results in the recruitment of {gamma}E-crystallin from the cytoplasm to the plasma membrane.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
MIP C-Terminus as Bait for Yeast Two-Hybrid Screening
In this study, we used a rat lens cDNA yeast two-hybrid library to screen for lens proteins that interact with MIP. The high hydrophobicity of MIP, a membrane channel protein, may interfere with its transport to the nucleus, required for the yeast two-hybrid interaction. Therefore, the full-length MIP may not be a suitable bait for yeast two-hybrid screening. The relatively hydrophilic C-terminus of MIP is located in the cytoplasmic side of the plasma membrane and is most likely involved in the MIP interaction with other proteins. Therefore, the 74-amino-acid C-terminus of MIP, rather than the full-length MIP molecule, was used as the bait for the yeast two-hybrid screening. We identified the lens fiber-specific {gamma}E crystallin as a binding protein to the lens MIP/aquaporin-0 C-terminal domain.

Although the full-length MIP bait construct was not used for the yeast two-hybrid screening, it was tested in the yeast two-hybrid assay. In most cases, it gave significantly less qualitative interaction signal (lighter-blue–stained colonies or longer times required for appearance of blue in the colonies) than when the C-terminal MIP fragment was used as the bait. This confirms our hypothesis that the full-length MIP may not be a good bait molecule for screening the yeast two-hybrid library. The interaction between {gamma}E-crystallin and the full-length MIP molecule in mammalian cells was subsequently confirmed by both coimmunoprecipitation assays and confocal fluorescence microscopy.

MIP Integration in the Plasma Membrane with a Long Hydrophilic Tag at the MIP N- or C-Terminus
To visualize the cellular localization of MIP in mammalian cells, we inserted an EGFP (265 amino acids) tag either at the N- or C-terminus of MIP. The addition of this long hydrophilic tail—almost the same length as MIP—at either end of the MIP molecule, still allows the hybrid protein to be inserted in the plasma membrane of epithelial cells. Even though the blocking of the MIP N-terminal does not interfere with plasma membrane insertion, we also observed some dense structures in the cytoplasm, suggesting that a free N-terminal is necessary for an efficient insertion in the membrane. It is interesting that the six mutations reported in the MIP protein linked to genetic cataracts and that prevent MIP integration in the plasma membrane, localize to hydrophobic transmembrane domains.1 2 3 4 5 6 These results suggest that the MIP transmembrane domain amino acid sequence is critical for the ability of MIP to be inserted into the plasma membrane and that addition of hydrophilic amino acids at the N- or C-terminal cytoplasmic domains does not interfere with MIP insertion in the plasma membrane.

Involvement of MIP C-Terminus in the Interaction between MIP and {gamma}E-crystallin
The coimmunoprecipitation experiments showed that both myc-MIP and MIP-myc (i.e., MIP with a short myc-tag, a 10-amino-acid peptide, at either the N- or C-terminal end of MIP) interacted with {gamma}E-crystallin (Fig. 2) . However, EGFP-MIP (with the bulky EGFP moiety at the N-terminal side of MIP), but not MIP-EGFP (with the bulky EGFP moiety at the C-terminal side of MIP), interacted with {gamma}E-crystallin (Fig. 3) . These results were confirmed further by confocal fluorescence microscopy, which showed that EGFP-MIP, but not MIP-EGFP, interacted with {gamma}E-crystallin, although both EGFP-MIP and MIP-EGFP were able to insert into the plasma membrane when transfected into rabbit kidney epithelial cells. These data suggest that MIP integration in the plasma membrane is not sufficient for interaction with {gamma}E-crystallin; a bulky EGFP tag at the C-terminus of MIP interferes with this interaction. These results suggest that the C-terminus of MIP may play a key role in its interaction with {gamma}E-crystallin. In contrast, {gamma}E-crystallin is able to interact with MIP when a FLAG tag is attached to either the N- or C-terminus of {gamma}E-crystallin (Fan et al., unpublished data, 2003), suggesting the addition of the short FLAG tag (a nine-amino-acid peptide) may not interfere with the tridimensional structure of {gamma}E-crystallin necessary for the interaction with MIP.

Interaction of MIP with {gamma}E-crystallin but Not with {gamma}D-crystallin
Both the biochemical (Figs. 2 3) and confocal fluorescence microscopy (Fig. 4) data presented in this article show that myc-tagged MIP (either myc-MIP or MIP-myc) and EGFP-MIP transfected into rabbit kidney epithelial cells are able to interact with {gamma}E-crystallin but not with {gamma}D-crystallin. {gamma}E-crystallin is a member of the highly conserved family of {gamma}-crystallins,23 33 34 specifically expressed in the lens fibers and differentially regulated during lens development.35 36 The particular function of each member of the crystallin family in the lens is not known. {gamma}E- and {gamma}D-crystallins are 91% identical at the amino acid level and the nonconserved amino acids are mainly in the third Greek key motif.23 33 The 14 different amino acids in {gamma}E- and {gamma}D-crystallins result in differences in the intramolecular ion pairs on the surfaces of both {gamma}-crystallins.37 The clustering of four histidines between positions 111 and 125 in the rat {gamma}E-crystallin but the presence of only one histidine in the same region of rat {gamma}D-crystallin may result in differences in their intermolecular interactions.37 Therefore, different amino acids in {gamma}E- and {gamma}D-crystallins may be available for interaction with other proteins, with only {gamma}E-crystallin being able to interact with MIP under the experimental conditions we have used. In addition, our data are consistent with the view that closely related {gamma}-crystallin family members may have distinct functions.

Recruitment of {gamma}E-crystallin to the Plasma Membrane by MIP
The addition of an HcRed tail to {gamma}E-crystallin allowed us to visualize the recruitment of {gamma}E-crystallin to the plasma membrane by MIP. Our results show that integration of MIP into the plasma membrane and an unblocked MIP C-terminus is necessary for recruitment of {gamma}E-crystallin to the plasma membrane. A calcium-ion–binding site at the {gamma}-crystallin Greek key motif has been identified.38 As MIP water channel is calcium regulated,10 it is tempting to speculate that {gamma}E-crystallin calcium binding has a role in regulating MIP channel activity.

Functional Link between MIP and {gamma}-crystallins
Both MIP and {gamma}-crystallins are specifically expressed in the lens fibers. {gamma}E-crystallin plays a role in transparency of the mouse lens. Five mutations resulting in genetic cataracts with a dominant phenotype have been identified in the murine {gamma}E-crystallin gene.24 26 29 39 The tightly packed lens fiber structure is disrupted and appearance of vacuolated cells is observed when either MIP or {gamma}E-crystallin is mutated.1 2 3 4 26 29 39 It is also interesting to point out that perturbation of suture formation is observed when either MIP is not expressed9 or the {gamma}E-crystallin is mutated.29 39 Furthermore, {gamma}-crystallins are selectively lost in the Cat Fraser MIP mutant.40 The Elo cataract is a mouse genetic cataract that results from a mutation in the {gamma}E-crystallin gene, in which the cataract phenotype appears at embryonic days 12 to 13.41 42 43 It is noteworthy that this is the same embryonic stage at which MIP begins to be expressed in the lens primary fibers44 and is the same stage at which the MIP Cat Fraser mouse mutant shows the cataract phenotype.45 Indeed, we have found that MIP does not interact with the {gamma}E-crystallin Elo mutant, whereas it interacts with its wild-type counterpart (Fan et al., unpublished data, 2003). Our results demonstrate for the first time specific interaction between MIP and {gamma}E-crystallin, providing evidence of a functional link between MIP and {gamma}-crystallins. Our data also suggest that interaction between MIP and {gamma}E-crystallin may have important implications for how MIP and {gamma}-crystallins are involved in lens cataractogenesis.

It is intriguing that {gamma}E-crystallin is a pseudogene in humans, resulting in lack of expression of this gene.34 36 46 47 Our results suggest that subtle amino acid changes may affect the interaction of {gamma}-crystallins with MIP. Therefore, evolutionary changes in the amino acid sequences of {gamma}-crystallins and/or MIP expressed in humans may result in the interaction of human MIP with another member of the {gamma}-crystallin family instead of {gamma}E-crystallin. Future research will help elucidate this paradigm.


   Acknowledgements
 
The authors are grateful to Joram Piatigorsky for critical review of the manuscript.


   Footnotes
 
Submitted for publication July 9, 2003; revised October 9 and 30, 2003; accepted November 7, 2003.

Disclosure: J. Fan, None; A.K. Donovan, None; D.R. Ledee, None; P.S. Zelenka, None; R.N. Fariss, None; A.B. Chepelinsky, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Ana B. Chepelinsky, National Eye Institute, National Institutes of Health, 7 Memorial Drive, MSC 0704, Building 7, Room 105, Bethesda, MD 20892-0704; abc{at}helix.nih.gov.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Shiels A, Bassnett S. Mutations in the founder of the MIP gene family underlie cataract development in the mouse. Nat Genet. 1996;12:212–215.[CrossRef][ISI][Medline][Order article via Infotrieve]
  2. Shiels A, Mackay D, Bassnett S, Al-Ghoul K, Kuszak J. Disruption of lens fiber cell architecture in mice expressing a chimeric AQP0-LTR protein. FASEB J. 2000;14:2207–2212.[Abstract/Free Full Text]
  3. Sidjanin DJ, Parker-Wilson DM, Neuhauser-Klaus A, et al. A 76-bp deletion in the Mip gene causes autosomal dominant cataract in Hfi mice. Genomics. 2001;74:313–319.[CrossRef][ISI][Medline][Order article via Infotrieve]
  4. Okamura T, Miyoshi I, Takahashi K, et al. Bilateral congenital cataracts result from a gain-of-function mutation in the gene for aquaporin-0 in mice. Genomics. 2003;81:361–368.[CrossRef][ISI][Medline][Order article via Infotrieve]
  5. Berry V, Francis P, Kaushal S, Moore A, Bhattacharya S. Missense mutations in MIP underlie autosomal dominant "polymorphic" and lamellar cataracts linked to 12q. Nat Genet. 2000;25:15–17.[CrossRef][ISI][Medline][Order article via Infotrieve]
  6. Francis P, Chung JJ, Yasui M, et al. Functional impairment of lens aquaporin in two families with dominantly inherited cataracts. Hum Mol Genet. 2000;9:2329–2334.[Abstract/Free Full Text]
  7. Varadaraj K, Kushmerick C, Baldo GJ, Bassnett S, Shiels A, Mathias RT. The role of MIP in lens fiber cell membrane transport. J Membr Biol. 1999;170:191–203.[CrossRef][ISI][Medline][Order article via Infotrieve]
  8. Shiels A, Bassnett S, Varadaraj K, et al. Optical dysfunction of the crystalline lens in aquaporin-0-deficient mice. Physiol Genom. 2001;7:179–186.[Abstract/Free Full Text]
  9. Al-Ghoul KJ, Kirk T, Kuszak AJ, Zoltoski RK, Shiels A, Kuszak JR. Lens structure in MIP-deficient mice. Anat Rec. 2003;273A:714–730.
  10. Nemeth-Cahalan KL, Hall JE. pH and calcium regulate the water permeability of aquaporin 0. J Biol Chem. 2000;275:6777–6782.[Abstract/Free Full Text]
  11. Drake KD, Schuette D, Chepelinsky AB, Jacob TJ, Crabbe MJ. pH-Dependent channel activity of heterologously-expressed main intrinsic protein (MIP) from rat lens. FEBS Lett. 2002;512:199–204.[CrossRef][ISI][Medline][Order article via Infotrieve]
  12. Ehring GR, Lagos N, Zampighi GA, Hall JE. Phosphorylation modulates the voltage dependence of channels reconstituted from the major intrinsic protein of lens fiber membranes. J Membr Biol. 1992;126:75–88.[ISI][Medline][Order article via Infotrieve]
  13. Michea LF, de la Fuente M, Lagos N. Lens major intrinsic protein (MIP) promotes adhesion when reconstituted into large unilamellar liposomes. Biochemistry. 1994;33:7663–7669.[CrossRef][Medline][Order article via Infotrieve]
  14. Gruijters WT. A non-connexon protein (MIP) is involved in eye lens gap-junction formation. J Cell Sci. 1989;93:509–513.[Abstract/Free Full Text]
  15. Dunia I, Recouvreur M, Nicolas P, Kumar N, Bloemendal H, Benedetti EL. Assembly of connexins and MP26 in lens fiber plasma membranes studied by SDS-fracture immunolabeling. J Cell Sci. 1998;111:2109–2120.[Abstract]
  16. Fotiadis D, Hasler L, Muller DJ, Stahlberg H, Kistler J, Engel A. Surface tongue-and-groove contours on lens MIP facilitate cell-to-cell adherence. J Mol Biol. 2000;300:779–789.[CrossRef][ISI][Medline][Order article via Infotrieve]
  17. Takemoto L, Takehana M, Horwitz J. Covalent changes in MIP26K during aging of the human lens membrane. Invest Ophthalmol Vis Sci. 1986;27:443–446.[Abstract/Free Full Text]
  18. Takemoto L, Kuck J, Kuck K. Changes in the major intrinsic polypeptide (MIP26K) during opacification of the Emory mouse lens. Exp Eye Res. 1988;47:329–336.[CrossRef][ISI][Medline][Order article via Infotrieve]
  19. Schey KL, Fowler JG, Shearer TR, David L. Modifications to rat lens major intrinsic protein in selenite-induced cataract. Invest Ophthalmol Vis Sci. 1999;40:657–667.[Abstract/Free Full Text]
  20. Schey KL, Little M, Fowler JG, Crouch RK. Characterization of human lens major intrinsic protein structure. Invest Ophthalmol Vis Sci. 2000;41:175–182.[Abstract/Free Full Text]
  21. Girsch SJ, Peracchia C. Calmodulin interacts with a C-terminus peptide from the lens membrane protein MIP26. Curr Eye Res. 1991;10:839–849.[ISI][Medline][Order article via Infotrieve]
  22. Swamy-Mruthinti S. Glycation decreases calmodulin binding to lens transmembrane protein, MIP. Biochim Biophys Acta. 2001;1536:64–72.[Medline][Order article via Infotrieve]
  23. Wistow GJ, Piatigorsky J. Lens crystallins: the evolution and expression of proteins for a highly specialized tissue. Annu Rev Biochem. 1988;57:479–504.[CrossRef][ISI][Medline][Order article via Infotrieve]
  24. Klopp N, Favor J, Loster J, et al. Three murine cataract mutants (Cat2) are defective in different gamma-crystallin genes. Genomics. 1998;52:152–158.[CrossRef][ISI][Medline][Order article via Infotrieve]
  25. Stephan DA, Gillanders E, Vanderveen D, et al. Progressive juvenile-onset punctate cataracts caused by mutation of the gammaD-crystallin gene. Proc Natl Acad Sci USA. 1999;96:1008–1012.[Abstract/Free Full Text]
  26. Klopp N, Loster J, Graw J. Characterization of a 1-bp deletion in the gammaE-crystallin gene leading to a nuclear and zonular cataract in the mouse. Invest Ophthalmol Vis Sci. 2001;42:183–187.[Abstract/Free Full Text]
  27. Graw J, Neuhauser-Klaus A, Loster J, Favor J. A 6-bp deletion in the Crygc gene leading to a nuclear and radial cataract in the mouse. Invest Ophthalmol Vis Sci. 2002;43:236–240.[Abstract/Free Full Text]
  28. Santhiya ST, Shyam Manohar M, Rawlley D, et al. Novel mutations in the gamma-crystallin genes cause autosomal dominant congenital cataracts. J Med Genet. 2002;39:352–358.[Free Full Text]
  29. Graw J. Cataract mutations and lens development. Prog Retin Eye Res. 1999;18:235–267.[CrossRef][ISI][Medline][Order article via Infotrieve]
  30. Nandrot E, Slingsby C, Basak A, et al. Gamma-D crystallin gene (CRYGD) mutation causes autosomal dominant congenital cerulean cataracts. J Med Genet. 2003;40:262–267.[Abstract/Free Full Text]
  31. Smith RS, Hawes NL, Chang B, et al. Lop12, a mutation in mouse Crygd causing lens opacity similar to human Coppock cataract. Genomics. 2000;63:314–320.[CrossRef][ISI][Medline][Order article via Infotrieve]
  32. Graw J, Klopp N, Neuhauser-Klaus A, Favor J, Loster J. Crygf(Rop): the first mutation in the Crygf gene causing a unique radial lens opacity. Invest Ophthalmol Vis Sci. 2002;43:2998–3002.[Abstract/Free Full Text]
  33. Breitman ML, Lok S, Wistow G, et al. Gamma-crystallin family of the mouse lens: structural and evolutionary relationships. Proc Natl Acad Sci USA. 1984;81:7762–7766.[Abstract/Free Full Text]
  34. Aarts HJ, den Dunnen JT, Leunissen J, Lubsen NH, Schoenmakers JG. The gamma-crystallin gene families: sequence and evolutionary patterns. J Mol Evol. 1988;27:163–172.[CrossRef][ISI][Medline][Order article via Infotrieve]
  35. Goring DR, Breitman ML, Tsui LC. Temporal regulation of six crystallin transcripts during mouse lens development. Exp Eye Res. 1992;54:785–795.[CrossRef][ISI][Medline][Order article via Infotrieve]
  36. Brakenhoff RH, Aarts HJ, Reek FH, Lubsen NH, Schoenmakers JG. Human gamma-crystallin genes: a gene family on its way to extinction. J Mol Biol. 1990;216:519–532.[CrossRef][ISI][Medline][Order article via Infotrieve]
  37. Norledge BV, Hay RE, Bateman OA, Slingsby C, Driessen HP. Towards a molecular understanding of phase separation in the lens: a comparison of the X-ray structures of two high Tc gamma-crystallins, gammaE and gammaF, with two low Tc gamma-crystallins, gammaB and gammaD. Exp Eye Res. 1997;65:609–630.[CrossRef][ISI][Medline][Order article via Infotrieve]
  38. Rajini B, Shridas P, Sundari CS, et al. Calcium binding properties of gamma-crystallin: calcium ion binds at the Greek key bet