γE-crystallin recruitment to the plasma membrane by specific interaction between lens MIP/aquaporin-0 and γE-crystallin (original) (raw)
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Investigative Ophthalmology & Visual Science, 2004
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 ␥Ecrystallin 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. ␥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 ␥E-crystallin interacts specifically with full-length MIP in mammalian cells. MIP did not interact with ␥D-crystallin, another member of the highly conserved ␥-crystallin gene family. Confocal fluorescence microscopy demonstrated that MIP interacted with ␥E-crystallin in individual mammalian cells and that this interaction resulted in the recruitment of ␥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 ␥-crystallins in lens fibers. (Invest Ophthal
Membranes, 2021
α-crystallin is a major protein found in the mammalian eye lens that works as a molecular chaperone by preventing the aggregation of proteins and providing tolerance to stress in the eye lens. These functions of α-crystallin are significant for maintaining lens transparency. However, with age and cataract formation, the concentration of α-crystallin in the eye lens cytoplasm decreases with a corresponding increase in the membrane-bound α-crystallin, accompanied by increased light scattering. The purpose of this review is to summarize previous and recent findings of the role of the: (1) lens membrane components, i.e., the major phospholipids (PLs) and sphingolipids, cholesterol (Chol), cholesterol bilayer domains (CBDs), and the integral membrane proteins aquaporin-0 (AQP0; formally MIP26) and connexins, and (2) α-crystallin mutations and post-translational modifications (PTMs) in the association of α-crystallin to the eye lens’s fiber cell plasma membrane, providing thorough insight...
Journal of Molecular Biology, 1998
Lens major intrinsic protein (MIP) is the founding member of the MIP family of membrane channel proteins. Its isolation from ovine lens ®bre cell membranes and its two-dimensional crystallization are described. Membranes were solubilized with n-octyl-b-D-glucoside and proteins fractionated by sucrose gradient centrifugation containing decyl-b-D-maltoside. MIP was puri®ed by cation exchange chromatography, and homogeneity was assessed by mass analysis in the scanning transmission electron microscope. Puri®ed MIP reconstituted into a lipid bilayer at a low lipid-to-protein ratio formed highly ordered tetragonal two-dimensional crystals. The square unit cell had a side length of 6.4 nm, and exhibited in negative stain four stain-excluding elongated domains surrounding a central stain-®lled depression. Projection maps of freeze-dried crystals exhibited a resolution of 9 A Ê , and revealed a monomer structure of MIP consisting of distinct densities. Despite signi®cant differences in the packing of tetramers in the crystals, the projection map of the MIP monomer was similar to that of aquaporin-1 (AQP1), the ®rst member of the MIP family which had its structure resolved to 6 A Ê . Our protocols for the puri®cation and reconstitution of MIP establish the feasibility for future work to visualize structure elements which determine the diverse functional properties of the MIP family members.
Journal of Molecular Biology, 1998
Lens major intrinsic protein (MIP) is the founding member of the MIP family of membrane channel proteins. Its isolation from ovine lens ®bre cell membranes and its two-dimensional crystallization are described. Membranes were solubilized with n-octyl-b-D-glucoside and proteins fractionated by sucrose gradient centrifugation containing decyl-b-D-maltoside. MIP was puri®ed by cation exchange chromatography, and homogeneity was assessed by mass analysis in the scanning transmission electron microscope. Puri®ed MIP reconstituted into a lipid bilayer at a low lipid-to-protein ratio formed highly ordered tetragonal two-dimensional crystals. The square unit cell had a side length of 6.4 nm, and exhibited in negative stain four stain-excluding elongated domains surrounding a central stain-®lled depression. Projection maps of freeze-dried crystals exhibited a resolution of 9 A Ê , and revealed a monomer structure of MIP consisting of distinct densities. Despite signi®cant differences in the packing of tetramers in the crystals, the projection map of the MIP monomer was similar to that of aquaporin-1 (AQP1), the ®rst member of the MIP family which had its structure resolved to 6 A Ê . Our protocols for the puri®cation and reconstitution of MIP establish the feasibility for future work to visualize structure elements which determine the diverse functional properties of the MIP family members.
Molecular organization and structural stability of .beta.s-crystallin from calf lens
Biochemistry, 1990
&-Crystallin has been purified to homogeneity. Its structural features and conformational behavior have been studied in solution. Protein secondary structure was estimated by curve fitting of far-UV circular dichroism spectra, which gave 16% a-helix, 45% P-sheet, 12% bends, and 27% remainders. This result indicates that the structural organization of &-crystallin is reasonably similar to that of other p and 7 family members. A comparison assessed between &and 7,-crystallin by the use of predictive methods (flexibility and volume plots) reveals that the two proteins differ in respect to their local flexibility and packing, although they show similar overall organization. The interdomain and the C-terminal regions were found to be more flexible in &-crystallin. This finding can be explained by the presence of smaller amino acid residues within these structural districts. The location of one out of four tryptophans, i.e., Trp-162, in a flexible and exposed region of the protein was found to be the origin of the fluorescence heterogeneity. In fact, the fluorescence emission maximum of the native protein, centered at 328 nm, is due to two emitting centers, whose emission maxima are located at 323 and 330 nm, respectively, as evidenced by acrylamide quenching of fluorescence. The effect of perturbing agents, such as p H and guanidine hydrochloride, on the conformational behavior of p, has also been evaluated by numerous spectroscopic techniques. The range of pH stability was between 6.5 and 8. Above this interval, a conformational change takes place. In the acid region, the protein is unstable and precipitates irreversibly. The conformational resistance to guanidine hydrochloride has also been shown to be weak. GdnHCl denaturation curves were neither superimposable nor ascribable to a very cooperative transition. This result suggests a non-two-state denaturation equilibrium reflecting the presence of structural domains. The main conclusion of our work is that the protein shows a very narrow range of stability. This result indicates that an inherent structural stability may not be a general property of lens proteins. Therefore, the evolutive hypothesis about a specific recruitment of stable proteins in the lens architecture may need reconsideration.
Molecular vision, 2010
To study the relationship of pigment epithelium-derived factor (PEDF) expression with the expression of vimentin and αB-crystallin by lens epithelial cells. Methods: Lens epithelial cells adhering to anterior capsules taken from young donor eyes aged from 20 to 35 years were cultured and passaged. We designed small interfering RNA (siRNA) constructs to specifically downregulate the expression of PEDF by these primary lens epithelial cells. Quantitative PCR was used to confirm the downregulation of PEDF RNA expression following infection of lens epithelial cells. To determine whether altering the expression of PEDF would effect the expression of vimentin or αB-crystallin, we performed western blotting 48 h after expression of the PEDF-directed siRNA. Results: PEDF RNA expression in the human lens epithelial cells was strongly downregulated by the three separate siRNA constructs. Western blotting revealed that the downregulation of PEDF expression resulted in a concomitant decrease in expression of vimentin and an increase in αB-crystallin protein.
Classification of Rat Lens Crystallins and Identification of Proteins Encoded by Rat Lens mRNA
European Journal of Biochemistry, 2005
Endogenous rat lens crystallins have been separated by gel filtration into four fractions, a, fiH, Br. and 7-crystallin. Elution patterns of soluble lens proteins from animals of different ages show a relative decrease of /?H and ;-crystallin during aging. Conversely the relative amounts of r and jL-crystallin are enhanced. The rat crystallin subunits from the four fractions were characterized by one-dimensional and two-dimensional gel electrophoretic techniques. From the results a classification could be derived and a nomenclature for the soluble rat lens proteins is proposed. The products synthesized by rat lens mRNAs in a heterologous cell-free system have also been characterized. Co-electrophoresis of the radioactive products synthesized de novo together with the isolated unlabeled protein fractions on two-dimensional gels shows the relation between primary gene products and their posttranslationally modified derivatives.
Journal of Biological Chemistry, 2012
Background: The small heat shock proteins, ␣A-crystallin and ␣B-crystallin are considered to be two subunits of one single monolithic lens protein, ␣-crystallin. Results: ␣A-Crystallin and ␣B-crystallin fractionate independent of each other and in two separate membrane compartments. Conclusion: ␣A-Crystallin and ␣B-crystallin are two independent proteins in the lens. Significance: These data provide functional insight into why ␣A-crystallin and ␣B-crystallin null mice have disparate phenotypes.