Structural Analysis of a MIP Family Protein from the Digestive Tract of Cicadella viridis
1995; Elsevier BV; Volume: 270; Issue: 29 Linguagem: Inglês
10.1074/jbc.270.29.17414
ISSN1083-351X
AutoresFabienne Beuron, Franoise Le Cahérec, Marie‐Thérèse Guillam, Annie Cavalier, A. Garret, Jean‐Pierre Tassan, Christian Delamarche, Patrick Schultz, Véronique Mallouh, Jean‐Paul Rolland, Jean-Franois Hubert, Jean Gouranton, Daniel Thomas,
Tópico(s)Coenzyme Q10 studies and effects
ResumoHomopteran insects, and especially Cicadella viridis, display in their digestive tract a specialized epithelial differentiation, the filter chamber (FC) acting as a water-shunting complex. The main intrinsic membrane protein of the FC is a 25,000-Da polypeptide (P25). In this paper we demonstrate that this P25 polypeptide is a member of the MIP family of membrane channel proteins, and that P25 forms homotetramers in the native membranes.Using polymerase chain reaction, a 360-base pair cDNA, named cic, was isolated from RNA of the FC. cic encodes a 119-amino acid polypeptide (CIC) whose homologies with MIP26, AQP1 (CHIP), AQP2, and γ-TIP are 38, 38, 34, and 20%, respectively. Using a specific antibody raised against a 15-amino acid peptide from the CIC sequence, we concluded that CIC and P25 are identical entities, and hence that P25 belongs to the MIP family.We investigated the quaternary structure of P25 in the membranes of the FC using biophysical analysis of P25 nondenaturing detergent micelles, scanning transmission electron microscopy, and image processing of conventional transmission electron microscopic images. All those different approaches converged to the conclusion that P25 exists as an homotetramer forming a regular two-dimensional array in the membranes. Homopteran insects, and especially Cicadella viridis, display in their digestive tract a specialized epithelial differentiation, the filter chamber (FC) acting as a water-shunting complex. The main intrinsic membrane protein of the FC is a 25,000-Da polypeptide (P25). In this paper we demonstrate that this P25 polypeptide is a member of the MIP family of membrane channel proteins, and that P25 forms homotetramers in the native membranes. Using polymerase chain reaction, a 360-base pair cDNA, named cic, was isolated from RNA of the FC. cic encodes a 119-amino acid polypeptide (CIC) whose homologies with MIP26, AQP1 (CHIP), AQP2, and γ-TIP are 38, 38, 34, and 20%, respectively. Using a specific antibody raised against a 15-amino acid peptide from the CIC sequence, we concluded that CIC and P25 are identical entities, and hence that P25 belongs to the MIP family. We investigated the quaternary structure of P25 in the membranes of the FC using biophysical analysis of P25 nondenaturing detergent micelles, scanning transmission electron microscopy, and image processing of conventional transmission electron microscopic images. All those different approaches converged to the conclusion that P25 exists as an homotetramer forming a regular two-dimensional array in the membranes. Water crosses the plasma membranes of most cells by diffusion through the lipid bilayer. Particular cell types exhibit high water permeability due to water selective membrane proteins(1Finkelstein A. Water Movement Through Lipid Bilayers, Pores, and Plasma Membranes: Theory and reality. Wiley and Sons, New York1987Google Scholar). Such proteins have been recently identified and gathered in the aquaporin family(2Agre P. Preston G.M. Smith B.L. Jung J.S. Raina S. Moon C. Guggino W.B. Nielsen S. Am. J. Physiol. 1993; 265: F463-F476Crossref PubMed Google Scholar) : AQP1 (CHIP) in mammalian red cell membranes and proximal renal tubules(3Denker B.M. Smith B.L. Kuhadja F.P. Agre P. J. Biol. Chem. 1988; 263: 15634-15642Abstract Full Text PDF PubMed Google Scholar, 4Smith B.L. Agre P. J. Biol. 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Chem. 1994; 269: 5497-5500Abstract Full Text PDF PubMed Google Scholar, 11Jung J.S. Bhat R.V. Preston G.P. Guggino W.B. Baraban J.M. Agre P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 13052-13056Crossref PubMed Scopus (613) Google Scholar), AQP5 in rat salivary glands(12Raina S. Preston G.M. Guggino W.B. Agre P. J. Biol. Chem. 1995; 270: 1908-1912Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar), and γ-TIP in Arabidopsis thaliana(13Maurel C. Reizer I. Schroeder J.I. Chrispeels M.J. EMBO J. 1993; 12: 2241-2247Crossref PubMed Scopus (415) Google Scholar). These membrane proteins belong to a larger family of polypeptides, forming transmembrane channels and found in bacteria, plants, and animals (14Pao G.M. Wu L.-F. Johnson K.D. Höfte H. Chrispeels M.J. Sweet G. Sandal N.N. Saier Jr., M.H. Mol. Microbiol. 1991; 5: 33-37Crossref PubMed Scopus (148) Google Scholar) called the MIP family, from its archetype, MIP26, the major intrinsic protein of bovine lens fibers(15Gorin M.B. Yancey S.B. Cline J. Revel J.-P. Horwitz J. Cell. 1984; 39: 49-59Abstract Full Text PDF PubMed Scopus (407) Google Scholar). The aquaporins are permeated by water, but fail to pass protons, or other ions, or uncharged solutes. The explanation for water-selective transport is unknown since only limited structural information exists. The understanding of the selectivity at the molecular level supports the quest for three-dimensional structural information. We previously investigated the filter chamber (FC)1 1The abbreviations used are: FCfilter chamberBSAbovine serum albuminPBSphosphate-buffered salinePCRpolymerase chain reactionOGn-octyl-β-D-glucopyranosidePAGEpolyacrylamide gel electrophoresisCTEMconventional transmission electron microscopySTEMscanning transmission electron microscopy. 1The abbreviations used are: FCfilter chamberBSAbovine serum albuminPBSphosphate-buffered salinePCRpolymerase chain reactionOGn-octyl-β-D-glucopyranosidePAGEpolyacrylamide gel electrophoresisCTEMconventional transmission electron microscopySTEMscanning transmission electron microscopy. of some homopteran sap sucking insects. In this highly specialized epithelial complex of the digestive tract, the large excess of water ingested with the sap is rapidly transferred from initial midgut to terminal midgut or Malpighian tubules down a transepithelial osmotic gradient. We described the morphology of this water shunting complex in Cicadella viridis(16Gouranton J. J. Microsc. (Paris). 1968; 7: 559-574Google Scholar). We showed that the whole surface of the plasma membranes from this highly water permeable FC is covered by a regular array of membrane particles, and that the major constituent of FC purified membranes is a 25,000-dalton hydrophobic polypeptide (P25) (17Hubert J.-F. Thomas D. Cavalier A. Gouranton J. Biol. Cell. 1989; 66: 155-163Crossref PubMed Scopus (20) Google Scholar). Finally, we demonstrated that FC is highly enriched in mRNA species encoding water channel proteins when microinjected into Xenopus oocytes(18Guillam M.-T. Beuron F. Grandin N. Hubert J.-F. Boisseau C. Cavalier A. Couturier A. Gouranton J. Thomas D. Exp. Cell Res. 1992; 200: 301-305Crossref PubMed Scopus (8) Google Scholar). filter chamber bovine serum albumin phosphate-buffered saline polymerase chain reaction n-octyl-β-D-glucopyranoside polyacrylamide gel electrophoresis conventional transmission electron microscopy scanning transmission electron microscopy. filter chamber bovine serum albumin phosphate-buffered saline polymerase chain reaction n-octyl-β-D-glucopyranoside polyacrylamide gel electrophoresis conventional transmission electron microscopy scanning transmission electron microscopy. As a result of its extremely high representation in the plasma membranes, it appears very likely that P25 takes an important part in the constitution of the regular array within the native membranes and is involved in the water transport function of the FC epithelia. We hypothesized that it could be a water specialized channel and thus belongs to the MIP family as all other previously characterized water channels do. Beside functional studies of P25, we focused our work on cloning the cDNA encoding P25 associated with its structural determination. In the first part of this work we demonstrate that the polypeptide P25 is a member of the MIP family. It was therefore interesting to investigate the structural organization of P25 in order to compare our observations with data relative to the structure of two previously characterized MIP proteins: MIP26 and AQP1 (CHIP). Due to its abundance in the native membranes, P25 constitutes two-dimensional crystals. This unique distribution for a MIP family protein is very favorable for a structural investigation since native membranes can be used directly for negative staining or cryoelectron microscopy. We report, in the second part of this work, the native structural organization of P25. Insects, C. viridis, were harvested from wet meadows from summer to autumn. After dissection, freshly collected filter chambers were homogenized in 10 mM Tris-HCl, pH 7.3, 0.4 mM phenylmethylsulfonyl fluoride. Membranes were purified over a discontinuous sucrose gradient as described(17Hubert J.-F. Thomas D. Cavalier A. Gouranton J. Biol. Cell. 1989; 66: 155-163Crossref PubMed Scopus (20) Google Scholar). The membrane fraction was then washed 18 h at 4°C in an alkaline buffer (5 mM glycine, 1 mM EDTA, 5 mM β-mercaptoethanol) to eliminate the extrinsic proteins(19Marchesi S.I. Steers E. Marchesi V.T. Tillack T.W. Biochemistry. 1970; 9: 50-57Crossref PubMed Scopus (222) Google Scholar). Electrophoretic analysis of membrane proteins were performed on SDS-polyacrylamide gels according to Laemmli(20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). Gels were stained with silver nitrate(21Sammons D.W. Adams L.D. Nishizama D.N. Electrophoresis. 1981; 2: 135Crossref Scopus (465) Google Scholar). For Western blotting studies, proteins were electrophoretically transferred onto nitrocellulose, incubated with rabbit antisera, and revealed by peroxidase-conjugated anti-rabbit IgG. Anti-P25 serum was used with a 1000-fold dilution and antipeptide serum was used with 40- and 100-fold dilutions. Purified P25 protein was also dotted on nitrocellulose in detergent solution and subsequently revealed as in Western blotting. Frozen C. viridis, stored at −70°C were embedded in historesine, and then plunged in liquid nitrogen-cooled isopentane. Cryostat sections of 16 μm were obtained at −25°C, they were then deposited on slides treated with 2% 3-aminopropyltriethoxysilane in acetone. After 4 h, sections were fixed for 5 min with a solution of 4% paraformaldehyde, 0.05% glutaraldehyde in 0.1 M phosphate buffer then rinsed with the same buffer. For labeling, sections were treated for 30 min with PAS (0.02 M phosphate buffer, pH 7.5, 0.03% saponin), then with 1% BSA in PAS. They were incubated for 2 h at 37°C in a solution of anti-P25 serum diluted 500-fold in BSA-PAS. Sections were then washed with PAS and incubated for 30 min at 37°C in a solution of GAR/IgG/fluorescein isothiocyanate diluted 20-fold in BSA-PAS. After washing in a solution of Evan's Blue-phosphate buffer, preparations were mounted in a solution of Evan's Blue glycerol phosphate buffer. Observations were carried out with a fluorescent light microscope. Filter chambers were fixed for 4 h in a solution of 4% paraformaldehyde, 0.01% glutaraldehyde in 0.1 M phosphate buffer saline (PBS). After rinsing, samples were dehydrated and embedded in Lowicryl according to Roth et al.(22Roth J. Bendayan M. Carlemalm E. Villiger W. Garavito M. J. Histochem. Cytochem. 1981; 29: 663-671Crossref PubMed Scopus (508) Google Scholar). Ultrathin sections were picked up on collodion carbon-coated nickel grids then immediately deposited on a solution of 1% BSA in PBS and incubated overnight at 4°C. Sections were then incubated for 2 h at room temperature with primary antibody diluted 300-fold in PBS-BSA, rinsed 3 times with PBS-BSA, and incubated 1 h at room temperature with a 10-nm GAR-gold secondary antibody diluted 40-fold in PBS-BSA. After washing, grids were stained with 2% uranyl acetate. Alternatively, freshly isolated membranes were deposited on glow-discharged carbon-coated nickel grids and incubated in the same solutions as for sections, 1 h at room temperature. After rinsing, the grids were fixed on a drop of 2.5% glutaraldehyde for 5 min, then negatively stained with 2% uranyl acetate. A pair of degenerate primers (sense, 5′-ATC AAC CC(AGTC) GCC GT(AGCT) ACC-3′, and antisense, 5-′CAG (AGCT)GA (GCA)CG GGC (AGCT)GG GTT-3′) were designed according to the two highly conserved NPA boxes found in cloned MIP family proteins. Total RNA was isolated by tissue homogenization in a lithium chloride-urea solution (23Auffray C. Rougeon F. Eur. J. Biochem. 1980; 107: 303-314Crossref PubMed Scopus (2084) Google Scholar) followed by phenol extraction and alcohol precipitation. RNA was reverse transcribed using random hexamers as primers (Life Technologies, Inc.). 100 ng of cDNA were used as template for PCR amplification (94°C, 1 min; 35°C, 1 min; 72°C, 1 min; 35 cycles) using 100 pmol of degenerated primers and 1.75 mM MgCl2. The PCR products were resolved on 1.8% agarose gel and stained with ethidium bromide. The PCR products were cloned in pBluescript (Stratagene) vector and sequenced by the double-strand dideoxynucleotide termination method (Pharmacia kit). Digestive tracts of C. viridis were frozen in liquid nitrogen. 10-μm sections were realized with a cryostat and mounted onto coated glass slides. Sections were dried for 2 h at room temperature and treated for 5 min with saline buffer, 5 min with Tris-HCl (10 mM), EDTA (1 mM), pH 7.6, and 15 min in the same buffer with 1 μg/ml proteinase K at 37°C. Sections were then fixed for 5 min with a solution of 4% paraformaldehyde, 0.1% glutaraldehyde, rinsed with PBS, dehydrated with successive gradated alcohol baths, then dried under vacuum. Hybridization was performed for 16 h at 50°C. The hybridization medium was as following: 50% formamide, 4 × SSC, 10% dextran sulfate, 1 × Denhardt's solution, 10 mM dithiothreitol, 0.5 mg/ml yeast tRNA, and 0.1 mg/ml salmon DNA. α-35S-UTP-labeled RNA probes were prepared after linearization by SmaI for antisense or by HindIII for sense, of the pBluescript vector containing the cic insert. Transcription by 2.5 units of T7 RNA polymerase for antisense probe or T3 for sense probe was performed in 20 mM Tris-HCl, pH 8.25, 6 mM MgCl2, 2 mM spermidine, 10 mM dithiothreitol, 40 units of RNasin, for 1 h at 37°C. Plasmid DNA was eliminated by a 15-min incubation in 1 unit of DNase I. Probes were precipitated and dissolved in hybridization buffer at 2 × 106 cpm/ml. Following hybridization, slides were rinsed for 1 h at room temperature with 2 × SSC, 50% formamide, then 1 h in 1 × SSC, 1 h at 37°C in 20 μg/ml RNase, 0.5 × SSC for 1 h and 45 min at 45°C, and then 0.5 × SSC for 15 min at room temperature. Slides were then dehydrated and covered with an autoradiographic emulsion. Exposure time was 14 days at 4°C. A peptide RVQGHSLYDESRPRC from the cic deduced amino acid sequence was synthesized. Rabbits were immunized by a first injection of the coupled peptide in complete Freund's adjuvant followed by 5 boosts at 3-week intervals in incomplete Freund's adjuvant. Preimmune or immune sera were assayed for reactivity with homogenates of whole filter chambers or with chromatographically purified P25 polypeptide. Membranes were incubated in 1% Triton X-100, 10 mM Tris-HCl, 150 mM NaCl, pH 7.4, for 12 h at 4°C or in 2% n-octyl-β-D-glucopyranoside (OG), 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, at room temperature for 2 h. Insoluble material was eliminated by a 105,000 × g centrifugation for 1 h at 4°C. Stokes radius of protein-detergent complexes were obtained by gel filtration. Aliquots of 20 μl, containing 1-2 μg of membrane proteins were chromatographed at room temperature on a AcA 34 UltroGel column (when OG was used) or on a Protein Pack SW300 HPLC column (with the Triton X-100) calibrated with protein markers of known Stokes radius. Elutions were performed in a 10 mM Tris-HCl, 150 mM NaCl, pH 7.4, containing either 1% Triton X-100 or 2% OG. Fractions were collected and their content analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Linear 2-20% (w/v) sucrose density gradients were prepared from 2 and 20% stock solution of sucrose in H2O or D2O (98%) containing 10 mM Tris-HCl, 150 mM NaCl, and 1% Triton X-100 or 2% OG. 100-μl samples were layered on top of gradients and ultracentrifugation to equilibrium was performed for 18 h at 4°C at 100,000 × g. Calibration curves for the determination of the apparent sedimentation coefficient were constructed using cytochrome c (s20,w = 1.17 S), bovine serum albumin (s20,w = 4.6 S), and IgG (s20,w = 9 S) as protein markers. After centrifugation, 20 fractions were collected from the bottom of each gradient. Protein content was analyzed by SDS-PAGE. All calculations were performed as described by Sadler et al.(24Sadler J.E. Rearick J. Paulson J. Hill R. J. Biol. Chem. 1979; 254: 4434-4442Abstract Full Text PDF PubMed Google Scholar). The molecular weight of the protein detergent complexes were calculated using the following Mr = [6 · Π · n · n20,w/(1−υ · ρ20,w) ]S20,wRS(Eq. 1) Where n is Avogadro's number, η20,w is the viscosity of water at 20°C (0.01002 g/(cm•s)), and ρ20,w is the density of water at 20°C (0.99823 g/ml). Stokes radius (Rs) were deduced from the calibration of chromatography column and s20,w from the calibration of H2O sucrose gradient. v, the partial specific volume of detergent protein micelles was calculated from data obtained by H2O and D2O gradient sedimentation. Values of v = 0.940 and v = 0.801 cm3/g were used for Triton X-100 and OG, respectively (4Smith B.L. Agre P. J. Biol. Chem. 1991; 266: 6407-6415Abstract Full Text PDF PubMed Google Scholar, 25Clarke S. J. Biol Chem. 1975; 250: 5459-5469Abstract Full Text PDF PubMed Google Scholar). The partial specific volume of the peptide moiety was assumed to be 0.735 cm3/g(26Martin R. Ames B. J. Biol. Chem. 1961; 236: 1372-1379Abstract Full Text PDF PubMed Google Scholar). Aliquots of membranes in buffer (5 μl) were applied to freshly glow-discharged 400-mesh collodion/carbon-coated grids and allowed to stand for 1 min. Grids were then quickly blotted, briefly rinsed with distilled water, and deposited on a 2% uranyl acetate drop. Excess stain was removed by blotting with filter paper and grids were then air-dried. Grids were observed with a Philips CM12 microscope operating at 80 kV. Approximately 10 μl of membrane suspension were placed on a copper grid coated with a holey carbon film freshly glow-discharged, blotted quickly with filter paper, and then plunged by a quick release mechanism into liquid ethane. The frozen grid was transferred to a Gatan cryoholder in a Philips CM12 microscope operating at 100 kV, the specimen was maintained below −160°C throughout the observations. On-line digital recording of pictures was carried out using a high resolution video camera CF 1500 ELCA (Sofretec, Bezons, France) linked to a microcomputer fitted with a digital acquisition card. Images of 512 × 512 pixels were integrated for 6 s on a 16 bit frame memory and saved as 256 gray levels image files. The image sampling was of 0.8 nm on the specimen scale and the electron dose was 25 e−/Å2. Corrections for dark current and uneven illumination were done by software(27Jericevic Z. Wiese B. Bryan J. Smith J.C. Methods Cell Biol. 1989; 30: 47-83Crossref PubMed Scopus (39) Google Scholar). Mass measurements were performed with the STEM at IGBMC in Strasbourg (France), using a Vacuum Generator HB5 microscope operating at 100 kV equipped with a cold-field emission gun and a dark-field annulus detector. All the observations were made at −130°C using a specially designed cold stage(28Homo J.-C. Electron Microsc. 1980; 1: 92Google Scholar). Dark-field images were recorded directly in digital form using the signal from the annular dark-field detector. Drops of membrane suspension were adsorbed to carbon film mounted on a microscope grid and allowed to stand for 2 min. Tobacco mosaic virus was added as an internal mass standard and the grid was washed 4 times with double distilled water. The grid was blotted to leave only a thin layer of fluid and immediately immersed in liquid N2. Freeze drying was carried out within the microscope for 2 h at −80°C. Processing of digitized images of the particle arrays of filter chambers membranes was achieved using the SPIDER software system(29Frank J. Shimkin B. Dowse H. Ultramicroscopy. 1981; 6: 343-358Crossref Google Scholar), running on SUN UNIX workstations. From a raw image, a suitable area was selected interactively on the image display and padded into a square field of 512 × 512 pixels. In order to calculate an initial reference image, the Fourier transform and power spectrum were calculated and the diffraction pattern indexed. The indexing was used to calculate a Fourier filter mask that was applied to the Fourier transform to produce a filtered image. A subarea of the filtered image was used as a reference in cross-correlation mapping of similar areas in the raw image. Areas centered on the peaks in the cross-correlation map were extracted from the raw image and averaged(30Saxton W.O. Baumeister W. Vigell W. Electron Microscopy at Molecular Dimensions. Springer, Berlin1980: 245-255Crossref Google Scholar, 31Frank J. Goldfarb W. Baumeister W. Vogell W. Electron Microscopy at Molecular Dimensions. Springer, Berlin1980: 261-269Crossref Google Scholar, 32Saxton W.O. Baumeister W. J. Microsc. 1982; 127: 127-138Crossref PubMed Scopus (680) Google Scholar). Rotational correlation coefficients were calculated for quantitative assessment of the symmetry and the resolution was estimated by calculating the radial correlation functions (32Saxton W.O. Baumeister W. J. Microsc. 1982; 127: 127-138Crossref PubMed Scopus (680) Google Scholar) and the phase residuals(33Unwin P.N.T. Klug A. J. Mol. Biol. 1974; 87: 641-656Crossref PubMed Scopus (67) Google Scholar, 34Frank A. Verschoor A. Boublik A.M. Science. 1981; 214: 1353-1355Crossref PubMed Scopus (304) Google Scholar). STEM images of membranes vesicles were displayed on the television monitor and areas were enclosed within squared contours. Masses were determined by integrating the densities enclosed within each contour with appropriate background subtraction. Recognizing that the observed membranes are flattened vesicles and thus are very often double-sided, the corresponding densities were divided by two and combined to densities from single sided areas to obtain the mass per unit area. Each mass integral was calibrated relative to corresponding integrals for tobacco mosaic virus. 89 measurements were conducted on membrane areas having on average a size of 10,000 nm2. When purified membranes from C. viridis filter chamber were analyzed by SDS-polyacrylamide gel electrophoresis in the absence of β-mercaptoethanol, 80% of the protein in alkali-stripped membranes is P25. Only a second membrane polypeptide of apparent molecular mass 150 kDa (P150) corresponding to 10-20% of the protein content was detected (Fig. 1A). In order to demonstrate the tissue specificity of P25, we conducted an immunofluorescence study on cryosections over the whole insect. A strong immunofluorescence related to the labeling of P25 by the anti-P25 serum was exclusively observed over the filter chamber, and no immunoreactivity was detected over the remaining parts of the insect (Fig. 2A). Subcellular localization of P25 was carried out by immunoelectron microscopy on ultrathin sections of filter chambers, incubated with the anti-P25 serum (whose specificity is reported in Fig. 1B) and decorated with GAR-Gold. The epithelial cells exhibited a strong immunoreactivity over the apical microvilli and the basal membrane infoldings (Fig. 2B). Isolated membranes were strongly labeled mainly on one side, thus inferring that P25 might be asymmetrically inserted into the membranes (Fig. 2C). This is indeed supported by the morphology of isolated freeze-dried membranes observed in CTEM after shadowing (Fig. 10B). Thus the filter chamber of C. viridis appears, in both structure and composition, as constituted by very specialized epithelia where P25 prevails as the major intrinsic membrane polypeptide. We designed degenerate oligonucleotides from the highly conserved two NPA boxes characteristic of the MIP family proteins. A 360-base pair cDNA fragment was amplified in the filter chamber of C. viridis by PCR using these primers (Fig. 3). We called cic (from Cicadella) the cDNA fragment amplified and CIC the deduced amino acid sequence. The homologies between members of the MIP family and CIC are described in Fig. 4. Between the two NPA boxes, sequence identity was 38% for CIC-MIP26 and for CIC-AQP1 (human or rat), 34% for CIC-AQP2 (rat), 29% for CIC-bib ("big brain" of Drosophila), and 20% for CIC-γTIP (A. thaliana). Thus, the sequence of CIC is closely related to the sequence the MIP channel family.Figure 4:Amino acid sequence alignment of MIP26, AQP2, AQP1, γTIP with CIC. At each position, amino acid residues identical with those of CIC are shaded. Accession numbers in data libraries are P06624, D13906, M77829, M84344, and X77957, respectively. The underlined sequence corresponds to the synthetic peptide used for raising a rabbit antiserum.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Sense and antisense probes were prepared from cic and used for in situ hybridization experiments in order to localize these mRNA, on sections of the digestive tract of C. viridis. In all experiments performed, hybridization was strong in the filter chamber and absent from other parts of digestive tract such as initial or terminal midgut (Fig. 5). No significant signal was obtained after incubation of tissue slices with the sense probe. These results indicated a selective tissue distribution of mRNA encoding the CIC polypeptide. The hydrophobic conserved domains identified in the sequence of MIP26 and AQP1 could correspond to putative transmembrane segments. This could also be the case for CIC. If so, the hydrophilic sequence RVQGHSLYDESRPRC (Fig. 4) should correspond to the "C loop" in the two-dimensional model previously proposed for AQP1 by Preston et al.(35Preston G.M. Caroll T.P. Guggino W.B. Agre P. Science. 1992; 256: 385-387Crossref PubMed Scopus (1649) Google Scholar). As reported on Fig. 6B, a positive signal is visualized on nitrocellulose membranes blotted with increasing quantities of chromatographically purified P25, and incubated with the antipeptide serum. On Western blots of whole homogenate from filter chambers, this serum recognizes a single 25-kDa polypeptide, while no signal was detected with the preimmune serum (not shown). The immunoreactivity of P25 with an antibody directed against an hydrophilic amino acid sequence of a protein of the MIP family (CIC), associated to their specific tissue localization in the filter chamber, leads us to conclude to an identity of those two polypeptides. Further understanding of the organization of P25 in the membranes were deduced from biophysical analysis of P25, nondenaturing detergent micelles. We report in this section results obtained after extraction of P25 with two distinct nondenaturing detergents, subsequent gel filtration, and sucrose gradient sedimentation. Fig. 7 shows a typical elution profile of a column loaded with OG filter chamber membrane extracts. Following Triton X-100 or OG extractions of filter chamber membrane proteins, only P25 and P150 were detected by silver staining of polyacrylamide gels. As in Fig. 7, a polypeptide of apparent molecular mass of 75 kDa was sometimes observed; its appearance corresponds to the cleavage of P150 disulfide bridge. Silver staining of electrophoresed fractions content following gel filtration revealed that P25-OG micelles are eluted in a single peak where no other polypeptide is coeluted. Qualitatively identical results were obtained with Triton X-100 extracted proteins submitted to gel filtration; in all experiments maximal elution peaks of P25 and P150 were different. P25 is thus extracted as a single species in micelles in a monomeric or an homo-oligomeric form. The columns were calibrated with proteins of known stokes radius. The Kav determined experimentally for P25-detergent micelles permits extrapolation of their stokes radius from the calibration curves. P25-OG and P25-Triton X-100 micelles have stokes radii of 4.90 and 4.75 nm, respectively (Fig. 8, A and B). In some cases, protein extraction and gel filtration were carried out with 0.1% SDS. The average value thus obtained for Stokes radius of P25-SDS micelles was 2.90 nm. The differences observed for P25 stokes radii in denaturing and nondenaturing detergent suggest that if, as one can expect, the monomeric form of P25 is present in SDS, the nondenaturing detergent-extracted P25 is in an oligomeric form. The hydrodynamic properties of the P25-detergent solubilized complexes were further analyzed by sucrose gradient centrifugation to provide an estimate of their sedimentation coef
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