©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Analysis of a MIP Family Protein from the Digestive Tract of Cicadella viridis(*)

(Received for publication, December 28, 1994; and in revised form, April 28, 1995)

Fabienne Beuron (1), Franoise Le Cahérec (1), Marie-Thérèse Guillam (1), Annie Cavalier (1), Annick Garret (1), Jean-Pierre Tassan (2), Christian Delamarche (1), Patrick Schultz (3), Véronique Mallouh (3), Jean-Paul Rolland (1), Jean-Franois Hubert (1), Jean Gouranton (1), Daniel Thomas (1)(§)

From the  (1)Laboratoire de Biologie Cellulaire, (2)Laboratoire de Biologie et Génétique du Développement, URA CNRS No 256, Université de Rennes 1, Campus de Beaulieu, 35000 Rennes, France, and the (3)IGBMC, 1 rue Laurent Fries, 67400 Illkirch, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 -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.


INTRODUCTION

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(1) . Such proteins have been recently identified and gathered in the aquaporin family(2) : AQP1 (CHIP) in mammalian red cell membranes and proximal renal tubules(3, 4, 5) , AQP2 in rat renal collecting tubules(6) , AQP3 in rat renal collecting ducts(7, 8, 9) , AQP4 in rat brain(10, 11) , AQP5 in rat salivary glands(12) , and -TIP in Arabidopsis thaliana(13) .

These membrane proteins belong to a larger family of polypeptides, forming transmembrane channels and found in bacteria, plants, and animals (14) called the MIP family, from its archetype, MIP26, the major intrinsic protein of bovine lens fibers(15) . 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)()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(16) . 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) (17) . Finally, we demonstrated that FC is highly enriched in mRNA species encoding water channel proteins when microinjected into Xenopus oocytes(18) .

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.


MATERIALS AND METHODS

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(17) . The membrane fraction was then washed 18 h at 4 °C in an alkaline buffer (5 mM glycine, 1 mM EDTA, 5 mM -mercaptoethanol) to eliminate the extrinsic proteins(19) .

Electrophoresis and Immunoblotting

Electrophoretic analysis of membrane proteins were performed on SDS-polyacrylamide gels according to Laemmli(20) . Gels were stained with silver nitrate(21) . 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.

Immunolocalization of P25

Immunofluorescence

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.

Gold Immunolabeling

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.(22) . 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.

Design of Primers and PCR

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 (23) 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 MgCl. 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).

In Situ Hybridization

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. -S-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 MgCl, 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 10 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.

Peptide Synthesis and Antiserum Production

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.

Protein Detergent Extraction

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--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.

Hydrodynamic Studies

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 HO or DO (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 (s = 1.17 S), bovine serum albumin (s = 4.6 S), and IgG (s = 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.(24) . The molecular weight of the protein detergent complexes were calculated using the following equation:

Where n is Avogadro's number, is the viscosity of water at 20 °C (0.01002 g/(cms)), and is the density of water at 20 °C (0.99823 g/ml). Stokes radius (R) were deduced from the calibration of chromatography column and s from the calibration of HO sucrose gradient.

v, the partial specific volume of detergent protein micelles was calculated from data obtained by HO and DO gradient sedimentation. Values of v = 0.940 and v = 0.801 cm/g were used for Triton X-100 and OG, respectively (4, 25) . The partial specific volume of the peptide moiety was assumed to be 0.735 cm/g(26) .

Conventional Transmission Electron Microscopy (CTEM)

Negative Staining

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.

Cryoelectron Microscopy

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.

Image Recording

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. Corrections for dark current and uneven illumination were done by software(27) .

Scanning Transmission Electron Microscopy (STEM)

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(28) . 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 N. Freeze drying was carried out within the microscope for 2 h at -80 °C.

Image Processing

Conventional Electron Microscopy Images

Processing of digitized images of the particle arrays of filter chambers membranes was achieved using the SPIDER software system(29) , 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(30, 31, 32) . Rotational correlation coefficients were calculated for quantitative assessment of the symmetry and the resolution was estimated by calculating the radial correlation functions (32) and the phase residuals(33, 34) .

STEM Mass Calculation

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 nm.


RESULTS

P25 Is the Major Integral Polypeptide of the Filter Chamber

When purified membranes from C. viridis filter chamber were analyzed by SDS-polyacrylamide gel electrophoresis in the absence of -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).


Figure 1: Polyacrylamide gel electrophoresis and immunoblot of the membrane proteins. A, silver-stained polyacrylamide gel showing the protein content of purified plasma membranes isolated from the C. viridis filter chamber. The P25 polypeptide represents 80-90% of total proteins. B, Western blot. Purified membranes were electrophoresed as in A, blotted, and incubated with the rabbit anti-P25 serum.



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).


Figure 2: Immunolocalization of P25. A, immunofluorescence staining of P25 on C. viridis cryosections incubated with anti-P25 serum. A strong labeling was strictly limited to the FC; E, esophagus; C, cuticula, exhibiting a strong nonspecific endogenous fluorescence. Magnification 40. B, immunogold localization of P25 on ultrathin sections of the filter chamber. This image shows two extremely thin neighboring epithelia in cross-section. Gold particles are abundantly present on microvilli of the brush border (Mv). A less intense labeling is obtained on the lamellar invaginations (L inv) and on tubular invaginations (T inv) at the basal pole of the cells. The scale bar represents 1 µm. C, immunolabeling of purified membranes of the filter chamber of C. viridis (same antibody as in B). Numerous gold particles are located in clearly delineated zones corresponding to one side. The scale bar represents 0.5 µm.



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).


Figure 10: Mass measurement by STEM of freeze-dried unstained filter chamber purified membranes. A, dark-field image of a membrane preparation. Higher signal intensities correspond to double layer membranes. The scale bar represents 0.1 µm. B, similar vesicle as in A observed after freeze-drying. A smooth and a rough surface are associated to the outer face (OF) and inner face (IF), respectively. The scale bar represents 0.3 µm. C, histogram showing the distribution of values of mass per unit area calculated from 89 measurements. The histogram exhibits two peaks: a single layer of 2425 Da/nm and a double layer of 4551 Da/nm.



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.

In the Filter Chamber, a mRNA Population Encodes a Protein of the MIP Family

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-TIP (A. thaliana). Thus, the sequence of CIC is closely related to the sequence the MIP channel family.


Figure 3: Agarose gel electrophoresis of the result of PCR. A product of 360 base pairs is clearly seen on the lane corresponding to the FC. Control lane without DNA (C).




Figure 4: Amino acid sequence alignment of MIP26, AQP2, AQP1, TIP 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.



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.


Figure 5: In situ hybridization using an antisense probe prepared from cic. On sections of the digestive tract of Cicadella, a strong signal was observed on the filter chamber only (fc). im, initial midgut. The scale bar represents 100 µm.



P25 Belongs to the MIP Family

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.(35) . 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).


Figure 6: Analysis of a synthetic peptide derived from CIC. A, SDS-gel electrophoresis of chromatographically purified P25. B, the dot blot shows an increasing signal with increasing quantities of P25 after incubation with the antipeptide serum.



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.

Physical Properties of P25 in Detergent Solution

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. 7shows 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.


Figure 7: Analysis of column fractions by SDS-polyacrylamide gel electrophoresis. Gel filtration analysis on a Protein Pack SW300 HPLC column of filter chamber membrane proteins extracted by 2% OG. Elutions were performed in a 2% OG buffer. Fractions were collected and aliquots diluted in Laemmli SDS-PAGE sample buffer. The gel was stained with silver nitrate. In fraction number 16, P25-OG micelles are eluted in a pure state.



The columns were calibrated with proteins of known stokes radius. The K 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.


Figure 8: Physical properties of the P25-detergent micelles. Purified C. viridis filter chamber membranes were incubated either in 2% OG (A and C) for 2 h or in 1% Triton X-100 for 12 h (B and D). After 100,000 g centrifugation, supernatants were analyzed by: gel chromatography to determine the stokes radius of the detergent-solubilized P25 (A and B), velocity sedimentation in linear 2-20% sucrose density gradients prepared in HO or in DO to determine s and v (C and D). Following ultracentrifugation, fractions were collected and analyzed by SDS-PAGE. Arrows indicate sedimentation position of P25-detergent micelles.



The hydrodynamic properties of the P25-detergent solubilized complexes were further analyzed by sucrose gradient centrifugation to provide an estimate of their sedimentation coefficient and molecular weight. For that purpose, membrane extracts were subjected to ultracentrifugation on linear 2-20% sucrose gradients made up in HO or in DO and calibrated with marker proteins of known sedimentation coefficient. Representative sedimentation experiments are shown in Fig. 8, C and D. The apparent sedimentation coefficient (s) deduced for P25-OG micelles is 6.8 S and for P25-Triton X-100 micelles 5.35 S. The amount of detergent bound to the peptide can be estimated from a determination of the partial specific volume, v, of the complexes by the method of Sadler et al.(24) . From the data obtained by the sedimentation behavior of the micelles in HO and DO gradients, the partial specific volumes of P25-OG and P25-Triton X-100 complexes are 0.808 and 0.799 cm/g, respectively.

The sedimentation data together with the gel filtration data allow the calculation of the molecular weight of the P25-detergent complexes by the application of the formula given under ``Materials and Methods'' (Table 1). This gives a molecular mass of 139,000 Da for P25-Triton X-100 micelles and 188,000 Da for P25-OG micelles. Assuming that v of the peptidic portion of the micelle is 0.735 cm/g, that the v of proteins and detergent are additive, and that the native bound lipid has been replaced by detergent(24) , the amount of detergent bound to the peptide can be calculated to be 0.41 g/g of protein for Triton X-100 and 0.801 g/g of protein for OG. Taking into account the amount of detergent bound to P25, the molecular mass of the protein part of the micelles are in both cases evaluated to 100,000 Da. As demonstrated by gel filtration and SDS-polyacrylamide gel electrophoresis, no other polypeptide than the 25,000 Da one constitutes the protein part of the micelles. Thus, we can conclude that in the two nondenaturing detergents used in this study, P25 is extracted as homotetramer. This reflects the organization of the peptide in the native membrane.



Structural Organization of P25 in the Membranes

A high magnification image of a negatively stained purified membrane is shown in Fig. 9A. Native membranes display a faint regular square array of particles. The unit cell dimensions were calculated from the diffraction pattern (Fig. 9A, inset), and gave values of a = b = 9.6 nm. The presence of distinct sets of spots slightly rotated one with respect to the other indicates that most of these sheets are double layered. Such an image was used for correlation averaging and 680 subimages were averaged. The resulting average, after 4-fold symmetrization, is shown in Fig. 9B. Most of the protein mass is contained in a tetrameric core around the 4-fold crystallographic axis which encloses a central stain-filled pit. The tetrameric core, delineated 5.4 5.4 nm frame, has four stain-excluding elongated domains of 4.5 nm length and 3.0 nm width. The calculated resolution is 3.0 nm according to the radial correlation function criterion and 3.5 nm according to the phase residual criterion.


Figure 9: Electron microscopy and image analysis of purified membranes. A, electron micrograph of isolated membranes from the filter chamber, negatively stained with 2% uranyl acetate. Membranes display a regular two-dimensional array of particles. Inset shows the power spectrum calculated from this area (scale bar, 0.1 µm). The unit cell dimensions are: 9.6 9.6 nm; 90°. B, two-dimensional projection map of a membrane protein tetramer calculated by correlation averaging. The resulting image corresponds to the average of 680 unit cells from a single membrane crystal as in A with 4-fold symmetry imposed (scale bar, 2.5 nm). C, electron micrograph of membranes isolated from the filter chamber and embedded in vitreous ice. A native regular array is distinguishable (scale bar, 0.1 µm). Inset shows the calculated computer transform of this image. D, averaged image from C obtained by correlation averaging (n = 290). Contrast has been inverted in order to get proteins displayed in bright (scale bar, 2.5 nm).



In frozen-hydrated specimens, the unfixed and unstained P25 protein appeared electron-dense and the vitreous ice surrounding the membranes electron transparent (Fig. 9C). The diffraction pattern reveals an intensity distribution and lattice parameters very similar to the negatively stained preparation (lattice constant of 9.3 nm, Fig. 9C, inset). Computer image processing carried out on such images confirmed the tetrameric state of the membrane particles as seen on the average of 290 subimages in Fig. 9D. Each monomer has a clear asymmetric distribution of mass with a mass core of size 3.5 2.5 nm. The resolution associated with this projection map have been estimated at 2.4 nm according to the radial correlation functions and 2.9 nm according to the phase residual criterion.

Mass per unit area were calculated from STEM dark-field images of purified freeze dried membranes as shown in Fig. 10A. The mass data presented on Fig. 10C conforms to a bimodal distribution. The first peak at 2425 ± 262 Da/nm (n = 40) represents a single layer membrane, the second peak 4551 ± 443 Da/nm (n = 49) represents double layers. The latest value corresponds to the strong signal of double sided membranes as we observed in many freeze-dried shadowed vesicles (Fig. 10B). Consequently we divided by two this value and averaged it with the first value, to give a resulting mass per unit area of 2357 ± 270 Da/nm (n = 89).

From the CTEM data, we measured the unit cell area to be 92.16 nm and containing 2 tetramers. Therefore with an average mass of 2357 Da/nm, the unit cell mass can be calculated to be 217,221 and 27,152 Da for the monomer. This value can only fit with one molecule of P25 (M = 25,000 Da) associated with 2,152 Da of lipid.


DISCUSSION

Our interest in understanding the molecular origin of water transport led us to characterize P25, an abundant, tissue-specific, and conserved intrinsic protein of the MIP family found in the FC of the digestive tract of an homopteran insect.

Based on functional observations of FC of the sap sucking insect C. viridis, we postulated that the passive high water movement occurring in those epithelia could be related to the presence of specific channels. We have previously shown that the membranes of these cells are covered with particles forming a regular array(17) , evidenced by the large amount of an intrinsic polypeptide: P25. These epithelial cells were also shown to contain mRNA whose expression induced an increase of water permeability(18) . Those data suggested that P25 may constitute a water channel.

The water channels are now referred to as aquaporins(2) , a subgroup of the larger MIP family of proteins whose model is MIP26(15) . MIP26 is a membrane channel with undefined specificity but seems to allow the passage of small molecules except water(15, 36, 37) .

Each protein of this family carries in its sequence two NPA boxes. PCR performed on cDNA prepared from C. viridis filter chamber total RNA reveals that this tissue encodes a CIC protein which possesses the two NPA boxes and whose sequence between the two boxes has 38% homology with MIP26 and AQP1 (CHIP), 20% with -TIP; the most conserved part being the hydrophobic sequence where 6 bilayer-spanning domains are predicted(5, 14) . A subsequent determination of the 3` and 5` failing region of the cDNA will permit a complete comparison with the MIP family members. Nevertheless, it is obvious that in the FC, a protein of the MIP family is expressed. The tissue specificity of P25, documented by in situ hybridization (CIC being a fragment of P25), immunoblotting, and immunocytochemistry data, can be related to the unique function of those hyperspecialized water-shunting epithelia.

Having demonstrated that P25 is a member of MIP family it appeared interesting to investigate its structure, for papers concerning the structural organization of MIP family proteins are rare. Zampighi et al.(38) proposed a two-dimensional projection map of the MIP26 lens fiber protein showing a rough square shape of the unit particle. However, the model failed to resolve the monomers organization. Recently relevant structural information was reported for AQP1 (CHIP)(39, 40) . The results were obtained from proteoliposomes loaded with AQP1 which do not reproduce the native conditions notably concerning the orientation of the protein in the membrane.

The two-dimensional membrane crystals constituted in the FC by an extraordinary abundant P25 allow microscopic observations directly on native membranes. Under these conditions it is clear that in each membrane the proteic channels are similarly oriented in contrast with the proteoliposomes. It was therefore of interest to use this model for a structural study of a MIP protein.

In the present work we report studies of the quaternary structure of the 25,000-Da polypeptide. From hydrodynamic and electron microscopic studies we conclude that this protein is organized as homotetramers in the native membrane.

Nonionic detergents are widely known not to disturb protein-protein interactions(25, 41, 42) . We have used Triton X-100 and OG for the extraction of filter chamber membrane proteins. Such experiments have been carried out with AQP1-Triton X-100 (4) and MIP26-OG (43) and both reveal a tetrameric structure of the solubilized proteins. The results obtained for P25 lead to the conclusion of the existence of P25 in a tetrameric native form.

The structure of the filter chamber's membranes constitutes an interesting field of investigation of the native organization of P25. A dense packing of intramembrane particles was previously reported for those membranes; it accounts for the relatively high density (1.23) measured in sucrose gradients(17) . The two-dimensional lattice depicted over the whole surface of the membranes in electron microscopy was analyzed using image processing. The basic motif of the lattice was found, from both negatively stained and frozen-hydrated specimens, to be composed of 4 elongated bilobed domains arranged around a central pit. Combined with the mass informations provided by the STEM measurements, those averages were interpreted as tetramers of P25 only. Despite the poor order encountered in the membranes (resolution of the diffraction patterns limited to the second order), we propose an informative two-dimensional projection map of the P25 tetramer. Our results are consistent with the two-dimensional structure reported for AQP1 (CHIP)(39) . Whereas the CHIP tetramer is composed of 1 glycosylated polypeptide for 3 nonglycosylated monomers(4) , P25 bears no carbohydrate residues.

The crystalline arrays observed in frozen-hydrated membranes can be assumed to represent closely their native state since no dehydration occurs during preparation and no fixatives or stains were employed. The slightly higher resolution associated with the frozen-hydrated specimen can be related to a refinement of the shape of the monomer on the correlation average.

Isolated membranes of the FC are clearly asymmetrical as observed in electron microscopy after freeze-drying and shadowing and after freeze-fracture(17) . This fact should reflect an asymmetry of P25 at the molecular level, as it was shown for AQP1 (CHIP) from structure prediction and three-dimensional reconstruction(44, 40) . For P25, immunolabeling experiments using the anti-15-mer synthetic peptide antibody should enable the precise mapping of the putative C extramembraneous loop pointing in the extracellular domain(35) .

As a conclusion, the structural investigation of P25 in its native environment may contribute to the elucidation of new elements concerning the conformation of the MIP family proteins. P25 shares common structural and biochemical features especially with AQP1 and MIP26. It also shows original particularities related to the striking specialization of the epithelial complex constituting the filter chamber. The functional state of P25 in vivo is obviously a dense packing of oriented tetramers in the membranes. Its function and specificity as a water channel are under investigation. Whether the functional unit is a monomer or a tetramer also remains to be elucidated.


FOOTNOTES

*
This work was supported by INSERM Contrat externe 930202. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) X77957[GenBank® Link].

§
To whom correspondence should be addressed: Laboratoire de Biologie Cellulaire, Université de Rennes 1, Campus de Beaulieu, 35042 Rennes Cedex, France. Tel.: 33-99-28-61-22; Fax: 33-99-28-67-94; dthomas{at}univ-rennes1.fr

The abbreviations used are: FC, filter chamber; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; OG, n-octyl--D-glucopyranoside; PAGE, polyacrylamide gel electrophoresis; CTEM, conventional transmission electron microscopy; STEM, scanning transmission electron microscopy.


ACKNOWLEDGEMENTS

We thank Dr. Katherine Le Guellec for helpful advice and Louis Communier for photography. Electron microscopy was performed at the Centre Commun de Microscopie Electronique Transmission of Rennes University.


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