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Am J Physiol Renal Physiol 280: F505-F512, 2001;
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Vol. 280, Issue 3, F505-F512, March 2001

Membrane topology and immunolocalization of CHIF in kidney and intestine

Haikun Shi1, Rivi Levy-Holzman1, Francoise Cluzeaud2, Nicolette Farman2, and Haim Garty1

1 Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100 Israel; and 2 Institut National de la Santé et de la Recherche Médicale U478, Faculte de Medecine Xavier Bichat, Paris, 75870 France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Corticosteroid hormone-induced factor (CHIF) is an aldosterone-induced gene, the function of which is yet unknown. It is specifically expressed in kidney collecting duct (CD) and distal colon and is upregulated by either Na+ deprivation or K+ loading. Hence, it may play a role in epithelial electrolyte transport. Previous studies have characterized regulation and tissue distribution of CHIF mRNA but provided no information on the protein itself. The present paper addresses this issue by using Western blotting, immunochemistry, and in vitro translation. CHIF is an ~8-kDa membranal protein, and protease digestion experiments suggest that its COOH tail faces the cell interior. The protein is abundant in distal colon, kidney medulla, and papilla but cannot be detected in a variety of other tissues. Confocal immunocytochemistry demonstrates that CHIF is present in the basolateral membrane of CD principal cells and distal colon surface cells, with occasional intracellular staining. Dexamethasone and low Na+ intake increase the abundance of CHIF. Unlike previous Northern data, induction of CHIF protein by low-Na+ intake was apparent not only in the distal colon but also in the kidney.

corticosteroid hormone-induced factor; phospholemman; gamma-sodium-potassium-adenosine 5'-triphosphatase; collecting duct; membrane topology; aldosterone


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CORTICOSTEROID HORMONE-INDUCED factor (CHIF) is a new cDNA with an as yet unknown function cloned by differential screening for aldosterone-induced transcripts (2). Its deduced amino acid sequence predicts a short transmembrane polypeptide (87 amino acids) that shares 30-50% amino acid identity with three polypeptides reported to be involved in the regulation or mediation of ion transport. These are the gamma -subunit of Na+-K+-ATPase (12), phospholemman (14), and Mat-8 (13).

Two lines of evidence provide circumstantial evidence for the involvement of CHIF in epithelial ion transport. First, it is an epithelium-specific gene. CHIF mRNA is specifically expressed in kidney collecting duct (CCD) < OMCD < inner medullary collecting duct (IMCD) and in distal colon surface cells (top 20% of the crypt) (3, 15). It cannot be detected in many other epithelial and nonepithelial tissues. These include other segments of the kidney tubule and intestine, lung, stomach, uterus, mammary gland, salivary gland, heart, brain, muscle, liver, and skin. Second, CHIF appears to be independently upregulated by a low-Na+ intake (through changes in plasma aldosterone) and by a high-K+ intake (irrespective of changes in plasma aldosterone) (3, 15, 16). The response to aldosterone appears to be tissue specific. In the distal colon, CHIF mRNA is strongly induced by aldosterone, dexamethasone, or a low-Na+ diet (3, 15). In the kidney, however, these effectors do not influence the level of CHIF mRNA, and only regulation by K+ intake is apparent. The same differential effects of aldosterone in kidney and colon have been reported before for the beta - and gamma -subunits of ENaC as well as the alpha -subunit of the colonic H+-K+-ATPase (1, 7). It has been suggested that this may reflect differences between rapidly and slowly differentiating cells (11).

Although the regulation and tissue distribution of CHIF mRNA are well established, no information exists regarding the protein translated by this gene. The present paper characterizes the protein expressed by CHIF in reticulocyte lysate and in native epithelia. The data demonstrate that CHIF is an ~8-kDa membrane protein that apparently does not undergo core glycosylation or signal peptide cleavage. The protein is specifically located in the basolateral membrane of collecting duct principal cells, and its distribution along the nephron matches the one reported before for CHIF mRNA. CHIF is upregulated by dexamethasone and low Na+. However, unlike its mRNA, the protein is upregulated by low Na+ in both kidney and distal colon.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

cDNA constructs. For expression in reticulocyte lysate,the coding region of CHIF was subcloned between 5' and 3' untranslated regions of Xenopus laevis beta -globin in a pBluscript KS-derived vector (6). In addition, the original initiating sequence (GUUGAUAUGG) has been modified to the sequence TTCACCAUGG, which is more compatible with consensus initiation sequences (9). Two additional constructs that translate truncated proteins have been generated. In the first (Delta N), the hydrophobic NH2 terminal likely to be a signal peptide (amino acids 2-21) was deleted. In the second (Delta C), a stop codon was introduced after amino acid 64, truncating the last 23 hydrophilic residues. For preparing glutathione S-transferase (GST) fusion protein, a CHIF cDNA fragment corresponding to amino acids 21-87 (i.e., the whole protein lacking the putative signal peptide) was subcloned into the BamHI/EcoRI site of pGEX3X. For transfection into IMCD-3 cells, the coding region of CHIF was subcloned into the mammalian expression vector pEGFP-N1. Two constructs were prepared. In the first, the whole coding region (lacking the stop codon) was subcloned upstream and in frame with enhanced green fluorescent protein (EGFP), generating a fluorescent fusion protein. In the second, full-length CHIF was cloned into the EcoRI/NotI site of this vector, replacing EGFP. All manipulations were done by standard recombinant DNA methods and verified by sequencing.

In vitro transcription and translation. Aliquots of 4 µg linearized cDNA were incubated for 60 min at 37°C with 40 mM Tris · HCl, pH 7.9, 6 mM MgCl2, 10 mM 1,4-dithiothreitol, 2 mM spermidine, 0.5 mM ATP, GTP, CTP, and UTP, and 40 U T7 RNA polymerase. Reaction mixtures were extracted with phenol-chloroform, precipitated in ethanol, and suspended in sterile water to a final concentration of 1 µg/µl. Translation was performed by using nuclease-treated rabbit reticulocyte lysate, with or without canine pancreatic microsomal membranes (Promega Biotech, Piscataway, NJ). A typical 50-µl reaction contained 35 µl lysate, 2 µg cRNA, 20 mM methionine-free amino acid mixture, 40 µCi [35S]methionine (1,000 Ci/mmol), and either 3.6 µl microsomes or diluent. The mixture was incubated for 60 min at 30°C and then subjected to various experimental manipulations detailed below. Proteins were resolved on a 15% polyacrylamide gel and visualized by autoradiography. Dissociation of proteins adsorbed but not incorporated into microsomes was done by alkaline lysis and high-speed sedimentation (17). The translation mixtures were diluted with 100 mM HEPES, brought to pH 11-11.5 with 0.1 N NaOH, and incubated for 10 min at 0°C. Aliquots of 50 µl were layered onto 0.2 M sucrose cushions and sedimented at 125,000 g for 40 min. The pellet and supernatant were collected and further analyzed. To hydrolize glycosyl groups, microsomal samples were denatured by boiling 10 min in 0.5% SDS and 1% beta -mercaptoethanol. They were then incubated for 60 min at 37°C with 500 U peptide N-glycosidase F (New England Biolabs, Beverly, MA) in 50 mM sodium phosphate (pH = 7.5) and 1% NP-40.

Antibodies. Rabbit polyclonal antibodies against a synthetic peptide (antibody 2531) and GST-CHIF fusion protein (antibody 3247) have been raised. The COOH-terminal peptide 63C R R N H T P S S L P E, predicted to be highly antigenic (8), was synthesized and coupled to keyhole limpet hemocyanin through its NH2-terminal cysteine. GST-CHIF fusion protein was expressed in Escherichia coli and affinity purified on reduced glutathione agarose beads. Rabbits were immunized by standard procedures, and sera were titrated by ELISA using the fusion protein and GST.

Animal treatment and membrane isolation. Rats (Wistar, 8-10 wk old) were used. To modulate expression of CHIF, the animals were either injected with dexamethasone (3 daily injections of 6 mg dexamethasone/kg body wt) or fed a normal, high-K+, and low-Na+ diet for 2 wk, as described previously (1, 15, 16). Rats were killed by cervical dislocation. The kidneys, distal colon, and other tissues were excised and washed in homogenizing buffer composed of (in mM) 90 KCl, 45 sucrose, 5 MgCl2, 10 EGTA, and 5 Tris · HCl (pH = 7.8). The distal colon was cut longitudinally and rinsed well. The epithelial layer was scraped off the connective tissue with a glass slide. Kidneys were dissected into cortex medulla and papilla, and other organs were cut into small pieces. Cells and tissue segments were suspended in the above homogenization buffer plus protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin A, and 1 µM leupeptin) and homogenized for 10 s at 4°C by using a Polytron (Kinematica, Lucerne, Switzerland). Unbroken cells, nuclei, and debris were removed by sedimentation at 1,000 g for 5 min. The cloudy supernatants were centrifuged at 30,000 g for 1 h. Pellets were suspended in the above homogenization buffer to a final concentration of 10-30 mg protein/ml, stored in liquid N2, and used over a period of several weeks.

Tissue culture. IMCD-3 cells (passage 8) were purchased from American Type Culture Collection and grown in 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium. Cells were transfected by electroporation (30 µg plasmid/107 cells) and replated. They were cultivated for another 48-72 h, analyzed by fluorescent microscopy, and harvested for membrane isolation and Western blotting. Expression of CHIF-EGFP was detected in ~40% of the cells.

Western blotting. Cell membranes were dissolved in SDS sample buffer, and aliquots of 40-100 µg protein were resolved electrophoretically on 15% polyacrylamide gels. Proteins were transferred to nitrocellulose membranes in a Trans-Blot semidry transfer cell (1 h at 20 mV, Bio- Rad). Nonspecific binding sites were blocked with 5% low-fat dry milk dissolved in PBS containing 0.05% Tween-20 (PBS-T). The nitrocellulose sheets were first overlaid with 1:1,000 dilution of the rabbit anti-sera in PBS-T (2 h, room temperature) and incubated with a 1:10,000 dilution of a horseradish peroxidase-conjugated goat anti-rabbit antibody (1 h, room temperature, Biolab). Bound antibody was detected by enhanced chemiluminesence.

Immunolocalization of CHIF in the kidney. Kidneys and distal colonic segments were excised from male Sprague-Dawley rats, frozen in liquid N2, and stored at -80°C. Cryostat sections (10 µm) were placed on superfrost slides, fixed in methanol (10 min, -20°C), and immersed in PBS. Immunolocalization was performed by incubating sections with the anti-peptide-CHIF antibody (1:100) overnight at 4°C. After several washes in PBS, a secondary antibody (goat anti-rabbit Fab fraction, Jackson, 1:200) coupled to the fluorochrome CY3 (red fluorescence) was incubated for 2 h in the dark at room temperature. For colocalization experiments, the CY3-labeled sections were incubated for 4 h at room temperature with antibodies against different protein markers and then overlaid with FITC-labeled (green fluorescence) goat anti-rabbit IgG Fab fraction or donkey anti-sheep antibody. The following polyclonal antibodies have been used for colocalization: 1) anti-aquaporin 2 (AQP2) anti-serum, (1:100) (4); 2) anti-Tamm-Horsfall protein antibody (sheep anti-oromucoid, 1:50, Biodesign, Kennebunk, ME); and 3) antibody raised against the alpha -subunit of Na+-K+-ATPase, 1:50) (5). After washes in PBS, slides were mounted by using immumount (Shandon) and examined in a confocal microscope (TCS 4D, Leica).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Topological characterization of CHIF in reticulocyte lysate. Hydropathy plot analysis of CHIF's deduced amino acid sequence predicts two hydrophobic domains (2). The first (amino acids 1-20) could be a cleavable signal peptide, and the second (amino acids 38-58) should be transmembranal. Thus it was suggested that the mature protein has an extracellular NH2 terminal, a single transmembrane domain, and a cytoplasmic COOH tail. The predicted topology was assessed by translating CHIF in reticulocyte lysate in the presence and absence of canine pancreatic microsomes. The major polypeptide synthesized had an electrophoretic mobility of ~8 kDa (Fig. 1). However, several additional higher bands were observed. These higher species were seen only if the protein was translated in the absence of microsomes (Fig. 1A) and were not detected in native epithelia (see Fig. 4). Hence, they are likely to reflect SDS-resistant aggregates formed by the expression of hydrophobic polypeptides in the absence of a lipid phase.


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  		Fig. 1.   In vitro translation of corticosteroid hormone-induced factor (CHIF). A: CHIF was translated in the absence (lane 1) and presence (lanes 2 and 3) of canine pancreatic microsomes. Microsomal samples were denatured and incubated with (lane 3) or without (lane 2) 500 U peptide N-glycosidase F (Endo F.), as described in MATERIALS AND METHODS. B: CHIF was translated in the presence of microsomes. The translation mixture was exposed to alkaline pH, and particular and soluble proteins were separated as described in MATERIALS AND METHODS. The supernatant (Sup.) and pellet were incubated for 60 min with or without 0.25 ng/ml proteinase K (Prot. K) and resolved on a SDS-PAGE gel. C: microsomes were added only at the end of the translation reaction. The mixture was separated to microsomal pellet and supernatant and resolved on a SDS-PAGE gel, as above. D: full-length and N-truncated (Delta N) CHIF were translated in reticulocyte lysate in the absence of microsomes.

To verify that CHIF is indeed incorporated into microsomes, samples were subjected to alkaline lysis, (which dissociates proteins that are adsorbed but not inserted into the microsomes) and then sedimented at 125,000 g (17). At least half of the polypeptide synthesized was associated with the membrane pellet (Fig. 1B). Such association was not observed if microsomes were added to the reaction mixture after the translation was completed (Fig. 1C). Moreover, the membrane-associated protein was largely protected from digestion by proteinase K whereas the soluble fraction was fully degraded by the enzyme (Fig. 1B). Thus association of CHIF with the microsomal membrane reflects a cotranslational insertion and not a posttranslational adsorption.

Translation of CHIF in the presence of microsomes did not alter the electrophoretic mobility of the major ~8-kDa polypeptide (Fig. 1A). Such a change is expected if the first hydrophobic domain suggested to be a signal peptide is cleaved, and/or core glycosylation takes place. Lack of glycosylation was further established by demonstrating that treatment with peptide N-glycosidase F does not alter the electrophoretic mobility of the translated protein (Fig. 1A). This enzyme hydrolyzes nearly all types of N-glycan chains from glycoproteins (10). The fact that signal peptide cleavage could not be seen was somewhat surprising because such cleavage does take place in phospholemman (14). In principle, it is possible that microsomes cleave the initial hydrophobic domain without a resulting measurable change in CHIF's apparent molecular mass, due to some abnormal electrophoretic mobility. To exclude such a possibility, a truncated construct that lacks the sequence corresponding to the first 20 hydrophobic amino acids was prepared and translated. Translation of this N-truncated construct (Delta N) was inefficient, and its incorporation into microsomes could not be detected. Nevertheless, the Delta N construct did translate a polypeptide that had an apparent molecular mass clearly smaller than that of full-length CHIF (Fig. 1D). Also, the microsomal membranes used in these experiments were active and could cleave the signal peptide of beta -lactamase, shifting its apparent molecular weight from ~31.5 to ~29 (not shown).

To further assess the membrane topology of CHIF, we have prepared and expressed a CHIF construct that lacks the hydrophilic COOH tail (Delta C). This construct translated a significantly smaller protein that was incorporated into microsomes (Fig. 2A). Treating microsomes with proteinase K reduced the full-length protein by ~3 kDa to a size similar to that of the Delta C protein. On the other hand, the electrophoretic mobility of the truncated protein was only slightly affected by the proteolytic digestion (Fig. 2A). Thus it appears that the COOH tail is likely to face the microsomal exterior, i.e., the cytoplasmic domain of the cell. The membrane topology suggested for CHIF is illustrated in Fig. 2B and is further described in DISCUSSION.


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  		Fig. 2.   Membrane topology of CHIF. A: full-length and C-truncated (Delta C) CHIF were expressed in the presence of microsomes. Microsomes were pelleted under isotonic conditions, incubated with and without proteinase K, and resolved on a SDS-PAGE gel. B: suggested topology of CHIF.

Characterization of CHIF in native membranes. Two polyclonal anti-CHIF antibodies have been raised. The first (denoted 2531) was directed against a COOH-terminal peptide, and the second (3247) against the whole protein (lacking the putative signal peptide) expressed as a GST fusion protein. Antibody specificity was tested by blotting membranes from transfected and nontransfected IMCD-3 cells. This kidney-derived cell line does not contain a significant amount of CHIF mRNA (data not shown) and presumably also does not express CHIF protein. Cells were transfected with a plasmid expressing a CHIF-EGFP fusion gene, and translation of the antigen was verified by recording cell fluorescence. Both antibodies labeled a ~36-kDa polypeptide present in transfected but not in nontransfected cells (Fig. 3A). Cells transfected with CHIF alone expressed an ~8-kDa epitope not detected in nontransfected or CHIF-EGFP-transfected cells (Fig. 3B). Another unknown ~60-kDa polypeptide was detected irrespective of transfection. Antibody 3247 appeared to give stronger signals in Western blots and was used in these experiments. For cellular localization by immunocytochemistry, the anti-peptide antibody gave better results.


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  		Fig. 3.   Detection of CHIF by Western blotting in transfected cells. A: inner medullary collecting duct (IMCD)-3 cells were transiently transfected with CHIF-enhanced green fluorescent protein (EGFP; +) or nontransfected (-). Membranes were isolated, and aliquots of 20 µg of protein were resolved on a SDS-PAGE gel and blotted with the anti-fusion protein (3247; left) and anti-peptide (2531; right) antibodies. B: IMCD-3 cells were transfected with vectors expressing CHIF alone (CHIF) and CHIF-EGFP, or were nontransfected (-). Membranes were isolated and blotted with the anti-fusion protein antibody.

Next, we used antibody 3247 to test for the presence of CHIF in native epithelia shown before to express its mRNA. An ~8-kDa polypeptide similar to the one observed in transfected cells and reticulocyte lysate was detected in rat distal colon (Fig. 4A). It was present in the membrane but not cytosolic fraction of the tissue. The wavy nature of the band visualized may suggest tight binding of lipids. The antibody also reacted with kidney, and the abundance profile was cortex < medulla < papilla (Fig. 4B). No signal was observed in a variety of other organs, with the possible exception of a faint band in the lung (Fig. 4C). Thus the ~8-kDa polypeptide detected by the antibodies in native epithelia has a tissue distribution that correlates well with that previously described for CHIF mRNA (3, 16).


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  		Fig. 4.   Western blotting of CHIF in native epithelia. Membrane and cytosolic fractions were extracted from various rat organs, resolved on a SDS-PAGE gel, and blotted with antibody 3247. A: membranal (2 µg protein) vs. cytosolic (20 µg protein) fractions in rat distal colon. B: membranal fractions from distal colon (2 µg protein), kidney cortex, medulla, and papilla (20 µg protein). C: membrane fractions from various organs (100 µg protein/lane).

To further characterize the cellular and nephron localization of CHIF, its expression was assessed by immunolocalization. CHIF was readily detectable all along the collecting duct, with no apparent change in abundance from CCD to IMCD (Fig. 5, A-D). Restriction of the expression to the collecting duct was further established by colocalization experiments. CHIF was found to colocalize with AQP2, an established marker of collecting duct principal cells (Fig. 5, I-L). On the other hand, immunostaining with antibody against uromucoid (Tamm-Horsfall protein), known to be expressed in the thick ascending limb of Henle's loop, showed no overlap with CHIF immunostaining (Fig. 5, M-P). The CH4 antibody (raised against the alpha -subunit of Na+-K+-ATPase) showed extensive labeling of the thick ascending limb of Henle's loop and, to a lesser extent, proximal tubules and collecting duct (Fig. 5, E-H). This pattern reflects the well-documented variable levels of the enzyme in these structures. Immunostaining by this antibody colocalized with the expression of CHIF in the cortical collecting duct (Fig. 5, E and F) but not in the other nephron segments. It also appears that the relative abundance of CHIF and alpha -Na+-K+-ATPase varies along the cortical collecting duct.


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  		Fig. 5.   Immunolocalization of CHIF in the kidney. Sections of kidney cortex, outer medulla, and inner medulla, were labeled with the anti-CHIF antibody (2351; red) and with antibodies against other marker proteins (green). Yellow indicates colocalization. A-D: CHIF alone. E-H: CHIF plus the alpha -subunit of Na+-K+-ATPase. I-L: CHIF plus aquaporin-2 (AQP2). M-P: CHIF plus uromucoid. Bar in A corresponds to 50 µm.

The cellular distribution of CHIF in collecting duct and its location in principal cells were further studied by examining higher magnifications of confocal images obtained from sections that were coimmunostained with anti-CHIF and anti-AQP2 antibodies (Fig. 6). These images revealed that CHIF is specifically located in the basolateral membrane of the principal cells of the collecting duct. On the other hand, the apical membrane is decorated by the anti-AQP2 but not by the anti-CHIF antibody. In some cells, CHIF immunostaining was also intracellular. Figure 7 depicts immunolocalizetion of CHIF in the distal colon. The protein was found in approximately the upper 30% of the crypt and, in particular, in the surface cells. Higher magnification shows that it primarily stains the basolateral membrane (Fig. 7B).


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  		Fig. 6.   Double immunofluorescence of collecting ducts. Kidney sections were labeled with the anti-CHIF antibody (2351; red) and an anti-AQP2 antibody (green). Two collecting ducts are shown. As expected, the anti-AQP2 antibody decorates the apical membrane of collecting duct principal cells. CHIF is seen in the basolateral membrane of the same cells. Bar, 10 µm.



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  		Fig. 7.   Immunolocalization of CHIF in the distal colon. Sections of colonic tissue were labeled with anti-CHIF antibody 2531. A: low-magnification image (bar = 125 µm). B: high-magnification image (bar = 15 µM). *, Lumen.

Finally, we examined whether treatments shown before to upregulate CHIF mRNA have a similar effect on the abundance of the protein. Accordingly, membranes were isolated from matched groups of rats exposed to various treatments and assayed for the abundance of CHIF by Western blotting. Figure 8 summarizes data obtained in three different experiments testing the effects of low-Na+ intake and dexamethasone. Both treatments, shown to elevate CHIF mRNA, were found to elevate protein abundance as well. A similar activation was seen after a high-K+ diet, but this effect was more variable (data not shown). As discussed immediately below, the above results differ somewhat from the previously described modulation of CHIF mRNA.


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  		Fig. 8.   Regulation of CHIF by dexamethasone and low-Na+ intake. Matched groups of rats (3 rats/group) were exposed to various treatments as detailed in MATERIALS AND METHODS. Membranes were isolated from distal colon and kidney medulla and blotted with antibody 3247. The figure depicts 3 different experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CHIF is an epithelial-specific gene with an as yet unknown function. A role in ion transport is inferred by the following findings: 1) independent induction by aldosterone and by K+ loading (3, 15, 16); 2) specific expression in kidney collecting duct principal cells and distal colon surface cells (3, 15); and 3) sequence homology to three other polypeptides thought to function as regulators of channels and pumps (12-14). Previous work studied distribution and induction of CHIF mRNA but provided no information on the protein translated by it. The present study addressed this issue by observations in protein translated in reticulocyte lysate and native epithelia.

In vitro translation in reticulocyte lysate produced an ~8-kDa polypeptide that is incorporated into microsomes. The calculated molecular weight of CHIF is 9077. However, considerable difference between the electrophoretic mobility and the protein mass may occur for small hydrophobic polypeptides. Because incorporation into microsomes and treatment with N-glycosidase F did not alter the electrophoretic mobility of CHIF, we concluded that this protein does not undergo signal peptide cleavage or N-glycosylation. The lack of glycosylation is expected because the only potential N-glycosylation site (66N) is in the COOH tail, thought to be intracellular. Because expression in the presence of microsomes was without effect on the electrophoretic mobility of CHIF, it is suggested that the putative signal peptide is not removed. However, it is also possible that the signal peptide is removed but the reduction in mass is compensated for by other posttranslational modifications (e.g., O-glycosylation, lipid acylation, etc.).

Membrane topology was further examined by comparing the accessibility of full-length and COOH-truncated CHIF to proteinase K. Proteolytic digestion of intact microsomes significantly decreased the size of the full-length protein but had a much smaller effect on the COOH-truncated CHIF (Fig. 2A). Thus the COOH tail appears to face the microsomal exterior and hence, the cell interior. This would suggest that CHIF has two transmembrane domains, a very short external loop and a cytoplasmic COOH tail (Fig. 2B). However, present data do not exclude the possibility that the putative extracellular loop is buried in the membrane and only the COOH tail sticks into the cytoplasm.

In the second part of this study, the cellular and tissue distribution of CHIF were characterized in native epithelia by using specific anti-CHIF antibodies. The observed tissue distribution of the protein matched well the one previously determined for its mRNA. A major finding in these experiments is that the anti-CHIF antibody preferentially decorates the basolateral membrane of collecting duct principal cells. In some cells, some additional intracellular staining is visible. The basolateral localization of CHIF may provide an important clue in elucidating the cellular role of this protein (see below).

Finally, we have studied the modulation of CHIF protein by salt intake and dexamethasone, shown before to upregulate its mRNA. The two stimuli evoked a significant increase in the abundance of CHIF. Yet, two significant differences were noted between the previously described regulation of CHIF mRNA and the modulation of CHIF protein. First, low-Na+ intake was shown to induce CHIF mRNA in the distal colon but not kidney (15). In the present study, however, upregulation of CHIF by Na+ deprivation is apparent in both kidney medulla and distal colon. This observation suggests an additional posttranscriptional regulation and lends support to the notion that it may be involved in the physiological response to aldosterone. A second difference was that the salt- and corticosteroid-induced changes in the abundance of CHIF protein monitored in this study were generally smaller than the previously described modulation of CHIF mRNA.

A major unresolved issue is the cellular role or biological activity mediated by CHIF. A previous study reported an "IsK-like" K+ channel activity in X. laevis oocytes injected with CHIF cRNA (2). This effect, however, turned out to be irreproducible, and many batches of oocytes failed to evoke it. The basolateral localization of this protein, together with the fact that it is induced by both low Na+ and high K+, may provide important clues to its cellular role. Two basolateral-specific transporters are the Na+-K+-ATPase and the basolateral K+ channel. Of these, only the pump should be activated by both Na+ deprivation and K+ loading. The sequence homology between CHIF and the gamma -subunit of Na+-K+-ATPase raises the possibility that it has a "gamma -like" function. Obviously, such a hypothesis will have to be assessed by more direct methods.


    ACKNOWLEDGEMENTS

This study was supported by research grants from the Minerva Foundation and the Samuel H. Epstein Fund for Renal Research (to H. Garty) and from Institut National de la Santé et de la Recherche Médicale funds (to N. Farman).


    FOOTNOTES

Address for reprint requests and other correspondence: H. Garty, Dept. of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100 Israel (E-mail: h.garty{at}weizmann.ac.il).

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

Received 20 June 2000; accepted in final form 7 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Asher, C, Wald H, Rossier BC, and Garty H. Aldosterone-induced increase in the abundance of Na+ channel subunits. Am J Physiol Cell Physiol 271: C605-C611, 1996[Abstract/Free Full Text].

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Am J Physiol Renal Fluid Electrolyte Physiol 280(3):F505-F512
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