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
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ABSTRACT |
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
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INTRODUCTION |
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
-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
- and
-subunits of ENaC as well as the
-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 |
cDNA constructs.
For expression in reticulocyte lysate,the coding region of CHIF was
subcloned between 5' and 3' untranslated regions of Xenopus laevis
-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 (
N),
the hydrophobic NH2 terminal likely to be a signal peptide
(amino acids 2-21) was deleted. In the second (
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%
-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
-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 |
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
( N) CHIF were translated in reticulocyte lysate in the absence of
microsomes.
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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 (
N) was inefficient, and its
incorporation into microsomes could not be detected. Nevertheless, the
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
-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 (
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
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 ( 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.
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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.
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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).
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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
-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
-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 -subunit of
Na+-K+-ATPase. I-L:
CHIF plus aquaporin-2 (AQP2). M-P: CHIF plus
uromucoid. Bar in A corresponds to 50 µm.
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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.
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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.
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 |
DISCUSSION |
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
-subunit of
Na+-K+-ATPase raises the possibility that it
has a "
-like" function. Obviously, such a hypothesis will have
to be assessed by more direct methods.
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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).
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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.
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REFERENCES |
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Asher, C,
Wald H,
Rossier BC,
and
Garty H.
Aldosterone-induced increase in the abundance of Na+ channel subunits.
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