Am J Physiol Cell Physiol
280: C166–C174, 2001.
The ⌬F508 mutation shortens the biochemical half-life
of plasma membrane CFTR in polarized epithelial cells
GHANSHYAM D. HEDA,1,2 MRIDUL TANWANI,2 AND CHRISTOPHER R. MARINO2,3,4
1
Research and 3Medical Services, Veterans Affairs Medical Center and Departments of
2
Medicine and 4Physiology and Biophysics, The University of Tennessee Health
Sciences Center, Memphis, Tennessee 38163
Received 24 May 2000; accepted in final form 9 August 2000
cystic fibrosis; regulation; membrane protein; endocytosis;
chloride channel; cystic fibrosis transmembrane conductance
regulator
(CF) is caused by a gene mutation
that results in the deletion of the phenylalanine at
position 508 of CFTR, the cystic fibrosis transmembrane conductance regulator (CFTR) protein (13). The
⌬F508 deletion results in a misfolding of the nascent
CFTR molecule in the endoplasmic reticulum (ER),
leading to its retention in the ER by chaparone proteins and its subsequent ubiquitination and premature
degradation by the cytoplasmic proteosome complex
(reviewed in Ref. 16). As a consequence, little if any
⌬F508 CFTR is processed to its mature, fully glycosylated form or trafficked to the plasma membrane where
CFTR normally functions as a cAMP-activated chloride
channel. Because the ⌬F508 mutant retains some in-
MOST CYSTIC FIBROSIS
Address for reprint requests and other correspondence: C. R.
Marino, Medical Service (111), VA Medical Center, 1030 Jefferson
Ave., Memphis, TN 38104 (E-mail: [email protected]).
C166
trinsic chloride transport function (8, 18), interest in
approaches that drive ⌬F508 CFTR past the ER quality control system and to the cell surface has grown.
Several in vitro studies have shown that surface
expression of ⌬F508 CFTR can be upregulated by
chemical or physical means. Butyrate compounds have
been shown to create cAMP-activated chloride currents
in cultured ⌬F508 cells, presumably by overwhelming
the ER quality control system through an increase in
gene transcription (4, 28). Glycerol (30) and low temperature (8) stabilize the conformation of newly synthesized ⌬F508, allowing some of it to bypass the ER
quality control system and reach biochemical maturity.
After each of these treatments, an increase in cAMPactivated chloride transport can be detected in the
mutant cells. These findings suggest that pharmacological agents that downregulate the ER quality control system might be useful in correcting the chloride
transport defect in ⌬F508 cells.
For this approach to be clinically effective, the ⌬F508
protein must maintain some degree of biochemical
stability after reaching the cell surface. Little is
known, however, of the fate of the CFTR protein after
it reaches the cell surface. Initial work suggested that
⌬F508 and wild-type CFTR had similar plasma membrane half-lives (8), but a subsequent in vitro study has
shown that cAMP-activated chloride currents in nonpolarized ⌬F508 cells are less stable than those in
wild-type cells (23). Because plasma membrane expression of the CFTR proteins was not directly examined in
that study, the molecular basis for this observation is
not known. The functional instability in ⌬F508 cells
could have been due to more rapid inactivation of
⌬F508 channels at the cell surface or to more rapid
internalization and/or degradation of the mutant protein. Functional inactivation is supported by data from
several studies that show the conduction properties of
⌬F508 CFTR differ from those of wild-type CFTR (6, 9,
11). In fact, a recent electrophysiological study has
since confirmed that ⌬F508 CFTR is more rapidly
inactivated than wild-type CFTR in excised membrane
patches (31).
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.
http://www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 18, 2017
Heda, Ghanshyam D., Mridul Tanwani, and Christopher R. Marino. The ⌬F508 mutation shortens the biochemical half-life of plasma membrane CFTR in polarized
epithelial cells. Am J Physiol Cell Physiol 280: C166–C174,
2001.—Although the biosynthetic arrest of the ⌬F508 mutant of cystic fibrosis transmembrane conductance regulator
(CFTR) can be partially reversed by physical and chemical
means, recent evidence suggests that the functional stability
of the mutant protein after reaching the cell surface is compromised. To understand the molecular basis for this observation, the current study directly measured the half-life of
⌬F508 and wild-type CFTR at the cell surface of transfected
LLC-PK1 cells. Plasma membrane CFTR expression over
time was characterized biochemically and functionally in
these polarized epithelial cells. Surface biotinylation,
streptavidin extraction, and quantitative immunoblot analysis determined the biochemical half-life of plasma membrane
⌬F508 CFTR to be ⬃4 h, whereas the plasma membrane
half-life of wild-type CFTR exceeded 48 h. This difference in
biochemical stability correlated with CFTR-mediated transport function. These findings indicate that the ⌬F508 mutation decreases the biochemical stability of CFTR at the cell
surface. We conclude that the ⌬F508 mutation triggers more
rapid internalization of CFTR and/or its preferential sorting
to a pathway of rapid degradation.
PLASMA MEMBRANE HALF-LIFE OF CFTR
MATERIALS AND METHODS
Cell line. Pig kidney epithelial cells (LLC-PK1), stably
transfected with human wild-type or ⌬F508-CFTR cDNA
were kindly provided by Dr. Seng Cheng (Genzyme, Boston,
MA). Cells were grown in low-glucose DMEM supplemented
with 10% FBS and 400 ␮g/ml Genticin (Life Technologies,
Grand Island, NY) at 37°C on plastic dishes coated with
human placental type IV collagen (Sigma Chemical, St.
Louis, MO). For 125I efflux experiments, cells were grown on
clear transwell membrane inserts coated with type IV collagen (Corning Costar, Cambridge, MA). For immunocytochemical experiments, cells were grown on collagen-coated
glass coverslips.
Upregulation of surface CFTR expression. Transfected
LLC-PK1 cells grown to ⬃70% confluence as described above
were treated for 60 h at 27°C in the presence of 5 mM sodium
butyrate to upregulate surface CFTR expression (12). Because low temperature partially inhibits cell proliferation,
cell overgrowth did not occur during this 60-h treatment, and
confluency was rarely achieved.
Biochemical determination of surface CFTR expression
over time. After upregulation of surface CFTR expression,
cells were washed with butyrate-free media and were incubated at 37°C in media containing 20 ␮g/ml of cycloheximide,
an inhibitor of protein synthesis, for designated time intervals up to 48 h. Cells were then washed with ice-cold PBS, pH
7.4, containing 0.1 mM CaCl2 and 1 mM MgCl2 to inhibit
vesicle trafficking, and surface biotinylation was performed
as described previously (19). Briefly, the glycosidic moieties
of surface membrane proteins were derivatized with sodium
periodate and biotinylated using biotin-LC-hydrazide according to company protocol (Pierce, Rockford, IL). The efficiency
of surface biotinylation was tested by examining the effect of
increasing biotin-LC-hydrazide exposure times on surface
CFTR expression. No time-dependent increase in surface
CFTR expression occurred with prolonged reagent exposure
(data not shown).
After surface biotinylation, cells were lysed with 1% SDS
containing 0.2 mM phenylmethylsulfonyl fluoride and 1 mM
benzamidine, sonicated to shear DNA molecules, and centrifuged at 10,000 g for 10 min at 4°C to remove cellular debris.
The clear supernatants, normalized to 50 ␮g of total protein,
were nutated for 30 min with an excess of streptavidin-coated
agarose (SA) beads or uncoated control agarose (CN) beads
(Sigma). After incubation, the beads were pelleted, and the
supernatants were subjected to 6.5% SDS-PAGE followed by
transfer to Hybond-P polyvinylidene difluoride membrane
(Amersham, Sunnyvale, CA). The transfer was blocked with
5% nonfat dry milk in 137 mM NaCl, 0.1% Tween 20, and 20
mM Tris 䡠 HCl, pH 7.6, and immunoblotted with 1:100 dilution of R3194, an affinity-purified polyclonal anti-CFTR antibody. The specificity of R3194 for CFTR has been characterized previously in transgenic mice (37), in CFTR- and
mock-transfected HEK-293 cells (17), and by COOH-terminal
CFTR peptide competition experiments (37). CFTR bands
were visualized by enhanced chemifluorescence (Amersham)
and were quantified using a STORM 860 imaging system
with ImageQuant software (Molecular Dynamics, Sunnyvale,
CA).
With the use of the above approach, biotinylated surface
proteins were irreversibly extracted by the SA bead processing. The efficiency of this extraction step was examined by
stripping the immunoblots and reblotting with streptavidinconjugated horseradish peroxidase followed by Sigma Fast
diaminobenzidine color development to confirm the absence
of biotinylated proteins in SA bead processed samples (see
Fig. 3). Thus the post-SA bead supernatants contain only
intracellular (unbiotinylated) proteins, whereas CN bead supernatants contain all cellular proteins (biotinylated and
unbiotinylated). Surface CFTR expression was then determined by subtracting the intensity of the CFTR signal after
SA bead extraction from the intensity of the total CFTR
signal from CN bead processed samples.
Functional determination of surface CFTR expression over
time. Chloride secretion was determined by isotopic efflux of
125
I from preloaded cells as previously described (33). Briefly,
membrane inserts containing LLC-PK1 cells treated with 20
␮g/ml of cycloheximide at 37°C were excised, and cells were
washed with efflux buffer (140 mM NaCl, 4.7 mM KCl, 1.2
mM CaCl2, 10 mM glucose, and 10 mM HEPES, pH 7.4). The
cells were then loaded with 125I by incubation with 3 ␮Ci of
125
I-labeled Na (New England Nuclear, Boston, MA) for 1 h
at 37°C. Cell monolayers were washed free of excess 125I, and
the time-dependent efflux of the isotope into the media was
measured after stimulation with the cAMP agonists forskolin
(10 ␮M) and isobutyl methylxanthine (IBMX, 1.5 mM). Cells
were then solubilized with 0.1 N NaOH, and 125I radioactivity in the media samples and the final cell lysate was determined by counting gamma emissions (Packard Minaxi
gamma counter, series 5000). The rate coefficient of iodide
efflux (r) was calculated using the following formula
r ⫽ 关ln 共R 1 兲 ⫺ ln 共R 2 兲兴/共t 1 ⫺ t 2 兲
where R1 and R2 are the percent of counts remaining in the
cell layer at times t1 and t2. Data are presented as means ⫾
SE of replicate experiments. Differential inhibition of 125I
efflux by DIDS and diphenylamine-2-carboxylic acid was
used to confirm that the cAMP-stimulated efflux from these
cells was CFTR mediated.
Immunocytochemical determination of surface CFTR expression. Butyrate/low-temperature-treated cells, grown on
collagen-coated coverslips, were fixed for 1 h with 4% paraformaldehyde and then permeabilized with 0.25% saponin in
PBS for 10 min. Free aldehyde residues were quenched for 30
min with 50 mM NH4Cl, and cells were blocked with 1% BSA,
both prepared in PBS. Cells were then incubated overnight
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 18, 2017
Documented differences in vesicle trafficking of
⌬F508 and wild-type CFTR (2, 32) suggest a potential
role for membrane trafficking in the regulation of
plasma membrane CFTR function. However, technical
difficulties in getting measurable quantities of the
⌬F508 protein to the cell surface have hindered efforts
to compare plasma membrane ⌬F508 and wild-type
CFTR protein expression. The current study overcame
that obstacle by the simultaneous treatment of cells
with low temperature and sodium butyrate, which act
synergistically to markedly upregulate surface CFTR
expression (12). With readily detectable levels of
⌬F508 CFTR at the cell surface, it became possible to
test the hypothesis that the ⌬F508 protein is more
biochemically unstable than wild-type CFTR. Performed in a polarized epithelial cell line, the experiments presented confirm that the biochemical half-life
of plasma membrane ⌬F508 CFTR is much shorter
than that of wild-type CFTR. These differences in biochemical half-life correlate with transport function,
suggesting that rapid internalization and/or degradation of ⌬F508 CFTR alone can account for the rapid
loss of chloride transport function characteristic of
⌬F508 cells.
C167
C168
PLASMA MEMBRANE HALF-LIFE OF CFTR
RESULTS
Because there is no natural cell type that expresses
both ⌬F508 and wild-type CFTR, transfected cell lines
have served as the standard model system for studying
differences in the processing and trafficking of CFTR.
The most widely used cell lines (CHO, 3T3, C127, and
HEK), however, are either unpolarized or of undetermined polarity. Because vesicle trafficking in polarized
and unpolarized cells may differ, we sought to examine
CFTR surface expression in a transfected mammalian
cell line with evidence of cellular polarity. LLC-PK1
cells are transformed epithelial cells from the proximal
tubule of pig kidney. Morphological evidence for polarity was obtained by electron microscopy, which demonstrated the appearance of apical tight junctions in
monolayers of transfected cells (Fig. 1A). Functional
polarity was demonstrated by double-label immunofluorescent microscopy experiments using apical and basolateral membrane markers. As shown in Fig. 1B,
Na⫹-K⫹-ATPase expression in LLC-PK1 cells is primarily surface and basal in location, consistent with its
known distribution along the basolateral membrane of
polarized epithelial cells. Although CFTR has both a
surface and an intracellular signal in these high-expressing cells, the surface signal is more apically distributed and does not colocalize with that of Na⫹-K⫹ATPase. The prominent intracellular CFTR signal
seen in these cells is discussed in greater detail below.
These experiments indicate that CFTR-transfected
LLC-PK1 cells have both morphological and functional
features characteristic of polarized epithelial cells.
For direct biochemical study of ⌬F508 expression at
the plasma membrane, sufficient quantities of the protein had to be driven to the cell surface. To accomplish
this, cells were treated at 27°C in the presence of 5 mM
sodium butyrate to markedly upregulate surface CFTR
expression (12). Previous time course experiments
identified 60 h of treatment as optimal for the upregulation of CFTR expression (12). Although wild-type
cells did not require butyrate or low temperature treatment for the detection of plasma membrane CFTR, we
chose to control our experiments by treating all cells
identically. As shown in Fig. 2, mature (band C) ⌬F508
CFTR was not detected in LLC-PK1 cells grown at 37°C
in the presence or absence of sodium butyrate, nor was
it detected by immunoblot analysis in cells grown at
27°C alone. When sodium butyrate and low-temperature treatments were combined, however, there was a
marked increase in total ⌬F508 CFTR expression that
was accompanied by the appearance of the mature,
fully glycosylated form (band C). The upregulation of
CFTR expression under these experimental conditions
was even more pronounced in wild-type LLC-PK1 cells.
Although mature (band C) CFTR has routinely been
used as a marker of plasma membrane CFTR expression (5, 8, 30), studies in both native (26, 36) and
transfected (21) cells have shown that not all mature
CFTR resides at the cell surface. To confirm that
⌬F508 CFTR was driven to the surface of LLC-PK1
cells by butyrate and low-temperature treatment,
plasma membrane CFTR expression was measured
biochemically, functionally, and cytochemically. Surface biotinylation and streptavidin extraction were
used to determine how much ⌬F508 protein actually
reached the cell surface and what molecular form(s) of
CFTR were targeted there. As shown in Fig. 3, most of
the CFTR in treated ⌬F508 and wild-type LLC-PK1
cells did not reach the cell surface. Based on replicate
experiments (n ⫽ 5), ⬃35% of CFTR was found to
reside at the cell surface (33.9 ⫾ 4.7% for ⌬F508 and
35.5 ⫾ 3.4% for wild-type CFTR). These biotinylation
experiments also demonstrated that only the mature
band C form of CFTR was expressed at the plasma
membrane. The immature band B form was unaffected
by streptavidin extraction, indicating that it was not
biotinylated and therefore not present at the cell surface. Surface expression of ⌬F508 CFTR after butyrate
and low-temperature treatment was subsequently confirmed cytochemically and functionally (Fig. 4). The
relative distribution of CFTR between surface and intracellular compartments, including the prominent intracellular signal, correlated with the above biotinylation data. Functional evidence for surface CFTR
expression in both cell lines was obtained by forskolin/
IBMX-stimulated 125I efflux assay (Fig. 4).
With the use of the surface biotinylation procedure,
the half-life of plasma membrane CFTR in ⌬F508 and
wild-type cells was examined next (Fig. 5). In these
experiments, surface CFTR expression was upregulated by pretreatment with sodium butyrate and low
temperature. Cells were then transferred to physiological temperature (37°C) and treated for up to 48 h in
the presence of cycloheximide. Cells were then surface
biotinylated, solubilized, and streptavidin-extracted,
with immunoblot analysis being used to quantify
CFTR protein expression. The rapid fall in immature
(band B) CFTR expression in the ⌬F508 cells was due
to the inhibition of new protein synthesis by cycloheximide, combined with ER degradation and some conversion to the mature band C form. Although not
shown in Fig. 5, band B CFTR was also rapidly degraded in wild-type cells (the signal was undetectable
by 4 h, the earliest time point we examined). These
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 18, 2017
at 4°C with R3194 (polyclonal anti-CFTR) and/or monoclonal
antibody 6H (monoclonal against the ␤-subunit of rat Na⫹K⫹-ATPase, generously provided by Michael J. Caplan, Yale
School of Medicine, New Haven, CT). Anti-CFTR labeling
was detected with FITC-conjugated goat anti-rabbit F(ab⬘)2
fragments, and anti-Na⫹-K⫹-ATPase labeling was detected
with tetramethylrhodamine isothiocyanate-conjugated goat
anti-mouse F(ab⬘)2 fragments (Jackson ImmunoResearch
Labs, West Grove, PA). For double-label experiments, primary antibodies were applied simultaneously as were the
fluorescent-conjugated secondary antibodies. Fluorescent
signals were visualized on an Axiophot fluorescent microscope (Zeiss) and digitally stored using Photoshop 4.01 software (Adobe Systems, Mountain View, CA). Photoshop was
not used to modify images other than to adjust contrast for
improved signal definition. No signal was detected in the
absence of primary antibody, indicating that background
labeling was low under the experimental conditions employed (data not shown).
PLASMA MEMBRANE HALF-LIFE OF CFTR
C169
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 18, 2017
Fig. 1. Analysis of LLC-PK1 cell polarity. Wild-type LLC-PK1 cells were grown on permeable supports, and their
polarity was determined both morphologically and functionally. A: low (left)- and high (right)-magnification
electron micrographs showing tight junctions at the apical domain of adjacent cells (arrowheads). Original
magnifications were ⫻12,500 and ⫻24,000, respectively. Bars, 1 ␮m. B: double-label immunofluorescence micrographs of cystic fibrosis transmembrane conductance regulator (CFTR; green) and Na⫹-K⫹-ATPase (red) distribution in the same LLC-PK1 cells. The distribution of both membrane proteins was captured through the apical,
subapical, and basal regions of cells by epifluorescence microscopy. CFTR labeling is seen throughout the cell, but
membrane labeling is most prominent in the apical and subapical regions. Na⫹-K⫹-ATPase is localized to the
plasma membrane at the basal part of the cell. The plasma membrane labeling pattern differs for each.
findings are consistent with the observations of Ward
and Kopito (34), who demonstrated that the immature
forms of ⌬F508 and wild-type CFTR have similar rates
of degradation.
In ⌬F508 cells, total band C CFTR expression was
nearly eliminated by 6 h. In contrast, total band C
CFTR expression in the wild-type cells persisted for
48 h. Although most of the band C CFTR was intracel-
C170
PLASMA MEMBRANE HALF-LIFE OF CFTR
half-life values for ⌬F508 and wild-type CFTR were
obtained (data not shown). Thus several lines of evidence indicate that the difference in plasma membrane
expression of ⌬F508 and wild-type CFTR reflects the
biology of these two protein species and not simply
overexpression.
DISCUSSION
lular, the persistence of a streptavidin-extractable pool
for as long as 48 h provides biochemical evidence for
the presence of plasma membrane CFTR expression in
wild-type cells for at least this length of time. Because
the relative distribution of CFTR between intracellular
and plasma membrane compartments did not change
appreciably over time (data not shown), the plasma
membrane half-life of each protein was estimated by
quantifying the rate of degradation of total band C
CFTR. From replicate experiments (n ⫽ 7), we calculated the biochemical half-life of plasma membrane
CFTR in ⌬F508 cells to be ⬃4 h, whereas the biochemical half-life of plasma membrane CFTR in wild-type
cells exceeded 48 h.
To rule out the possibility that the relatively long
plasma membrane half-life of wild-type CFTR was an
artifact of overexpression, these biochemical and functional studies were repeated under conditions of comparable total CFTR expression in the two cells lines.
Based on data from Fig. 2, wild-type cells treated with
sodium butyrate at 37°C had CFTR expression levels
that were comparable to those found in ⌬F508 cells
treated with sodium butyrate at 27°C. Thus cells were
pretreated in this manner and then processed and
analyzed as described previously. Under these conditions of comparable total CFTR expression, the biochemical half-life of band C CFTR in wild-type cells
continued to exceed 24 h. Surface biotinylation experiments also confirmed the presence of plasma membrane CFTR for at least this length of time (Fig. 6).
This contrasts with the near absence of any detectable
⌬F508 protein after 6 h of chase. Although we were
able to detect some functional ⌬F508 CFTR at these
early time points by the qualitative 125I efflux assay, no
functional CFTR expression was detected in ⌬F508
cells after 24 h of chase, which contrasts sharply with
the persistence of functional CFTR in the wild-type
cells at this time. Similar kinetic studies were performed in transfected (unpolarized) C127 cells, where
⌬F508 expression exceeds that of wild-type CFTR after
butyrate and low-temperature treatment, and similar
Fig. 3. Plasma membrane expression of CFTR. ⌬F508 and wild-type
LLC-PK1 cells were grown to ⬃70% confluence and then were treated
with 5 mM sodium butyrate at 27°C for 60 h to upregulate plasma
membrane CFTR expression. Cells were then surface biotinylated as
described in MATERIALS AND METHODS. Detergent lysates of the biotinylated cells were normalized to total protein (50 ␮g) and then
reacted with streptavidin-conjugated agarose beads (SA) to remove
all surface-biotinylated proteins or with plain agarose (control, CN)
beads, which remove no protein. Lysate proteins after bead treatment were separated by SDS-PAGE and were transferred to polyvinylidene difluoride membranes. Top: transfer membrane was immunoblotted with R3194 to detect CFTR protein expression. CN lanes
depict total CFTR expression, whereas SA lanes show only the
unbiotinylated (intracellular) proteins in those samples. Arrows
identify CFTR bands B and C. Bottom: the same transfer membrane
was stripped of antibodies and reblotted with streptavidin-conjugated alkaline phosphatase followed by diaminobenzidine reaction to
detect the presence of biotinylated proteins in each lane. The absence
of biotinylated proteins in the SA lanes shows the efficiency of the
streptavidin bead extraction step.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 18, 2017
Fig. 2. Effect of sodium butyrate (SB) and temperature on CFTR
protein expression. ⌬F508 and wild-type LLC-PK1 cells were grown
to ⬃70% confluence and then were treated for 60 h in the absence (⫺)
or presence (⫹) of 5 mM sodium butyrate at 37 or at 27°C. After
treatment, cells were harvested, lysed, and normalized to 50 ␮g total
protein before SDS-PAGE and immunoblotting with the anti-CFTR
antibody R3194. Arrows identify CFTR bands B and C.
Until the long-term goals of gene therapy are realized, strategies designed to upregulate plasma membrane expression of ⌬F508 CFTR remain an important
potential therapeutic option in CF (16, 29). Much emphasis has been placed on the biogenesis of ⌬F508
CFTR, with the goal being to overcome its biosynthetic
arrest and thereby increase the delivery of functional
⌬F508 CFTR channels to the cell surface. Effective
correction of the CF defect, however, will require stable
expression of ⌬F508 after reaching the plasma membrane, and one study has shown that the functional
half-life of plasma membrane ⌬F508 in intact cells is
PLASMA MEMBRANE HALF-LIFE OF CFTR
C171
less than that of wild-type CFTR (23). The current
study sought to test the hypothesis that the short
functional half-life in ⌬F508 cells results from more
rapid removal of CFTR channels from the cell surface.
To study the biochemical stability of plasma membrane ⌬F508 CFTR, sufficient quantities of the mutant
protein needed to be driven to the cell surface. The
current study took advantage of combination therapy
with sodium butyrate and low temperature to increase
surface ⌬F508 protein expression to levels sufficient for
the direct measurement of CFTR protein half-life by a
quantitative immunoblot technique (12). Thus, in all
Fig. 5. Biochemical half-life of plasma membrane CFTR. ⌬F508 and
wild-type LLC-PK1 cells were pulsed with butyrate and low temperature and then were chased at 37°C in the presence of 20 ␮g/ml
cycloheximide for the times indicated. At the completion of the chase,
cells were transferred to 4°C, and surface proteins were biotinylated,
solubilized, and processed through either streptavidin (SA) or control
(CN) agarose beads. Postbead lysates, normalized to 50 ␮g total
protein, were immunoblotted with R3194 anti-CFTR antibody. Arrows identify CFTR bands B and C.
experiments, cells were pretreated with sodium butyrate and low temperature to upregulate CFTR protein expression and then were followed at 37°C to
examine the plasma membrane half-life of each CFTR
species. Because cell polarity can affect membrane
trafficking, all experiments were performed in a polarized epithelial cell line (LLC-PK1).
After butyrate and low-temperature treatment, most
of the CFTR in these cells was intracellular. Surface
biotinylation experiments demonstrated that only the
mature, fully glycosylated (band C) form of CFTR
reached the cell surface. No evidence for the direct
targeting of immature (electrophoretic bands A and B)
CFTR to the plasma membrane was found. Quantitative analysis of CFTR distribution in butyrate/lowtemperature-treated LLC-PK1 cells demonstrated that
⬃65% of band C CFTR remained intracellular. This
was true for both ⌬F508 and wild-type CFTR and is
similar to observations made in other cell systems (26,
36). Although changes in band C CFTR expression
correlated with changes in plasma membrane CFTR
expression in transfected LLC-PK1 cells, this may not
be true in all cell types. Thus surface protein labeling
techniques remain the most accurate means of measuring plasma membrane CFTR expression.
The biochemical finding of a prominent intracellular
compartment of CFTR was confirmed immunocytochemically. After butyrate/low-temperature treatment,
both ⌬F508 and wild-type cells labeled similarly, with
modest surface but prominent intracellular signals.
The identity of the intracellular signal in each cell type
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 18, 2017
Fig. 4. Cytochemical and functional
detection of plasma membrane CFTR.
Expression of ⌬F508 (left) and wildtype (right) CFTR at the plasma membrane after butyrate and low-temperature treatment was determined by
immunofluorescence microscopy (top)
and 125I efflux assay (bottom). Arrows
in the immunofluorescence micrographs denote plasma membrane
CFTR labeling. Arrows in the 125I efflux graphs note the time when cells
were stimulated with forskolin/isobutyl methylxanthine (IBMX). E, Control
condition (cells grown at 37°C without
any butyrate treatment); F, 27°C ⫹ SB.
125
I efflux values represent means ⫾
SE from replicate experiments.
C172
PLASMA MEMBRANE HALF-LIFE OF CFTR
remains unknown. In the wild-type cells, surface biotinylation demonstrated that most of the unbiotinylated CFTR is of the band C form and therefore is
presumably in a post-ER compartment (Golgi, endosomes, transport vesicles, etc.). In ⌬F508 cells, where
most of the unbiotinylated CFTR is of the band B form,
a large ER component to the intracellular signal would
be expected. Identification of the sources of intracellular CFTR labeling in these cells will, however, require
much additional study.
The kinetics of ⌬F508 and wild-type CFTR degradation over time was examined at physiological temperature and under conditions of protein synthesis inhibition. The rate of CFTR degradation was quantified
biochemically and confirmed by functional assay. Replicate experiments indicate that the biochemical halflife of plasma membrane ⌬F508 CFTR is ⬃4 h,
whereas the biochemical half-life of plasma membrane
wild-type CFTR exceeds 48 h. These values correlate
with changes in 125I efflux and are remarkably similar
to the functional data generated by Lukacs et al. (23) in
nonpolarized C127 cells. This time-dependent correlation between the rate of plasma membrane CFTR degradation and the loss of cAMP-stimulated 125I efflux
indicates that the functional instability in ⌬F508 cells
can be theoretically attributed to more rapid degradation of plasma membrane ⌬F508 CFTR. The data,
however, do not exclude a contributory role for channel
inactivation in this process.
These findings establish that the ⌬F508 mutation
has a negative effect on the biochemical stability of
CFTR at the cell surface. This biochemical instability
must be due to more rapid internalization of mutant
protein and/or its selective targeting for rapid degradation. CFTR is endocytosed in clathrin-coated vesicles
(1, 22), and the molecular signal for CFTR internalization may reside in its cytoplasmic tail (10, 24, 25). The
⌬F508 mutation, however, is in the first nucleotidebinding domain and is some distance from the cytoplasmic end of the molecule. Thus CFTR must have
another internalization signal, or the ⌬F508 mutation
must have an indirect effect on the COOH-terminal
signal(s). Based on our understanding of CFTR folding
during biogenesis, it is attractive to hypothesize that
the ⌬F508 mutation affects CFTR folding in such a
manner that the COOH-terminal internalization signal is altered. The fundamental question is what is the
signal? COOH-terminal tyrosine-based sequences have
been implicated in the positioning of membrane proteins in coated pits (7, 20), and one recent study has
implicated phosphorylation of tyrosine-1424 in the regulation of CFTR endocytosis (25). On the other hand,
the rate of internalization of Ste6, a yeast homolog of
CFTR, and Ste3p, another yeast membrane protein,
are regulated by ubiquitination (14, 27). Because ubiquitination is the major signal for the degradation of
both wild-type and ⌬F508 CFTR during biogenesis
(35), it is provocative to speculate that it is also involved in plasma membrane CFTR degradation. During CFTR biosynthesis, ubiquitination targets the immature forms of both ⌬F508 and wild-type CFTR to
rapid proteosome degradation with similar kinetics
(34). A pool of wild-type CFTR, however, escapes this
fate, becomes fully glycosylated, and reaches the
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 18, 2017
Fig. 6. Half-life of plasma membrane
CFTR under conditions of comparable
levels of CFTR expression. To confirm
that the long plasma membrane halflife of wild-type CFTR was not an artifact of overexpression, an experiment
was performed under conditions of comparable total CFTR expression in both
cell types. Based on data in Fig. 2,
⌬F508 cells treated with 5 mM sodium
butyrate at 27°C were compared with
wild-type cells treated with 5 mM sodium butyrate at 37°C. After treatment,
all cells were transferred to 37°C and
were incubated in the presence of 20
␮g/ml cycloheximide for the times
shown. Top: biochemical half-life of
⌬F508 and wild-type CFTR after surface biotinylation, SA or CN bead extraction, and immunoblotting with
R3194. Bottom: 125I efflux from forskolin/IBMX-stimulated cells at baseline
(F) and after 24 h of chase at 37°C (■). E,
Control condition (cells grown at 37°C
without any butyrate treatment or
chase period). The biochemical and
functional stability of wild-type CFTR
continues to exceed 24 h, even when
relative overexpression is corrected.
PLASMA MEMBRANE HALF-LIFE OF CFTR
We thank Dr. Raymond A. Frizzell for helpful discussions and
acknowledge the technical assistance of Virginia Jeanes.
This work was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs
(C. R. Marino) and by the Cystic Fibrosis Foundation (C. R. Marino).
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
REFERENCES
1. Bradbury NA, Cohn JA, Venglarik CJ, and Bridges RJ.
Biochemical and biophysical identification of cystic fibrosis
transmembrane conductance regulator chloride channels as
components of endocytic clathrin-coated vesicles. J Biol Chem
269: 8296–8304, 1994.
2. Bradbury NA, Jilling T, Berta G, Sorscher EJ, Bridges RJ,
and Kirk KL. Regulation of plasma membrane recycling by
CFTR. Science 256: 530–532, 1992.
3. Chavrier P and Goud B. The role of ARF and Rab GTPases in
membrane transport. Curr Opin Cell Biol 11: 466–475, 1999.
4. Cheng SH, Fang SL, Zabner J, Marshall J, Piraino S,
Schivi SC, Jefferson DM, Welsh MJ, and Smith AE. Functional activation of the cystic fibrosis trafficking mutant ⌬F508-
20.
21.
22.
CFTR by overexpression. Am J Physiol Lung Cell Mol Physiol
268: L615–L624, 1995.
Cheng SH, Gregory RJ, Marshall J, Paul S, Souza DW,
White GA, O’Riordan CR, and Smith AE. Defective intracellular transport and processing of CFTR is the molecular basis of
most cystic fibrosis. Cell 63: 827–834, 1990.
Dalemans W, Barbry P, Champigny G, Jallat S, Dott K,
Dreyer D, Crystal RG, Pavirani A, Lecocq J-P, and Lazdunski M. Altered chloride ion channel kinetics associated with
the ⌬F508 cystic fibrosis mutation. Nature 354: 526–528, 1991.
Davis CG, Goldstein JL, Sudhof TC, Anderson RGW, Russell DW, and Brown MS. Acid-dependent ligand dissociation
and recycling of LDL receptor mediated by growth factor homology region. Nature 326: 760–765, 1987.
Denning GM, Anderson MP, Amara JF, Marshall J, Smith
AE, and Welsh MJ. Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature 358: 761–764, 1992.
Drumm ML, Wilkinson DJ, Smit LS, Worrell RT, Strong
TV, Frizzell RA, Dawson DC, and Collins FS. Chloride conductance expressed by ⌬F508 and other mutant CFTRs in Xenopus oocytes. Science 254: 1797–1799, 1991.
Haardt MH, Benharouga M, Lechardeur D, Kartner N,
and Lukacs GL. C-terminal truncations destabilize the cystic
fibrosis transmembrane conductance regulator without imparing its biogenesis. J Biol Chem 274: 21873–21877, 1999.
Haws CM, Nepomuceno IB, Krouse ME, Wakelee H, Law T,
Xia Y, Nguyen H, and Wine JJ. ⌬F508-CFTR channels: kinetics, activation by forskolin, and potentiation by xanthines. Am J
Physiol Cell Physiol 270: C1544–C1555, 1996.
Heda GD and Marino CR. Surface expression of the cystic
fibrosis transmembrane conductance regulator mutant, ⌬F508,
is markedly up-regulated by combination treatment with sodium
butyrate and low temperature. Biochem Biophys Res Commun
271: 659–664, 2000.
Kerem B, Rommens JM, Buchanan JA, Markiewicz D, Cox
TK, Chakravarti A, Buchwald M, and Tsui L-C. Identification of the cystic fibrosis gene: genetic analysis. Science 245:
1073–1080, 1989.
Kolling R and Hollenberg CP. The ABC-transporter Ste6
accumulates in the plasma membrane in a ubiquitinated form in
endocytosis mutants. EMBO J 13: 3261–3271, 1994.
Kolling R and Losko S. The linker region of the ABC-transporter Ste6 mediates ubiquitination and fast turnover of the
protein. EMBO J 16: 2251–2261, 1997.
Kopito RR. Biosynthesis and degradation of CFTR. Physiol Rev
79, Suppl 1: S167–S173, 1999.
Lee MG, Whigley WC, Zeng W, Noel LE, Marino CR,
Thomas PJ, and Muallem S. Regulation of Cl⫺/HCO3⫺ exchange by cystic fibrosis transmembrane conductance regulator
expressed in NIH 3T3 and HEK 293 cells. J Biol Chem 274:
3414–3421, 1999.
Li C, Ramjeesingh M, Reyes E, Jensen T, Chang X-B,
Rommens JM, and Bear CE. The cystic fibrosis mutation
(delta F508) does not influence the chloride channel activity of
CFTR. Nat Genet 3: 311–316, 1993.
Lisanti MP, Le Bivic A, Sargiacomo M, and RodriguezBoulan E. Steady-state distribution and biogenesis of endogenous Madin-Darby canine kidney glycoproteins: evidence for
intracellular sorting and polarized cell surface delivery. J Cell
Biol 109: 2117–2127, 1989.
Lobel P, Fujimoto K, Ye RD, Griffiths G, and Kornfeld S.
Mutations in the cytoplasmic domain of the 275 kd mannose
6-phosphate receptor differentially alter lysosomal enzyme sorting and endocytosis. Cell 57: 787–796, 1989.
Lukacs GL, Chang XB, Kartner N, Rotstein OD, Riordan
JR, and Grinstein S. The cystic fibrosis transmembrane regulator is present and functional in endosomes. Role as a determinant of endosomal pH. J Biol Chem 267: 14568–14572, 1992.
Lukacs GL, Segal G, Kartner N, Grinstein S, and Zhang F.
Constitutive internalization of cystic fibrosis transmembrane
conductance regulator ocurs via clathrin-dependent endocytosis
and is regulated by protein phosphorylation. Biochem J 328:
353–361, 1997.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 18, 2017
plasma membrane. If ubiquitination was also the signal for plasma membrane CFTR degradation, one
would have to postulate that the wild-type and mutant
proteins were differentially ubiquitinated at the cell
surface to account for their different rates of degradation. Because the yeast data suggest that the proteasome complex is not the final target of ubiquitinated
plasma membrane Ste6 (15), one must also consider
the possibility that ubiquitination of plasma membrane CFTR serves a different role (e.g., signaling for
internalization and/or sorting rather than proteosome
degradation). Although provocative, a role for ubiquitination in the downregulation of surface CFTR expression remains highly speculative at this time.
Alternatively, mutant and wild-type CFTR may have
similar rates of internalization but are differentially
sorted in the endocytic compartment, with ⌬F508 being targeted for rapid lysosomal degradation while
wild-type CFTR is recycled back to the plasma membrane. In this case, the ⌬F508 mutation might be
affecting the interaction of CFTR with specific GTPbinding (Rab) proteins that regulate vesicle trafficking
(reviewed in Ref. 3). Rab4 has been most closely associated with sorting endosomes, so an understanding of
its interactions with ⌬F508 and wild-type CFTR would
be of particular interest. Last, one must consider the
effect of phosphorylation on CFTR trafficking, particularly in light of data showing that the rate of endocytosis differs in ⌬F508 and wild-type cells after cAMP
activation (2). Although the current study did not examine the biochemical stability of plasma membrane
CFTR under stimulatory conditions, the basal phosphorylation state of these two CFTR species may differ,
and this, in turn, could expose different internalization
signals.
In conclusion, the ⌬F508 mutation dramatically reduces the residence time of CFTR at the cell surface.
This must be due to an effect of the mutation on the
rate of CFTR internalization and/or its intracellular
targeting. Although little is known about this aspect of
CFTR biology, it has clear implications for any therapeutic strategies that rely on delivering more ⌬F508
protein to the cell surface.
C173
C174
PLASMA MEMBRANE HALF-LIFE OF CFTR
30. Sato S, Ward CL, Krouse ME, Wine JJ, and Kopito RR.
Glycerol reverses the misfolding phenotype of the most common
cystic fibrosis mutation. J Biol Chem 271: 635–638, 1995.
31. Schultz BD, Frizzell RA, and Bridges RJ. Rescue of dysfunctional deltaF508-CFTR chloride channel activity by IBMX. J
Membr Biol 170: 51–66, 1999.
32. Schweibert EM, Gesek R, Ercolani L, Wjasow C, Gruenert
DC, Karlson K, and Stanton BA. Heterotrimeric G proteins,
vesicle trafficking, and CFTR Cl⫺ channels. Am J Physiol Cell
Physiol 267: C272–C281, 1994.
33. Venglarik CJ, Bridges RJ, and Frizzell RA. A simple assay
for agonist-regulated Cl and K conductances in salt-secreting
epithelial cells. Am J Physiol Cell Physiol 259: C358–C364,
1990.
34. Ward CL and Kopito RR. Intracellular turnover of cystic
fibrosis transmembrane conductance regulator. J Biol Chem
269: 25710–25718, 1994.
35. Ward CL, Omura S, and Kopito RR. Degradation of CFTR by
the ubiquitin-proteasome pathway. Cell 83: 121–127, 1995.
36. Webster P, Vanacore L, Nairn AC, and Marino CR. Subcellular localization of CFTR to endosomes in a ductal epithelium.
Am J Physiol Cell Physiol 267: C340–C348, 1994.
37. Zeng W, Lee MG, Yan M, Diaz J, Benjamin I, Marino CR,
Kopito RR, Freedman S, Cotton CU, Muallem S, and
Thomas P. Immuno and functional characterization of CFTR in
submandibular and pancreatic acinar and duct cells. Am J
Physiol Cell Physiol 273: C442–C455, 1997.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 18, 2017
23. Lukacs GL, Xiu-Bao C, Bear C, Kartner N, Mohamed A,
Riordan JR, and Grinstein S. The ⌬F508 mutation decreases
the stability of cystic fibrosis transmembrane conductance regulator in the plasma membrane. J Biol Chem 268: 21592–21598,
1993.
24. Mukherjee S, Ghosh RN, and Maxfield FR. Endocytosis.
Physiol Rev 77: 759–803, 1997.
25. Prince LS, Peter K, Hatton SR, Zaliauskiene L, Cotlin LF,
Clancy JP, Marchase RB, and Collawn JF. Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator requires a tyrosine-based signal. J Biol Chem 274: 3602–
3609, 1999.
26. Prince LS, Tousson A, and Marchase RB. Cell surface labeling of CFTR in T84 cells. Am J Physiol Cell Physiol 264: C491–
C498, 1993.
27. Roth AF and Davis NG. Ubiquitination of the yeast a-factor
receptor. J Cell Biol 134: 661–674, 1996.
28. Rubenstein RC, Egan ME, and Zeitlin PL. In vitro pharmacologic restoration of CFTR-mediated chloride transport
with sodium 4-phenylbutyrate in cystic fibrosis epithelial
cells containing ⌬F508-CFTR. J Clin Invest 100: 2457–2465,
1997.
29. Rubenstein RC and Zeitlin PL. A pilot clinical trial of oral
sodium 4-phenylbutyrate (Buphenyl) in deltaF508-homozygous cystic fibrosis patients: partial restoration of nasal epithelial CFTR function. Am J Respir Crit Care Med 157: 484–
490, 1998.