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J. Biol. Chem., Vol. 277, Issue 19, 16419-16425, May 10, 2002
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From the
Received for publication, December 25, 2001, and in revised form, February 26, 2002
Mutations in the cystic fibrosis
transmembrane conductance regulator protein (CFTR) cause cystic
fibrosis. The most common disease-causing mutation, The cystic fibrosis transmembrane conductance regulator protein
(CFTR)1 mediates
transepithelial chloride transport across epithelial cells in the
airways, intestine, pancreas, and sweat gland. Some mutations in CFTR
cause the lethal genetic disease cystic fibrosis, which produces
chronic lung infection, progressively impaired pulmonary function, and
pancreatic insufficiency. The most common CFTR mutant, CFTR biosynthesis is an inefficient process. Newly synthesized wild
type (wt)-CFTR and CFTR folding in the ER appears to be facilitated by interactions with
the cellular quality control machinery. Wild type CFTR and Here we have used fluorescence recovery after photobleaching to measure
the diffusional mobility of wt-CFTR and Cell Culture, Plasmids, Transfection, and Treatments--
COS7
(ATCC CRL-1651) and CHO-K1 (ATCC CCL-61) cells (obtained from the
University of California, San Francisco Cell Culture Facility) were
cultured in DMEH-21 and Ham's F12 media, respectively, supplemented
with 10% fetal bovine serum, 100 units/ml penicillin and 100 µg/ml
streptomycin at 37 °C in a 5% CO2/95% air
atmosphere. Cells were grown on 18-mm diameter glass coverslips
in 12-well plates. Cells were transiently transfected with plasmid DNA
using 6 µl of lipofectamine (Invitrogen) and 1 µg of plasmid DNA
according to the manufacturer's instructions.
cDNA encoding wt-CFTR was fused downstream of the enhanced GFP
(CLONTECH) as described previously, and the
Photobleaching experiments were performed 2-3 days after transfection
for CFTR-transfected cells and 1-2 days after transfection for
cytoplasmic or ER-targeted unconjugated GFP. For most experiments GFP-CFTR-transfected cells were treated with brefeldin A (BFA) by
16-24 h of incubation at 5 µg/ml at 37 °C in the CO2
incubator. For ATP depletion, cells were incubated in
phosphate-buffered saline containing 6 mM 2-deoxyglucose
and 0.02% sodium azide at 37 °C for 30 min or 1 h.
Geldanamycin (Calbiochem) was used at 0.1 µg/ml for 90 min (14) and
tunicamycin (Calbiochem) at 5 µg/ml for 20 h. Clasto-lactacystin
Fluorescence Recovery after Photobleaching--
For spot
photobleaching, an argon ion laser beam (488 nm, Innova 70-4,
Coherent) was modulated by an acousto-optic modulator and the
first-order beam was directed onto cells through an oil immersion
(Nikon fluor) objective lens (×40, numerical aperture (N.A.) 1.3;
×60, N.A. 1.4; or ×100, N.A. 1.4) using an inverted epifluorescence
microscope. To visualize cells and to select cellular regions to be
bleached, full-field epi-illumination was accomplished using the
expanded zero-order laser beam directed onto the objective lens by a
fiber optic. The beam was reflected onto the objective by a dichroic
mirror (510 nm), and emitted fluorescence was filtered by serial
530 ± 15 nm bandpass and 515 nm longpass filters. Emitted fluorescence was detected by a photomultiplier and digitized by a
14-bit analog-to-digital converter. The photomultiplier was transiently
gated off during the bleach pulse. Fluorescence was sampled over 200 ms
before the bleach pulse, then at rates of up to 1 MHz. For measurement
of slow recoveries, an electronic shutter was used to collect
fluorescence at 1 Hz, averaging 10,000 acquisitions during the shutter
open time of 20 ms.
For photobleaching experiments with image detection, a Leitz upright
microscope with a cooled CCD camera detector (Photometrics) was used to
record full-field epifluorescence images. An electronically shuttered
bleach beam from the argon laser (488 nm) was directed onto the sample
(from below) using a Nikon ×25 long working-distance air objective.
Cells were viewed (from above) using an oil immersion ×60 objective
(N.A. 1.4) and GFP filter set (HQ filters, Chroma). Software was
written (in LabVIEW, National Instruments) to coordinate the bleach
pulse, illumination and camera shutters, and image acquisition.
Fluorescence loss in photobleaching (FLIP) experiments were performed
using the same apparatus in which repeated laser photobleaching was
performed with specified delays between bleach pulses.
All photobleaching experiments were done at 37 °C. Cultured cells
were mounted in custom built perfusion chambers designed to fit a
PDMI-2 microincubator (Harvard Apparatus) controlled by a TC-201A
temperature regulator (Harvard Apparatus). Objective lens temperature
was also maintained at 37 °C using a lens thermoregulator (Bioptechs
Inc., Butler, PA). For maneuvers on GFP-CFTR-expressing cells, single
recovery curves from 5-12 different cells were averaged to generate a
single averaged recovery curve, and this was repeated 4-10 times using
different coverslips. For cytoplasmic GFP and ER-targeted
GFP-expressing cells, each averaged recovery curve was generated from
10 individual recoveries, each from a different cell. For
diOC4(3) measurements, averaged recovery curves were generated from the average of 5-8 cells. Results are reported as
means ± S.E. for the number of different coverslips, and
representative averaged recovery curves from single coverslips are
shown in figures for the reader to evaluate curve shape and
signal-to-noise ratio. Statistical analysis was performed by
analysis of variance against control conditions, and values of
p < 0.05 were considered significant.
Analysis of Photobleaching Recovery Data--
Spot
photobleaching recovery curves were analyzed as described in Levin
et al. (27). Briefly, recovery half-times
(t1/2), the time for fluorescence to recover by 50%
due to diffusion, were determined from fluorescence recovery curves,
F(t), by non-linear regression. Fluorescence
recovery curves were taken to be a combination of reversible (as
determined from fixed cell analysis) and irreversible (due to
diffusion) processes, F(t) = a1(F(t)reversible + a2(F(t)irreversible) where a1 + a2 = 1. The diffusion-mediated
recovery was fitted using the semiempirical equation:
F(t)irreversible = F0 + (F0 + R(Finf The diffusion of wt-CFTR and Photobleaching experiments with image detection were done to evaluate
the gross mobility of GFP-CFTR chimeras in BFA-treated COS7 (Fig.
2A) and CHO (Fig.
2B) cells. A large region of the cell, ~5 µm in diameter
(marked by cross-hairs), was bleached by the laser pulse,
and fluorescence recovery was recorded by serial imaging. In each case
a darkened region is seen immediately postbleach (t = 0 s). Recovery into this bleached region is diffusive in nature from
the edges of the bleach spot inward and appears to be nearly complete.
The kinetics of fluorescence recovery were qualitatively similar for
cells expressing wt-CFTR and
Diffusional Mobility of the Cystic Fibrosis Transmembrane
Conductance Regulator Mutant,
F508-CFTR, in the Endoplasmic
Reticulum Measured by Photobleaching of GFP-CFTR Chimeras*
,¶
Departments of Medicine and Physiology,
Cardiovascular Research Institute, University of California, San
Francisco, California 94143-0521 and § Dartmouth Medical
School, Hanover, New Hampshire 03755-3835
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
F508, is
retained in the endoplasmic reticulum (ER) and is unable to function as
a plasma membrane chloride channel. To investigate whether the
ER retention of F508-CFTR is caused by immobilization and/or
aggregation, we have measured the diffusional mobility of green
fluorescent protein (GFP) chimeras of wild type (wt)-CFTR and
F508-CFTR by fluorescence recovery after photobleaching.
GFP-labeled F508-CFTR was localized in the ER and wt-CFTR in the
plasma membrane and intracellular membranes in transfected COS7 and
Chinese hamster ovary K1 cells. Both chimeras localized to the
ER after brefeldin A treatment. Spot photobleaching showed that CFTR
diffusion (diffusion coefficient ~10
9
cm2/s) was not significantly slowed by the F508 mutation
and that nearly all wt-CFTR and F508-CFTR diffused throughout the ER
without restriction. Stabilization of molecular chaperone interactions by ATP depletion produced remarkable F508-CFTR immobilization (~50%) and slowed diffusion (6.5 × 1010
cm2/s) but had little effect on wt-CFTR. Fluorescence
depletion experiments revealed that the immobilized F508-CFTR in
ATP-depleted cells remained in an ER pattern. The mobility of wt-CFTR
and F508-CFTR was reduced by maneuvers that alter CFTR processing or
interactions with molecular chaperones, including tunicamycin,
geldanamycin, and lactacystin. Photobleaching of the fluorescent ER
lipid diOC4(3) showed that neither ER restructuring nor
fragmentation during these maneuvers was responsible for the slowing
and immobilization of CFTR. These results suggest that
(a) the ER retention of F508-CFTR is not due to
restricted ER mobility, (b) the majority of F508-CFTR is
not aggregated or bound to slowly moving membrane proteins, and
(c) F508-CFTR may interact to a greater extent with
molecular chaperones than does wt-CFTR.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
F508-CFTR, is
retained at the endoplasmic reticulum (ER) and consequently is unable
to function as a plasma membrane chloride channel (1).
Electrophysiological studies indicate that the ER-retained F508-CFTR
is at least partially functional in excised ER membranes (2). Further,
growth of F508-CFTR-expressing cells under non-physiological
conditions (low temperature, Ref. 3 or with chemical chaperones, Ref.
4) indicates that under certain conditions F508-CFTR can be
transported to the plasma membrane and restore cell chloride permeability.
F508-CFTR are folded into the ER membrane where
they become core-glycosylated (5, 6). Only 20-30% of newly
synthesized wt-CFTR is transported to the Golgi where complex
glycosylation occurs, with the remaining protein ubiquinated and
degraded by the proteasome (7, 8). Wild type CFTR exists initially in a
protease-susceptible form (t1/2 ~30 min) that
becomes more stable upon transport from ER to Golgi (t1/2 ~24 h), whereas F508-CFTR is rapidly
degraded (t1/2 ~30 min) (5, 6). Inhibition of
proteasome function results in the formation of aggresomes, which are
detergent-insoluble, perinuclear protein aggregates common to many
degenerative diseases (9-11).
F508-CFTR
have been shown to interact with the molecular chaperones Hsc70, Hdj2,
Hsp70, Hsp90, and calnexin but not with BiP or Grp94 (12-15). Recent
data suggest that the Hsc70 co-chaperone CHIP (C-terminus of
Hsc70-interacting protein) targets immature CFTR for
proteasome-mediated degradation (16). Biochemical data indicate
that calnexin and Hsp70 bind F508-CFTR more avidly than wt-CFTR (12,
13). The differential processing of F508-CFTR and wt-CFTR, however,
is probably not accounted for by gross structural differences.
Proteolytic cleavage experiments have suggested that the conformation
of F508-CFTR is similar to that of the early, relatively unstable
form of wt-CFTR (17). In vitro folding studies of the
isolated nucleotide binding domain 1 (containing the phenylalanine 508 residue) support the view that the F508 mutation does not affect
structure (18, 19) but may affect folding kinetics.
F508-CFTR at the ER to
investigate whether F508-CFTR immobilization or interaction with
chaperones is responsible for its ER retention. Several mechanisms have
been proposed to explain the failure of F508-CFTR to be exported
from the ER: immobilization of F508-CFTR, potentially by association
with chaperones or by self-aggregation; efficient F508-CFTR
recycling from the Golgi; and/or failure of the ER export machinery to
recognize F508-CFTR. Photobleaching measurements were done on cells
expressing green fluorescent protein (GFP) chimeras of wt-CFTR and
F508-CFTR; a series of maneuvers was used to probe for interactions
with CFTR processing machinery. It was shown previously that GFP
fusion to the N terminus of CFTR did not effect CFTR localization,
processing, or function (9, 20-22). We used photobleaching previously
to quantify aqueous phase rheology in cellular and organellar
compartments (23-25) and to investigate protein-protein interactions
of aquaporin (AQP) water channels in the ER and plasma membranes (26,
27). We find here that the ER retention of F508-CFTR is not due to
immobilization, aggregation, or binding to slow moving membrane
components; however, a greater fraction of F508-CFTR than wild type
CFTR interacted with molecular chaperones after ATP depletion, and
inhibition of CFTR processing by a proteasome inhibitor resulted in
CFTR aggregation and immobilization.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
F508
mutation was introduced by site-directed mutagenesis (20, 22). A
glycosylation-null mutant of GFP-wt-CFTR, in which asparagine residues
were replaced by glutamines at positions 894 and 900, was also
generated by site-directed mutagenesis. For GFP expression in the ER
lumen, the GFP sequence was flanked by an N-terminal preprolactin
leader sequence and a C-terminal KDEL motif (25). For GFP expression in
the cytoplasm, the coding region of pEGFP
(CLONTECH) was ligated into pcDNA3.1 as a
HindIII/EcoRI fragment.
-lactone (the active form of lactacystin, Sigma) was used at 6 µM for 1.5, 6, or 14 h. In heat shock experiments,
cells were incubated at 42 °C for 1 h followed by 16-20 h
recovery at 37 °C. Paraformaldehyde (for GFP-CFTR immobilization)
was used at 4% for 30 min in phosphate-buffered saline at 23 °C. ER
membranes in untransfected cells (subjected to various treatments
above) were fluorescently labeled with 3,3'-dibutyloxacarbocyanine iodide (diOC4(3), Molecular Probes) by incubation for 10 min with 10 µM dye in phosphate-buffered saline at
37 °C. In some experiments transfected cells were identified by
their green fluorescence prior to diOC4(3) labeling, and
those cells were photobleached. The relatively small amount of GFP had
little effect on the recovery of the much brighter and more rapidly
diffusing diOC4(3).
F0))(t/t1/2)/(1 + (t/t1/2)) where F0
is prebleach fluorescence, Finf is fluorescence
at infinite time, and R is the fractional fluorescence
recovery. Absolute diffusion coefficients were calculated from
t1/2 using a mathematical model of ER diffusion (25,
28).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
F508-CFTR in the ER of living
cells was determined using GFP as a fluorescent reporter (Fig. 1A). Transfection of COS7 and
CHO cells with the GFP-wt-CFTR chimera showed GFP localization to the
plasma membrane and intracellular membranes (including Golgi and ER)
but exclusively to the ER after BFA treatment (Fig. 1B). The
GFP-F508-CFTR chimera was seen in an ER-like pattern without or
after BFA treatment (Fig. 1C). Similar patterns of ER
staining were observed in COS7 and CHO cells transfected with
ER-localized (aqueous phase) GFP or stained with the fluorescent ER
lipid probe diOC4(3) (not shown).
View larger version (36K):
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Fig. 1.
GFP-CFTR expression in transiently
transfected COS7 and CHO cells. A, GFP-CFTR construct
showing CFTR fused downstream of the GFP reporter. B,
fluorescence micrographs of transiently transfected COS7 and CHO cells
expressing GFP-wt-CFTR without (left) and after
(right) BFA treatment. C, fluorescence
micrographs of cells expressing GFP- F508-CFTR. Scale bar,
10 µm.
F508-CFTR and in both cell types. In
each case the fluorescence recovery was > 80% complete as
determined from the ratio of fluorescence intensity in the bleach spot
to the whole cell immediately after versus 4 min after the
bleach. Quantitative spot photobleaching was done in the brighter COS7
cells.
View larger version (78K):
[in a new window]
Fig. 2.
Photobleaching of GFP-CFTR chimeras in
transiently transfected COS7 (A) and CHO
(B) cells. Photobleaching with image detection in
cells expressing GFP-labeled wt-CFTR and F508-CFTR (after BFA
treatment). Images are shown before bleaching, with the bleach spot
indicated by white cross-hairs, and at the indicated times
after the bleach pulse. All experiments were done at 37 °C. Each
image series is one set of experiments representative of 4-8.
Scale bar, 10 µm.
Fig. 3A shows spot
photobleaching of GFP-labeled wt-CFTR (top) and F508-CFTR
(bottom) using a ×60 oil immersion objective to produce a
small spot (~1.2-µm diameter) in which GFP fluorescence was
bleached to ~50% of its initial value. To distinguish between processes that produce irreversible photobleaching (authentic GFP
diffusion) from those that produce reversible photobleaching (from
triplet-state relaxation or other photophysical phenomena), fluorescence recovery was measured in fixed cells (Fig. 3B)
and using different spot sizes (Fig. 3C). As found in prior
photobleaching studies (26, 27), the fluorescence recovery remaining
after fixation represents reversible (diffusion-independent)
photobleaching because fixation abolishes diffusion. Fig. 3B
(top curve) shows a small component (5-10%) of reversible
photobleaching with ~1-s exponential time constant. Similar
recoveries were found under the same photobleaching conditions for
unconjugated GFP in cytoplasm (middle curve) and aqueous
phase (unconjugated) GFP in the ER lumen (bottom curve). As
expected for reversible fluorescence recovery, the recovery rates did
not depend on spot size (not shown). Fig. 3C shows that (in
unfixed cells) the fluorescence recovery was spot
size-dependent as expected for diffusive processes, because
the recovery time for a larger spot increases approximately with the
square of the spot diameter. Using a regression procedure to determine
(t1/2) for irreversible fluorescence
recovery (see "Experimental Procedures"), t1/2 values for GFP-wt-CFTR were 1040 ± 60 ms (100×), 3.0 ± 0.4 s (60×), and 4.9 ± 0.2 s (40×) (n = 4-10 sets of measurements). The t1/2 values for
GFP-F508-CFTR (in BFA-treated cells) were 1170 ± 120 ms
(100×), 3.3 ± 0.5 s (60×), and 6.4 ± 0.3 s
(40×) (n = 4-10). The relative
t1/2 are as expected for a diffusive process.
|
To determine absolute diffusion coefficients, comparative
photobleaching measurements were done for the GFP-labeled CFTR chimeras (Fig. 3A) and for soluble GFP in the ER lumen. From a series
of experiments shown in Fig. 3A, t1/2 measured using a ×60 objective were 3.0 ± 0.4 s (wt-CFTR),
3.3 ± 0.5 s (F508-CFTR, +BFA), and 3.3 ± 0.6 s
(F508-CFTR, BFA) (n = 5-10 sets of experiments).
Fig. 3D (top) shows the substantially faster
fluorescence recovery of soluble GFP in the ER lumen of COS7 cells
(t1/2, 60 ± 4 ms; n = 12).
This value was similar to that measured in CHO-K1 cells under identical
conditions (t1/2, 65 ± 4 ms; n = 6, not shown). The absolute diffusion coefficient for luminal GFP in
COS7 cells was computed using a simple model to correct for ER
geometric effects (28) as (6.0 ± 0.5) × 108
cm2/s, similar to that previously measured in CHO cells
(25) and LLC-PK1 cells (27).
Fig. 3D (middle) summarizes diffusion
coefficients for the GFP-chimeras. There was no significant effect of
the F508 mutation on CFTR diffusion and/or of BFA treatment on
F508-CFTR mobility. Fig. 3D (bottom)
summarizes the percentage of mobile GFP, determined from the
completeness of the fluorescence recovery at long times. GFP-wt-CFTR fluorescence recovered almost completely (92 ± 2%, n = 10), whereas GFP-F508-CFTR recovery in
BFA-treated and untreated cells was slightly though significantly
smaller (85 ± 2% and 82 ± 1%).
To investigate interactions of wt-CFTR and F508-CFTR
with elements of the ER quality control machinery, we used a series of
maneuvers to modulate molecular chaperone interactions. Fig. 4A shows wt-CFTR mobility
after ATP depletion (a maneuver that increases interactions between
CFTR and chaperones in the ER), which was produced by 30 min of
incubation with 6 mM 2-deoxyglucose and 0.02% sodium azide
(top curve). The diffusion coefficient of GFP-wt-CFTR was
similar to control conditions, (10.6 ± 0.9) × 1010 cm2/s (n = 7), and the
percentage recovery was reduced significantly to 80 ± 2%. The
diffusion coefficient of GFP-F508-CFTR was reduced significantly
after a 30-min ATP depletion to (6.6 ± 0.5) × 1010 cm2/s (n = 5) (Fig.
4A, bottom curve), and substantially more
GFP-F508-CFTR became immobilized (mobile percentage, 55 ± 5%). ATP depletion for 60 min had no further effect. Table
I summarizes averaged diffusion
coefficients and percentages of mobile GFP. To investigate whether the
reduced F508-CFTR mobility in ATP-depleted cells was related to
restructuring or fragmentation of the ER, the mobility of the
fluorescent ER membrane marker diOC4(3) was measured. Fig. 4B shows that diffusion of diOC4(3) was
essentially complete with a diffusion coefficient of (2.4 ± 0.1) × 108 cm2/s (n = 10). diOC4(3) diffusion was unaffected by BFA treatment ((2.4 ± 0.1) × 108 cm2/s,
n = 5, middle curve) or ATP depletion
((2.7 ± 0.3) × 108 cm2/s,
n = 4, bottom curve). Further,
photobleaching of transfected cells expressing GFP-labeled wild type
CFTR or F508-CFTR and subsequently labeled with
diOC4(3) in situ showed that the expression of
GFP-CFTR chimeras did not affect ER structure or fluidity (not shown).
Image photobleaching experiments similar to those shown in Fig. 2 were
also performed on diOC4(3)-labeled cells (not shown), and
it was demonstrated that ATP depletion did not alter ER continuity or
gross structure.
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To identify the cellular location of the immobilized GFP-F508-CFTR
in ATP-depleted cells, fluorescence loss in photobleaching (FLIP)
experiments were performed (Fig. 4C). A fixed spot in the cell was repetitively bleached with delays between each bleach pulse to
permit diffusion of unbleached GFP-labeled CFTR into the location of
the bleach spot. Under these conditions GFP-wt-CFTR freely diffused
throughout the ER with ~10% of cell fluorescence remaining after 35 bleach pulses (top). In contrast, a substantial amount of
fluorescence remained after bleaching of GFP-F508-CFTR in
ATP-depleted cells (bottom) with the remaining fluorescence having an ER pattern. These findings suggest that ATP depletion does
not alter ER continuity or gross structure and that the immobilization of F508-CFTR in ATP-depleted cells results from interactions with ER proteins.
Additional maneuvers were carried out to modulate putative
CFTR interactions with molecular chaperones and elements of protein processing and degradation (Fig. 5 and
Table I). Heat shock, which nonspecifically up-regulates the expression
of molecular chaperones, produced small reductions in the diffusion
coefficients and percentage mobilities of wt-CFTR and F508-CFTR
(Fig. 5, top curves). Proteasome inhibition by lactacystin
resulted in mild slowing and immobilization of wt-CFTR and F508-CFTR
after 6 h of incubation (second curves), with complete
immobilization after 14 h (third curves). GFP was seen
in an ER pattern at 6 h but was concentrated in aggresomes at
14 h (not shown). The ansamysin compound geldanamycin, which
disrupts associations with Hsp90, caused comparable slowing of wt-CFTR
and F508-CFTR with somewhat greater immobilization of F508-CFTR
(fourth curves). Last, the role of glycosylation was
investigated by incubation with tunicamycin (to inhibit addition of
oligosaccharide chains), using a glycosylation-null mutant of wt-CFTR
(asparagines 894 and 900 replaced by glutamines). Somewhat more
immobilization of F508-CFTR than wt-CFTR was found with tunicamycin
(fifth curves). Although the glycosylation-null mutant of
wt-CFTR expressed poorly and a substantial proportion of
fluorescence was associated with aggresomes, the mobility of ER-associated CFTR could be measured in some cells and was found to be
similar to that produced by tunicamycin (sixth curve).
|
As was done for the ATP depletion experiments, the diffusion of the ER
marker diOC4(3) was measured in transfected cells to establish that ER restructuring or fragmentation was not responsible for reduced CFTR mobility. The diffusion of diOC4(3) in
cells that were heat-shocked or treated with lactacystin, geldanamycin, or tunicamycin was essentially complete (>96% mobile) and similar to
that measured in control cells, with diffusion coefficients of
2.2-2.7 × 108 cm2/s (n = 4 for each treatment, data not shown).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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This study has shown that although F508-CFTR is retained in the
ER, it diffuses at a comparable rate to wt-CFTR in BFA-treated cells.
Similar observations were made for the T126M mutant of AQP2 (a water
channel that is partially misfolded and similarly retained in the ER,
Ref. 27) and for a temperature-sensitive folding mutant of the
vesicular stomatitis virus G protein (29). Therefore, slowed or
restricted F508-CFTR mobility in the ER cannot account for its
failure to be exported. Efficient recycling from the Golgi or failure
to be recognized by the ER export machinery remain possible
explanations for the ER localization of F508-CFTR. The observation
of small amounts of F508-CFTR in post-ER membranes suggests that
efficient recycling may be responsible for the retention of
F508-CFTR in the ER (30).
The small immobile pool of F508-CFTR and the substantial
immobilization after ATP depletion suggest that a greater fraction of
F508-CFTR interacts with molecular chaperones than does wt-CFTR. Approximately 50% of F508-CFTR was immobile after ATP depletion, with only minor effects on the mobility of wt-CFTR. The simplest interpretation of this finding is that ATP depletion, which stabilizes protein interactions with molecular chaperones, is able to reveal F508-CFTR interactions that are not observable under normal
conditions. As discussed in the Introduction, F508-CFTR structure is
thought to be similar to that of an early intermediate of wt-CFTR, and biochemical evidence supports F508-CFTR associations with molecular chaperones.
Additional maneuvers were performed to investigate specific
interactions between wt-CFTR and F508-CFTR and the cellular protein processing machinery. Inhibition of N-linked glycosylation
by tunicamycin resulted in significant slowing and immobilization of
both wt-CFTR and F508-CFTR, similar to findings for the VSVG glycoprotein (29). Similar results were found with a glycosylation-null wt-CFTR mutant, which was poorly expressed in the plasma membrane as
determined by biotinylation studies (not shown). Protein glycosylation can facilitate folding, sorting, and quality control (31). Our data are
thus consistent with a role for glycosylation in CFTR folding.
Proteasome inhibition causes the accumulation of deglycosylated wt-CFTR
and F508-CFTR in detergent-insoluble, high molecular weight
aggregates (9, 32). Consistent with the observations here, Bebök
et al. (32) demonstrated an association between the Sec61
translocation system and CFTR that was enhanced by proteasome inhibition. After prolonged (14 h) incubation with lactacystin, wt-CFTR
and F508-CFTR were found in aggresomes (9, 10). Aggresomes are
vimentin-enclosed, pericentriolar, electron-dense structures that
contain aggregates of misfolded, often ubiquinated proteins associated
with molecular chaperones and proteasomes. As expected, the GFP-CFTR
chimeras were completely immobile in aggresomes.
Inhibition of interaction between Hsp90 and CFTR by geldanamycin also
slowed the diffusion of wt-CFTR and F508-CFTR, with greater
immobilization of F508-CFTR. Geldanamycin has been shown to disrupt
interactions of Hsp90 and CFTR, increasing the degradation rates of
wt-CFTR and F508-CFTR (14). Hsp90 has thus been proposed to have a
potential role in protecting CFTR from rapid degradation. The relative
immobilization of wt-CFTR and F508-CFTR after geldanamycin treatment
supports a role of Hsp90 for CFTR processing. As suggested for VSVG by
Nehls et al. (29), we believe that the physical state of
wt-CFTR and F508-CFTR after perturbation of CFTR processing by
tunicamycin or geldanamycin may be different from that produced by ATP
depletion. The slowing and immobilization of CFTR by ATP depletion may
represent CFTR interactions with molecular chaperones existing in large
complexes (33), whereas the immobilization after tunicamycin and
geldanamycin, or molecular deglycosylation, may represent CFTR
self-aggregation. Because of the logarithmic relationship between
molecular radius and diffusion rate for a protein in a bilayer,
substantial aggregates (10-100 units) must be formed to significantly
slow diffusion. The similar responses of wt-CFTR and F508-CFTR to
some of the pharmacological maneuvers are consistent with the
intrinsically inefficient folding of wt-CFTR, which when compared with
P-glycoprotein (another member of the ABC cassette family) is poorly folded.
The diffusion coefficient for CFTR diffusional mobility in the ER,
~10 × 1010 cm2/s, is within the range
of other ER membrane proteins that have been studied. The membrane
water channel AQP2 (retained in the ER with BFA) and the ER-retained
mutant AQP2-T126M have diffusion coefficients of 2.6-3.0 × 1010 cm2/s (27). Cytochrome P450 2C2, an
intrinsic ER protein, has a diffusion coefficient of 5.8 × 1010 cm2/s (34). VSVG in its native and
misfolded states and two ER resident transmembrane proteins (lamin B
receptor and the -subunit of the signal recognition particle
receptor) have diffusion coefficients of 26-50 × 1010 cm2/s, depending upon temperature (29).
Two Golgi resident transmembrane proteins (galactosyltransferase and
the KDEL receptor) retained in the ER with BFA have diffusion
coefficients of 21-48 × 1010 cm2/s,
depending upon cell type and temperature (29, 35). The diffusion
coefficient of the ER-retained MHC class 1 molecule H2Ld is
20-46 × 1010 cm2/s and that of TAP1
(transporter associated with antigen presentation 1) is 12 × 1010 cm2/s (36). The variation in
these diffusion coefficients probably reflects different protein sizes,
interactions with ER and cytoplasmic proteins, and ER composition in
different cells.
In summary, the photobleaching data provide the following evidence:
(a) the ER retention of F508-CFTR is not due to
restricted ER mobility, (b) the majority of F508-CFTR is
not aggregated or bound to slowly moving membrane proteins, and
(c) F508-CFTR may interact to a greater extent with
molecular chaperones than does wt-CFTR. Measurements of ER membrane
protein diffusion provide a unique in vivo approach to study
protein-protein associations that complements classical biochemical and
2-hybrid methods.
| |
ACKNOWLEDGEMENT |
|---|
We thank Katherin Karlson for assistance with the generation of the glycosylation-null mutant of EGFP-CFTR.
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FOOTNOTES |
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* This study was supported by National Institutes of Health Grants EB00415, DK43840, HL59198, DK35124, EY13574, and HL60288, Research Development Program Grant R613 from the Cystic Fibrosis Foundation (to A. S. V), and National Institutes of Health Grants DK45881 and DK34533 and a Research Development Program grant from the Cystic Fibrosis Foundation (to B. A. S).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.
¶ To whom correspondence should be addressed: 1246 Health Sciences East Tower, Cardiovascular Research Inst., University of California, San Francisco, San Francisco, CA 94143-0521. Tel.: 415-476-8530; Fax: 415-665-3847; E-mail: verkman@itsa.ucsf.edu.
Published, JBC Papers in Press, February 27, 2002, DOI10.1074/jbc.M112361200
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ABBREVIATIONS |
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The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; ER, endoplasmic reticulum; GFP, green fluorescent protein; wt, wild type; CHO, Chinese hamster ovary; BFA, brefeldin A; N.A., numerical aperture; VSVG, vesicular stomatitis virus G protein; AQP, aquaporin; diOC4(3), 3,3'-dibutyloxacarbocyanine iodide; FLIP, fluorescence loss in photobleaching.
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