From the Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521
Received for publication, March 1, 2001, and in revised form, April 10, 2001
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ABSTRACT |
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Mutations in the aquaporin-2 (AQP2) water channel
cause the hereditary renal disease nephrogenic diabetes insipidus
(NDI). The missense mutation AQP2-T126M causes human
recessive NDI by retention at the endoplasmic reticulum (ER) of renal
epithelial cells. To determine whether the ER retention of AQP2-T126M
is due to relative immobilization in the ER, we measured by
fluorescence recovery after photobleaching the intramembrane mobility
of green fluorescent protein (GFP) chimeras containing human wild-type and mutant AQP2. In transfected LLC-PK1 renal epithelial cells, GFP-labeled AQP2-T126M was localized to the ER, and wild-type AQP2 to
endosomes and the plasma membrane; both were localized to the ER after
brefeldin A treatment. Photobleaching with image detection indicated
that the GFP-AQP2 chimeras were freely mobile throughout the ER.
Quantitative spot photobleaching revealed a diffusion-dependent irreversible process whose recovery
depended on spot size and was abolished by paraformaldehyde fixation.
In addition, a novel slow reversible fluorescence recovery
(t1/2 ~2 s) was characterized whose recovery was
independent of spot size and not affected by fixation. AQP2
translational diffusion in the ER was not slowed by the T126M mutation;
diffusion coefficients were (in cm2/s × 10 A number of genetic disorders are caused by defective cellular
processing of proteins encoded by mutant genes. The human diseases cystic fibrosis, hereditary emphysema, and nephrogenic diabetes insipidus (NDI)1 are caused
by retention of mutant plasma membrane proteins at the endoplasmic
reticulum (ER) and their consequent failure to function at the plasma
membrane (1). Humans with NDI are unable to form a concentrated urine,
resulting in polyuria, polydipsia, and the potential for severe
dehydration if not given free access to fluids (2). Hereditary
non-X-linked NDI is caused by mutations in the aquaporin-2 (AQP2)
protein, a water channel that is normally expressed at the luminal
plasma membrane of kidney collecting duct epithelial cells after
stimulation by the antidiuretic hormone vasopressin (3). Collecting
duct water permeability is regulated by a membrane cycling mechanism in
which vasopressin-induced phosphorylation of the cytoplasmic AQP2 C
terminus results in its exocytic transport from an intracellular
vesicular pool to the cell plasma membrane (4, 5).
A number of AQP2 mutations cause autosomal recessive NDI by retention
at the ER (2, 3, 6-8). The missense AQP2 mutant T126M is ER-retained
in transfected mammalian cells where it is functional as a water
channel but appears to be mildly misfolded (9). Chemical chaperones
such as glycerol, which correct mutant protein mistrafficking in cell
culture models of cystic fibrosis and other diseases (10), correct the
AQP2-T126M processing defect and increase cell membrane water
permeability (11). Recently, a transgenic knock-in mouse model of NDI
created by AQP2-T126M gene replacement confirmed the ER retention of
AQP2-T126M in the in vivo kidney (12). Homozygous AQP2-T126M
mutant mice manifested severe polyuria and developed renal failure in
their first week of life.
The purpose of this study was to determine whether the ER retention of
AQP2 mutants results from restricted or slowed intramembrane mobility
caused by protein aggregation or interaction with components of the
quality control machinery such as molecular chaperones. AQP2 mobility
in living cells was measured by fluorescence recovery after
photobleaching (FRAP) using renal epithelial cells expressing green
fluorescent protein (GFP)-labeled wild-type and mutant AQP2. We
previously established photobleaching methods and analysis procedures
to quantify solute mobility in organelles including the ER and
mitochondria (13-15). The GFP label here did not affect AQP2 cellular
localization, and it was found previously that GFP-labeled wild-type
AQP2 had native tetrameric structure, water channel function, and
regulated cellular trafficking (16). Diffusion of wild-type and mutant
AQP2 were compared quantitatively, and the effects were characterized
of a series of maneuvers to modify putative AQP2 interactions.
The results here provide the first information about the ER retention
mechanism of a naturally occurring mutation in a membrane protein
associated with human disease. During the course of our experiments, a
paper by Nehls et al. (17) was published on the ER mobility
of a temperature-sensitive folding mutant of the vesicular stomatitis
virus G (VSVG) protein. Although some conclusions about VSVG and AQP2
mobility at the ER are similar, there are substantial differences in ER
diffusion rates and interactions with molecular chaperones (see
"Discussion").
Cell Culture and Transfections--
Missense point mutant T126M
of human AQP2 was generated by site-directed mutagenesis and confirmed
by sequence analysis. The cDNAs encoding wild-type and mutant AQP2
were fused downstream from EGFP (CLONTECH) as
described previously for wild-type AQP2 (16). LLC-PK1 renal epithelial
cells (obtained from the University of California, San Francisco Cell
Culture Facility) were grown at 37 °C in a 5% CO2/95%
air atmosphere using DMEH-21 medium. Cells were cultured on 18 mm-diameter round coverglasses in 12-well dishes in DMEH-21 medium
containing 500 µg/ml G418. For some studies cells were transiently
transfected with the GFP-labeled AQP2-T126M mutant by overnight
incubation with DMEH-21 medium containing 1 µg of plasmid and 6 µl
of EffecteneTM (Qiagen) per well. LLC-PK1 cells were also
transfected with unconjugated EGFP in the cytoplasm as described by
Kneen et al. (18) and in the ER as described by Dayel
et al. (14). In some experiments ER membranes were stained
with 3,3'-dibutyloxacarbocyanine iodide (diOC4 (3),
Molecular Probes) by incubation for 10 min at 37 °C using 10 µM dye concentration.
Cell Treatments--
Transfected cells were cultured on glass
coverslips until near confluence and mounted in a custom-built closed
perfusion chamber designed to fit in a PDMI-2 microincubator (Harvard
Apparatus). Perfusion chamber temperature was controlled by a TC-201A
temperature regulator (Harvard Apparatus) and objective temperature by
a lens thermoregulator (Bioptechs Inc., Butler, PA). Photobleaching
measurements were performed at 37 °C unless otherwise indicated.
Cell cultures were generally perfused with phosphate-buffered saline
containing 6 mM D-glucose at 1 ml/min, and
photobleaching measurements were made within 1 h of mounting the
cells in the perfusion chamber. In some experiments, cells were
preincubated with brefeldin A (1 µg/ml, 16 h, 37 °C) to give
AQP2 localization in the ER. In some experiments cells were incubated
with the fixative paraformaldehyde (4%, 30 min, 23 °C), the
metabolic inhibitors 2-deoxy-D-glucose (50 mM,
1 h, 37 °C) and sodium azide (0.05%) (for ATP depletion), the
cAMP agonist forskolin (50 µM, 15 min, 37 °C), or the
cytoskeleton-disrupting agent cytochalasin D (5 µg/ml, 45 min,
37 °C). Some cell cultures were heat-shocked by incubation at
42 °C for 1 h followed by 16-h incubation at 37 °C.
Fluorescence Recovery after Photobleaching--
Photobleaching
measurements were carried out on an apparatus described previously (19)
with modification. For spot photobleaching, an argon ion laser beam
(488 nm, Innova 70-4; Coherent) was modulated by an acousto-optic
modulator (Primrose) and directed onto the transfected cells through an
objective using a Nikon inverted epifluorescence microscope (Diaphot).
For cell visualization and selection of the spot to be bleached,
full-field epi-illumination was accomplished using the defocused
undiffracted beam delivered by a fiberoptic. The full-field and
nondefocused laser beams were reflected by a dichroic mirror (510 nm)
onto the sample using oil immersion objective lenses (Nikon oil Fluors;
100×, N.A. 1.4 or 40×, N.A. 1.3). Sample fluorescence was filtered by
a 530-nm interference filter, detected using a photomultiplier, and
digitized by a 14-bit analog-to-digital converter. The photomultiplier
was transiently gated off during the bleach pulse by reducing the voltage of the second dynode. Fluorescence was sampled over 1 s
before the bleach pulse, then at rates of up to 1 MHz. For measurement of slow recoveries, an electronic shutter was used to collect average
fluorescence every 0.5 s or slower. Each fluorescence value was
the average of 10,000 samples collected during the shutter open time of
20 ms.
For photobleaching experiments with image detection, a Nikon upright
microscope with cooled CCD camera detector (Photometrics) was used to
record full-field fluorescence images. An electronically shuttered
bleach beam from the argon laser was directed onto the sample from
below using a Nikon 25× extra long working distance air objective.
Cells were viewed from above by epifluorescence using a 60× oil
immersion objective and GFP filter set (HQ filters, Chroma). Software
was written to coordinate the bleach pulse, excitation and camera
shutters, and image acquisition.
Analysis of Photobleaching Recovery Data--
Recovery half-time
(t1/2), defined as the time when fluorescence
recovered by 50% due to diffusion, was determined from fluorescence
recovery curves, F(t), using a multiple-component
regression. Fluorescence recovery was treated as a combination of
reversible and irreversible processes: F(t) = a1[F(t)reversible] + a2[F(t)irreversible],
where a1+a2 = 1. F(t)reversible was fitted by
nonlinear least square exponential regression using
F(t) measured in paraformaldehyde-fixed cells. The diffusion-mediated recovery was fitted using the semiempirical equation: F(t)irreversible = Fo + [Fo + R(Finf Measurements were done on transfected LLC-PK1 cells expressing
wild-type and T126M AQP2 proteins fused downstream from a GFP reporter.
Fig. 1A shows the cDNA
constructs, indicating the amino acid sequence at the GFP-AQP2 fusion
site and the location of the T126M mutation. Fig. 1B shows
confocal fluorescence micrographs of cells expressing the GFP-AQP2
constructs. Without brefeldin A treatment, GFP-labeled AQP2-T126M was
localized in the ER (left) and wild-type AQP2 in endosomes
and the plasma membrane (middle). After incubation with
forskolin for 15 min, GFP-labeled wild-type AQP2 was seen mainly at the
plasma membrane, whereas the localization of AQP2-T126M was unchanged
(data not shown). After treatment with brefeldin-A, which
partially collapses the Golgi and prevents ER-to-Golgi vesicle traffic,
both GFP-AQP2 chimeras showed the characteristic ER staining pattern
(wild-type AQP2 shown at right), as also seen in cells
expressing KDEL-tagged unconjugated GFP or stained with cyanine
diOC4 (3) (not shown). Photobleaching studies of GFP-AQP2
diffusion in the ER were carried out on brefeldin A-treated cells or
cells expressing AQP2-T126M without brefeldin A treatment.
10) 2.6 ± 0.5 (wild-type) and
3.0 ± 0.4 (T126M). Much faster diffusion was found for a lipid
probe (diOC4(3), 2.7 × 10
8
cm2/s) in the ER membrane and for unconjugated GFP in the
aqueous ER lumen (6 × 10
8
cm2/s). ER diffusion of GFP-T126M was not significantly
affected by up-regulation of molecular chaperones, cAMP activation, or actin filament disruption. ATP depletion by 2-deoxyglucose and azide
resulted in comparable slowing/immobilization of wild-type and T126M
AQP2. These results indicate that the ER retention of AQP2-T126M does
not result from restricted or slowed mobility and suggest that the
majority of AQP2-T126M is not aggregated or bound to slowly moving
membrane proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Fo)](t/t1/2)/[1 + (t/ t1/2)], where Fo
is pre-bleach fluorescence, Finf is fluorescence
at infinite time, and R is the fractional fluorescence
recovery (20). For each maneuver, recovery curves were measured on at
least 40 different cells using three or more sets of cultures generated
on different days. Parameters are reported as mean ± S.E., where
sets of six individual recovery curves were averaged and fitted to
establish statistics. Absolute diffusion coefficients (D,
cm2/s) were calculated from t1/2 using a
mathematical model of ER diffusion (15) in which the effective spot
size of the bleached region was determined each day using thin layers
of solution standards (fluorescein in saline) as described previously
(20, 21).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Photobleaching of GFP-labeled AQP2 proteins
in the ER membrane of transfected LLC-PK1 cells at 37 °C.
A, cDNA construct showing fusion of AQP2-T126M
downstream from the GFP reporter. B, fluorescence
micrographs of transfected LLC-PK1 cells expressing the indicated
GFP-AQP2 proteins. C and D, series of
fluorescence micrographs are shown for brefeldin A-treated cells
expressing wild-type GFP-AQP2 (C) and cells expressing the
AQP2-T126M mutant (D). Images are shown before bleaching and
at indicated times after bleaching. See "Results" for
explanations. Temperature was maintained at 37 °C.
Cross-hairs indicate center of bleach spot. Scale
bar: 10 µm.
Photobleaching imaging experiments were done to examine the continuity of the ER membrane and to determine whether GFP-labeled AQP2-T126M is restricted in its ER mobility. A large region of the cell was bleached by the laser pulse. Fig. 1C shows pre-bleach and a series of post-bleach images of brefeldin A-treated LLC-PK1 cells expressing wild-type GFP-AQP2. The post-bleach image showed a darkened region corresponding to the site of the bleach pulse. There was progressive recovery of fluorescence over the first ~50 s. At 200 s, the ER was similar in appearance to the pre-bleach image, except for reduced intensity as a consequence of the substantial fraction of total cell GFP-AQP2 that was irreversibly bleached. Similar results for many different cells, different laser spot sizes, and different bleached regions of the cell indicated that with respect to diffusion of wild-type AQP2 the ER membrane is continuously connected throughout the cell. Similar findings were reported previously for unconjugated GFP in the aqueous lumen of ER, albeit with a substantially faster rate of diffusion (14).
Imaging photobleaching experiments were also done on cells expressing AQP2-T126M. Examination of serial images from many cells (representative example in Fig. 1D) showed that the GFP-labeled AQP2-T126M protein was able to move freely throughout the ER, with qualitatively comparable fluorescence recovery kinetics to wild-type AQP2.
Spot photobleaching measurements were done to characterize quantitatively the fluorescence recovery processes and to compare the diffusion of GFP-labeled wild-type AQP2 and AQP2-T126M. GFP fluorescence in a small spot was bleached: ~0.7-µm diameter produced by a 100× oil immersion objective, or ~2-µm diameter produced by a 40× objective. Bleach times were selected to reduce fluorescence intensity just after the bleach to ~60% of initial fluorescence.
Initial experiments done using a 100× objective showed nearly complete
fluorescence recovery in a few seconds in brefeldin A-treated cells
expressing GFP-labeled wild-type AQP2 (Fig.
2A, top curve).
"No bleach" control experiments were done for each set of studies
to prove that the attenuated probe beam did not produce bleaching. To
confirm that the recovery corresponded to diffusion, measurements were
done after paraformaldehyde treatment (to immobilize GFP-AQP2) and
using a 40× lens (to increase spot diameter and slow diffusion-related
recovery ~ [100/40]2 ~ 6-fold). Interestingly,
partial recovery was seen in paraformaldehyde-fixed cells with an
exponential time constant, , of 3.4 ± 0.8 s (second curve). Fluorescence recovery in the absence of diffusion defines a reversible recovery process, as created by triplet state or other
photophysical relaxation processes. For example, the decay of a GFP
molecule from an excited triplet state to the ground state would
produce fluorescence recovery because of restoration of the population
of excitable GFP molecules. Reversible GFP fluorescence recovery has
been described (14, 22), albeit at a much faster rate (1-5 ms,
depending on construct), and slow GFP flickering has been reported
(23). Examination of the early fluorescence time course after bleaching
in the paraformaldehyde-treated cells revealed a second apparent
reversible recovery process (
~ 4 ms) of low amplitude
(inset). In addition to insensitivity to fixation,
diffusion-independent reversible fluorescence recovery processes do not
depend on spot size. The recovery of the slow reversible process was
not affected by increasing objective spot diameter ~2.5-fold
(third curve) in the paraformaldehyde-treated cells
(3.0 ± 0.5 s), confirming reversible recovery. Similar
results were obtained using a 10× objective to illuminate whole cells (not shown). The remaining recovery after the reversible process seen
for the first curve in Fig. 2A was
diffusion-dependent, as shown by its disappearance after
paraformaldehyde treatment (second curve) and its
significant slowing when bleached and viewed with a larger spot size
(t1/2 ~ 12 s, fourth curve).
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The slow reversible fluorescence recovery processes for GFP-AQP2 were
not noted previously for GFP in cytoplasm, where recovery was measured
over much shorter times (22). Fig. 2B shows a series of
photobleaching measurements similar to those in Fig. 2A, but for unconjugated GFP in the cytoplasm of LLC-PK1 cells. The top curve shows substantially faster fluorescence recovery of the free
GFP in aqueous-phase cytoplasm compared with membrane diffusion of
GFP-AQP2. The second and third curves, where GFP
mobility was abolished by paraformaldehyde fixation, shows recovery
similar to that seen for GFP-AQP2 in paraformaldehyde-fixed cells in
Fig. 2A (, 3-5 s). The fourth curve shows
that the irreversible (diffusion-dependent) fluorescence
recovery process was strongly dependent on spot size as anticipated
(t1/2 10 ms, 100× versus 57 ms, 40×).
Thus the slow (3-5 s) reversible recovery process seen for GFP-AQP2
does not appear to be context-specific.
Quantitative spot photobleaching experiments were done to compare the
ER membrane diffusion of GFP-labeled wild-type AQP2 and AQP2-T126M. It
was postulated that interactions of the mutant ER-retained protein with
components of the quality control machinery might result in slowed
diffusion. Fig. 3A shows
representative fluorescence recovery data, and Fig. 3B
summarizes averaged diffusion coefficients. Qualitatively the recovery
curves for the brefeldin A-treated GFP-labeled wild-type and mutant
AQP2 were similar, as was the recovery curve for AQP2-T126M in
non-brefeldin A-treated cells. The averaged diffusion coefficients did
not differ significantly. Fitted percentage recoveries were generally
>90%, indicating that nearly all AQP2 is mobile, in agreement with
the conclusion from the image photobleaching studies in Fig. 1,
C and D. In addition a series of experiments were
done at 23 °C. Although the absolute diffusion coefficients were
lower than those at 37 °C, there was no significant effect of the
T126M mutation.
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The computation of absolute diffusion coefficients relied on a
Monte-Carlo random walk model, which takes into account the reticular
geometry of the ER and relates diffusion coefficients to fluorescence
recovery kinetics (15). Previously this model was used to compute the
diffusion coefficient of unconjugated GFP in the aqueous lumen of the
ER (14). A similar experiment was done to measure the diffusion of
unconjugated GFP in the ER lumen of the LLC-PK1 cells used here. GFP
fluorescence recovered much faster (Fig. 3A, bottom
curve), with a diffusion coefficient of (6.0 ± 0.7) × 108 cm2/s (Fig. 3B),
in agreement with that of 5-10 × 10
8
cm2/s for GFP diffusion in the ER of CHO cells. The
200-fold lower diffusion coefficient for GFP-AQP2 indicates the
remarkably slowed diffusion of integral membrane proteins compared with
aqueous-phase macromolecules.
ER membrane diffusion of GFP-labeled AQP2-T126M was measured in
response to a series of cell treatments designed to modulate AQP2
phosphorylation state (forskolin), interactions with molecular chaperones (ATP metabolic depletion and heat shock), and interactions with cytoskeleton (cytochalasin D). Fig.
4A shows representative fluorescence recovery curves for cells expressing GFP-labeled AQP2-T126M, with averaged diffusion coefficients for a series of
measurements summarized in Fig. 4C. There was no significant effect of forskolin treatment, heat shock, or cytochalasin treatment on
AQP2-T126M diffusion coefficients or mobilities. However, ATP depletion
by 2-deoxyglucose and azide produced a small but significant slowing of
diffusion, as well as a decrease in the mobile fraction (percentage
fluorescence recovery) from >90% to 64 ± 7%. To determine whether the effect of ATP depletion was specific for AQP2-T126M, similar measurements were done on GFP-labeled wild-type AQP2 (Fig. 4B). ATP depletion produced similar slowing and reduced
mobility of GFP-labeled wild-type AQP2 and unconjugated GFP. ATP
depletion also caused a significant reduction in the mobility of
unconjugated GFP in the ER lumen (Fig. 4B, middle
curves). To distinguish between an effect of ATP depletion on ER
"crowding," as proposed by Nehls et al. (17),
versus ER restructuring and geometric differences, the
diffusion of a small lipophilic ER membrane probe,
diOC4(3), was measured. Fig. 4, B (bottom
curves) and C, showed no effect of ATP depletion on
diOC4(3) diffusion, indicating that ATP depletion does not
affect ER geometry in a manner that influences the relationship between
fluorescence recovery and molecular diffusion.
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DISCUSSION |
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The principal conclusion of this study is that despite its retention at the endoplasmic reticulum, the human disease causing AQP2 mutant T126M is not slowed in its translational diffusion in the ER compared with wild-type AQP2. Both wild-type AQP2 and AQP2-T126M diffused freely throughout the ER without restrictions. Furthermore, AQP2-T126M diffusion was not changed significantly by maneuvers designed to modulate AQP2 phosphorylation and interactions with components of the cellular quality control machinery and the actin skeleton. Therefore slowed or restricted ER membrane diffusion of AQP2-T126M cannot account for its efficient retention in the ER. Formally our results indicate that the rate-limiting step for the ER diffusion of AQP2 is not sensitive to the T126M mutation. It is noted that interactions between AQP2-T126M and soluble macromolecules are unlikely to be detected by diffusion measurements, because the diffusion of intramembrane proteins is orders of magnitudes slower than that of aqueous-phase macromolecules. For example, the binding of large 30-nm diameter gold beads does not slow the diffusion of much smaller membrane receptors (24). The quantitatively comparable diffusion of wild-type AQP2 and AQP2-T126M makes unlikely significant AQP2-T126M aggregation or interactions with intramembrane structures that have diffusive rates comparable with or slower than AQP2. However, we cannot rule out the possibility of slowing or immobilization of a small fraction (<5-10%) of AQP2-T126M molecules that cannot be resolved by the photobleaching recovery measurements.
The approach here utilized fluorescence recovery after photobleaching of GFP-labeled AQP2 chimeras. The relatively polar GFP moiety is unlikely to affect the membrane diffusion of AQP2. We showed previously that GFP labeling did not alter the water permeability or tetrameric intramembrane structure of AQP1 and AQP2 (16), or the regulated membrane trafficking of AQP2 (25). Furthermore, the diffusion of GFP-labeled wild-type AQP2 at the plasma membrane was remarkably slowed by cAMP-induced phosphorylation of serine 256 in the AQP2 C terminus domain (16). The cAMP-induced slowing of AQP2 mobility at the plasma membrane was abolished by mutation of the phosphorylation site at serine 256 to alanine, and by cytochalasin D disruption of the actin skeleton. In the experiments here, cAMP agonists and cytochalasin D treatment did not affect AQP2 mobility in the ER. Although we have not determined whether the AQP2 C terminus can be phosphorylated in the ER, the results suggest that ER-associated AQP2 does not interact directly or indirectly with the actin skeleton. Recent two-hybrid experiments have identified a number of putative proteins that interact with the phosphorylated C terminus of AQP2 (26).
The data here represent the first study of the mobility of a
disease-causing mutant membrane protein that produces a defective cell
phenotype by ER retention. It will be interesting to determine the
generality of the conclusions here to other mutant membrane proteins,
such as the F508-CFTR protein that is retained at the ER and causes
the disease cystic fibrosis. In agreement with the conclusion here,
Nehls et al. (17) reported that the diffusion of GFP-labeled
VSVG was unimpeded, so that its ER retention was not the consequence of
slowed mobility. However, their diffusion coefficient for VSVG was
10-fold greater than that of AQP2 found here, and the
temperature-sensitive unfolded VSVG was largely immobilized after
cellular ATP depletion, which allowed a stable interaction with BiP.
Comparative studies of folded and unfolded VSVG required measurements
at 32 °C (with brefeldin A treatment) and 40 °C, respectively. We
find here that the diffusion of wild-type and mutant AQP2 in renal
epithelial cells at 37 °C was not quantitatively different following
ATP depletion or up-regulation of molecular chaperones by temperature
shock. It is likely that fundamentally different mechanisms apply for
the recognition of unfolded VSVG versus mutant AQP2. For
VSVG interactions with BiP, protein disulfide isomerase, and calnexin
have been reported, whereas thus far it has not been possible to detect
an interaction between mutant AQP2 and known components of the quality
control machinery.
The diffusion coefficients of AQP2 and AQP2-T126M at the ER, ~3 × 1010 cm2/s, are a few-fold
greater than that of wild-type AQP1 or AQP2 at the plasma membrane
(~0.9 × 10
10 cm2/s at
37 °C), which is consistent with the relatively low
cholesterol content and high membrane fluidity of the ER. The ER
diffusion of AQP2 was similar to another ER protein, cytochrome P450
(2.6-6.2 × 10
10 cm2/s)
(27), but much less than that reported for VSVG (4-6 × 10
9 cm2/s) (17). To date the
diffusional mobilities of other ER proteins have not been reported. For
comparison, unconjugated GFP in the aqueous-phase ER lumen diffused
substantially faster (6 × 10
8
cm2/s) than membrane-bound GFP chimeras. The small lipid
probe diOC4 (3) in the ER membrane also diffused
substantially faster (3 × 10
8
cm2/s), in agreement with previous measurements of the
diffusion of fluorescently labeled phospholipids (28).
An important unresolved issue is how proteins like AQP2-T126M are
retained at the ER. AQP2-T126M does not contain a classical ER
retention sequence (like KKXX), and wild-type AQP2
efficiently exits the ER. No evidence was found for a population of
AQP2-T126M that was discontinuous from the ER, providing further
evidence that retrieval from a downstream compartment is not the
mechanism of its ER retention. The photobleaching results indicate that AQP2-T126M aggregation or interactions (transient or sustained) with
slowly moving membrane components cannot account for its ER retention.
The ER may contain specialized membrane domains that bud off during the
formation of vesicles destined to fuse with the Golgi. The AQP2-T126M
protein might be excluded from these specialized submicroscopic domains
because of misfolding and inability to be recognized by as yet
unidentified proteins responsible for accumulation in transport
vesicles (29). The identification of ER retention mechanisms of
disease-causing protein mutations remains a major challenge.
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FOOTNOTES |
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* This work was supported by Grants DK43840, DK35124, HL59198, and HL60288 from the National Institutes of Health and RDP Grant R613 from the National Cystic Fibrosis Foundation.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.
Both authors contributed equally to this work.
§ Summer student scholar supported by the American Heart Association, Western States Affiliate.
¶ To whom correspondence should be addressed: 1246 Health Sciences East Tower, Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0521. Tel.: 415-476-8530; Fax: 415-665-3847; E-mail: verkman@itsa.ucsf.edu.
Published, JBC Papers in Press, April 10, 2001, DOI 10.1074/jbc.M101901200
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ABBREVIATIONS |
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The abbreviations used are: NDI, nephrogenic diabetes insipidus; ER, endoplasmic reticulum; AQP2, aquaporin-2; GFP, green fluorescent protein; EGFP, enhanced GFP; VSVG, vesicular stomatitis virus G.
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REFERENCES |
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1. | Aridor, M., and Balch, W. E. (1999) Nat. Med. 5, 745-751[CrossRef][Medline] [Order article via Infotrieve] |
2. | Bichet, D. G. (1998) Am. J. Med. 105, 431-442[CrossRef][Medline] [Order article via Infotrieve] |
3. | Deen, P. M., Verkijk, M. A., Knoers, N. V., Wieringa, B., Monnens, L. A., van Os, C. H., and van Oost, B. A. (1994) Science 264, 92-95[Medline] [Order article via Infotrieve] |
4. | Nielsen, S., Chou, C. L., Marples, D., Christensen, E. I., Kishore, B. K., and Knepper, M. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1013-1017[Abstract] |
5. |
Fushimi, K.,
Sasaki, S.,
and Marumo, F.
(1997)
J. Biol. Chem.
272,
14800-14804 |
6. | Deen, P. M. T., Croes, H., van Aubel, R. A. M. S., Ginsel, L. A., and van Os, C. H. (1995) J. Clin. Invest. 95, 2291-2296[Medline] [Order article via Infotrieve] |
7. | Mulders, S. M., Knoers, N. V., van Lieburg, A. F., Monnens, L. A., Leumann, E., Wuhl, E., Schober, E., Rijss, J. P., van Os, C. H., and Deen, P. M. T. (1997) J. Am. Soc. Nephrol. 8, 242-248[Abstract] |
8. |
Canfield, M. C.,
Tamarappoo, B. K.,
Moses, A. M.,
Verkman, A. S.,
and Holtzman, E. J.
(1997)
Hum. Mol. Genet.
6,
1865-1871 |
9. |
Tamarappoo, B. K.,
Yang, B.,
and Verkman, A. S.
(1999)
J. Biol. Chem.
274,
34825-34831 |
10. | Welch, W. J., and Brown, C. R. (1996) Cell Stress Chaperones 1, 109-115[Medline] [Order article via Infotrieve] |
11. |
Tamarappoo, B. K.,
and Verkman, A. S.
(1998)
J. Clin. Invest.
101,
2257-2267 |
12. |
Yang, B. A.,
Gillespie, A.,
Carlson, E. J.,
Epstein, C. J.,
and Verkman, A. S.
(2001)
J. Biol. Chem.
276,
2775-2779 |
13. |
Partikian, A.,
Ölveczky, B.,
Swaminathan, R.,
Li, Y.,
and Verkman, A. S.
(1998)
J. Cell Biol.
140,
821-829 |
14. |
Dayel, M.,
Hom, E. F.,
and Verkman, A. S.
(1999)
Biophys. J.
76,
2843-2851 |
15. | Ölveczky, B. P., Periasamy, N., and Verkman, A. S. (1997) Biophys. J. 73, 2836-2847[Abstract] |
16. |
Umenishi, F.,
Verbavatz, J. M.,
and Verkman, A. S.
(2000)
Biophys. J.
78,
1024-1035 |
17. | Nehls, S., Snapp, E. L., Cole, N. B., Zaal, K. J., Kenworthy, A. H., Roberts, T. H., Ellenberg, J., Presley, J. F., Siggia, E., and Lippincott-Schwartz, J. (2000) Nat. Cell Biol. 2, 288-295[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Kneen, M.,
Farinas, J.,
Li, Y.,
and Verkman, A. S.
(1998)
Biophys. J.
74,
1591-1600 |
19. | Kao, H. P., and Verkman, A. S. (1996) Biophys. Chem. 59, 203-210[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Periasamy, N.,
and Verkman, A. S.
(1998)
Biophys. J.
75,
557-567 |
21. | Kao, H. P., Abney, J. R., and Verkman, A. S. (1993) J. Cell Biol. 120, 175-184[Abstract] |
22. | Swaminathan, R., Hoang, C. P., and Verkman, A. S. (1997) Biophys. J. 72, 1900-1907[Abstract] |
23. |
Haupts, U.,
Maiti, S.,
Schwille, P.,
and Webb, W. W.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
13573-13578 |
24. | Lee, G. M., Zhang, F., Ishihara, A., McNeil, C. L., and Jacobson, K. A. (1993) J. Cell Biol. 120, 25-35[Abstract] |
25. | Gustafson, C. E., Levine, S., Katsura, T., McLaughlin, M., Aleixo, M. D., Tamarappoo, B. K., Verkman, A. S., and Brown, D. (1998) Histochem. Cell Biol. 110, 377-386[CrossRef][Medline] [Order article via Infotrieve] |
26. | Yang, B., and Verkman, A. S. (2000) J. Am. Soc. Nephrol. 11, 23A |
27. |
Szczesna-Skorupa, E.,
Chen, C. D.,
Rogers, S.,
and Kemper, B.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
14793-14798 |
28. | Lee, G. M., Ishihara, A., and Jacobson, K. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6274-6278[Abstract] |
29. | Klumperman, J. (2000) Curr. Opin. Cell Biol. 12, 445-449[CrossRef][Medline] [Order article via Infotrieve] |