1Department of Physiology, School of Medicine, University of California Los Angeles and Veterans Administration Greater Los Angeles Health Care System 90073; and 2Division of Nephrology, David Geffen School of Medicine, University of California Los Angeles Center for the Health Sciences, Los Angeles, California 90095
Submitted 20 February 2003 ; accepted in final form 20 May 2003
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ABSTRACT |
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sorting and trafficking; apical signals; plasma membrane targeting
In contrast to polarized cells, nonpolarized cells were thought to have a default pathway for surface delivery of plasma membrane proteins (29). However, there is increasing evidence that apical and basolateral sorting and trafficking pathways exist not only in polarized but also in nonpolarized cells (11). When apical and basolateral proteins are expressed in nonpolarized cells, in TGN they are sorted into different containers that travel separately until they fuse with the plasma membrane (12, 19, 23, 32). Therefore, at least for some proteins, surface delivery in nonpolarized cells does not occur by a default mechanism but is rather a result of a sorting event in TGN (12, 19, 32). However, it is unclear whether a signal-based sorting event in TGN is an absolute requirement for subsequent surface delivery of the proteins in nonpolarized cells.
If expressed in nonpolarized HEK-293 cells, the H,K-ATPase is localized on
the plasma membrane (13,
16). The -subunit is
not able to leave the endoplasmic reticulum (ER) without the
-subunit,
and, when expressed alone in HEK-293 cells, it does not reach the plasma
membrane (13,
16). In contrast, plasma
membrane delivery of the
-subunit does not require the presence of the
-subunit. Hence, it is the
-subunit that must be responsible for
plasma membrane delivery of the gastric H,K-ATPase in HEK-293 cells. It is
unknown whether surface delivery of the gastric H,K-ATPase
-subunit
occurs by a default mechanism or whether it depends on the presence of
intrinsic sorting or trafficking signals.
To address these issues, we examined intracellular trafficking of the
gastric H,K-ATPase -subunit, normally an apical protein, in nonpolarized
HEK-293 cells using a fusion protein of the
-subunit and yellow
fluorescent protein (YFP). By generating mutants with successive exclusion of
glycosylation sites, we found that six of the seven glycosylation sites in the
-subunit are essential for the plasma membrane targeting in HEK-293
cells. The data strongly indicate that plasma membrane delivery of the gastric
H,K-ATPase
-subunit in nonpolarized HEK-293 cells is sorting or
trafficking signal dependent.
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METHODS |
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Mutants were generated by using the QuikChange mutagenesis kit (Stratagene,
La Jolla, CA). The rabbit gastric H,K-ATPase -subunit has seven
N-glycosylation sites in the extracellular COOH terminus that we
numbered from one to seven from Asn99 (the first) to Asn 222 (the seventh).
Seven single mutants of YFP-
fusion protein lacking a single
N-glycosylation site, one double mutant lacking the first and the
second glycosylation sites, and one triple mutant lacking the first, the
second, and the fourth glycosylation sites were constructed using pEYFP-
as a template (Table 1). Two
single mutants of the gastric H,K-ATPase
-subunit lacking the fourth or
the seventh sites and one double mutant lacking the first and the second sites
were constructed using pcDNA(+)-
as a template
(Table 1). Some of the single
mutants were used as the templates for creation of double mutants. The double
mutant was used as the template for construction of a triple mutant. Amino
acid residues are numbered according to the sequence of the rabbit gastric
H,K-ATPase
-subunit.
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Transient transfection. HEK-293 cells were grown on 10-cm plates
till 80% confluent or on coverslips in six-well plates till 60% confluent and
transiently transfected with pECFP- alone, pEYFP-
alone, or with
both pECFP-
and pEYFP-
using FuGENE 6 transfection reagent
(Roche). The cells stably expressing the H,K-ATPase
-subunit
(16) were grown on coverslips
till 60% confluent and then transiently transfected with pECFP-
. After
24 h, cells from 10-cm plates were collected for membrane isolation. Cells on
coverslips were fixed with methanol for further confocal microscopic studies
(see Confocal microscopy studies).
Stable transfection. To obtain cell lines stably expressing
wild-type YFP-, mutant YFP-
fusion proteins, or mutant H,K-ATPase
-subunits, HEK-293 cells were grown on 10-cm plates till 50% confluent
and transfected with wild-type YFP-
, mutant pEYFP-
, or mutant
pcDNA3(+)
using FuGENE 6 transfection reagent (Roche). Stable cell lines
were selected by adding, 24 h after transfection, the eukaryotic selection
marker G-418 at a final concentration of 1.0 mg/ml. This concentration of
G-418 was maintained until single colonies appeared. Fifteen to twenty
colonies were isolated, expanded, and grown in the presence of 0.25 mg
G-418/ml medium. The stable expression of the YFP-
fusion protein or
H,K-ATPase
-subunit was confirmed by Western blot analysis using
monoclonal antibody against green fluorescent protein (GFP) (BD Bioscience
Clontech) and a 2B6 monoclonal antibody against the H,K-ATPase
-subunit
(MBL) as described in SDS-PAGE and Western blot analysis. Two clones
with the best ratio of expressed protein (YFP-
fusion protein or mutant
H,K-ATPase
-subunit) to total protein were grown on coverslips in
six-well plates for confocal studies and in the flasks for isolation of
membranes.
The cell lines expressing mutant H,K-ATPase -subunits were subjected
to a second transfection with pcDNA3.1-(zeo+)
. By addition of the
second selection marker zeocin at a concentration 0.4 mg/ml in addition to
maintenance concentration of G-418 of 0.25 mg/ml, 15-20 cell lines expressing
both
- and
-subunits were selected. Two clones with the best
ratio of expressed H,K-ATPase to total protein were expanded for isolation of
membranes. Maintenance concentration for zeocin was 0.1 mg/ml.
Confocal microscopy studies. Cells stably expressing wild-type or
mutant YFP- were grown on coverslips in six-well plates till 80%
confluent and fixed with methanol at -20°C. Cells subjected to transient
transfection on coverslips (see Transient transfection) were fixed
with methanol 24 h after transfection. Confocal microscopic images were
acquired using the Zeiss LSM 510 laser scanning confocal microscope.
Preparation of crude membranes. The cells stably expressing the
wild-type or mutant YFP- were grown till confluent in flasks and then
collected. The cells transiently expressing CFP-
and YFP-
fusion
proteins were collected from 10-cm plates. Cells were collected by suspending
them in the sodium-free buffer A (10 mM PIPES/Tris, pH 7.0, with 2 mM
EGTA and 2 mM EDTA) containing 250 mM sucrose. The cell suspension was
homogenized with a tight Dounce homogenizer (Wheaton, Millwille, NY). The
homogenate was collected, layered onto the 42% sucrose solution, and spun in
the Beckman SW28 swinging bucket rotor at 25,000 rpm for 1 h at 4°C. The
fraction at the buffer/sucrose interface was collected and diluted to the
total volume of 15 ml in buffer A. The membrane fraction was
collected by centrifugation in a Beckman 75Ti rotor (35,000 rpm, 4°C, 1
h). The pellet was resuspended in 2 ml of buffer A and homogenized
with a Teflon homogenizer (Wheaton). The total protein concentration was
determined by using modified Lowry protein assay reagent (Pierce). The typical
protein concentration was 5-10 µg/µl. The membranes were aliquoted,
flash frozen, and stored at -80°C. When indicated, the membrane fraction
was treated with PNGase F (New England BioLabs) or EndoH (Prozyme)
according to the manufacturers' instructions.
Biotinylation and estimation of surface YFP-
expression. HEK-293 cells stably expressing wild-type or mutant
YFP-
were grown for 2 days after becoming confluent in six-well plates.
Biotinylation of the plasma membrane proteins was performed by previously
described procedures (7,
15). Briefly, cell monolayers
were rinsed with cold phosphate-buffered saline containing 1 mM
Ca2+ and 1 mM Mg2+ (PBS2+). Cold
PBS2+ (2 ml) containing 1 mg/ml
sulfosuccinimidyl-6-(biotinamido)hexanoate (EZ-Link Sulfo-NHS-LC-biotin;
Pierce, Rockford, IL) was added to each well. After 30 min of incubation at
4°C, the PBS2+/biotin solution was discarded, and the
biotinylation reaction was quenched by adding cold PBS22+ with 50
mM NH4Cl (30 min, 4°C). Cells were washed twice with
PBS2+ and then incubated with 60 µl of lysing buffer (1% SDS, 4
mM EGTA, and 10 mM Tris, pH 8.0) for 30 min with agitation at room
temperature. Cell lysates were transferred into Eppendorf tubes and clarified
by centrifugation (15,000 g for 5 min). Samples containing 15 µl
of supernatant mixed with an equal volume of SDS-containing sample buffer were
loaded onto SDS-PAGE gel to determine the total YFP-
content in the
supernatant. To precipitate biotinylated proteins, 40 µl of each
supernatant were incubated with 100 µl of streptavidin-agarose beads
(Sigma) in a total volume of 800 µl of the lysing buffer for 1 h at 4°C
with continuous rotation. Precipitated complexes were washed twice in 15 mM
Tris, pH 8.0, containing 0.5% Triton X-100, 4 mM EGTA, and 0.5 M NaCl and then
in the same buffer without NaCl. Proteins were eluted from the beads by
incubation in 40 µl of SDS-PAGE sample buffer (4% SDS, 0.05% bromophenol
blue, 20% glycerol, 1%
-mercaptoethanol in 0.1 M Tris, pH 6.8) for 5 min
at 80°C. After centrifugation, 30 µl of supernatant were loaded onto an
SDS-PAGE gel next to the lane with the sample containing 15 µl of cell
lysate obtained from the same cell line.
After SDS-PAGE and Western blotting (see below), nitrocellulose membranes
were scanned and the bands were quantified using the AMBIS optical imaging
system (AMBIS, San Diego, CA). A ratio between the density of the biotinylated
YFP- band and the density of the YFP-
fully glycosylated band from
the total cell lysate for each cell line was calculated as a percentage of
that in the wild type.
SDS-PAGE and Western blot analysis. The microsomal membrane
fraction (5-15 µg), cell lysates, or biotinylated proteins were loaded on
the 10% Tris-glycine or 4-12% Nu-PAGE Bis-Tris gels (Invitrogen). As a
standard for native -and
-subunits, 40-170 ng of purified gastric
vesicles (G1 fraction)
(20) were used and molecular
weight standards (Bio-Rad Laboratories, Hercules, CA) were run on each
gel.
After SDS-PAGE, proteins were transferred to nitrocellulose membranes
(Bio-Rad Laboratories). The membranes were washed twice with TBS [10 mM Tris
· HCl, pH 7.4, 150 mM NaCl, 0.05% (vol/vol) Tween-20] and incubated in
TBS containing 1% (wt/vol) bovine serum albumin. After 30 min, the membranes
were incubated in the primary antibody solution [monoclonal antibody 12.18
against amino acids 666-689 of the H,K-ATPase -subunit diluted 1:10,000
in TBS, or monoclonal antibody 2B6 against amino acids 236-281 of the
H,K-ATPase
-subunit (MBL Bio-Rad Laboratories) diluted 1:1,000 in TBS,
or monoclonal anti-GFP antibody (Clontech) diluted 1:2,000]. After 1 h, the
membranes were washed twice with TBS and incubated with the secondary antibody
solution [anti-mouse IgG conjugated to alkaline phosphatase (Promega, Madison,
WI), diluted 1:2,000 in TBS]. Then, after 1 h, the membranes were washed twice
and incubated for 15 min in TBS. After a final wash, the membranes were
incubated for 5 min in AP buffer containing
5-bromo-4-chloro-3-indolylphosphate-p-toluidine salt (BCIP)/nitro
blue tetrazolium (NBT) color substrate according to the manufacturer's
instructions (Promega).
Quantification of the expressed H,K-ATPase. The content of the
H,K-ATPase in the membrane fraction of the HEK-293 cells was quantified by
using purified hog gastric vesicles (G1) containing 85%
H,K-ATPase (20) as a standard.
Three different amounts of the membrane fraction and three different amounts
of G1 were loaded on the same gel. SDS-PAGE and subsequent Western
blot analysis with antibody 12.18 against
-subunit were performed as
described above. All blots were scanned using the AMBIS optical imaging system
(AMBIS), and the optical density of the bands was measured. Purified
G1 (40-170 ng) yielded a linear calibration curve. For each
membrane preparation, a range of loaded amounts of membrane fraction giving
similar values of the optical density compared with the densities obtained for
G1 was chosen. The amount of the
-subunit of the H,K-ATPase
in the sample was calculated using the calibration curve for G1.
This allowed determination of the content of H,K-ATPase in crude membrane
preparations as the percentage of total membrane protein. The enzyme content
for each membrane preparation was calculated as the average of values obtained
for three samples of the respective membrane fraction.
NH4+-stimulated ATPase activity. Na+-free reaction buffer [40 mM Tris · HCl, pH 7.4, 2 mM MgCl2 containing the inhibitors of possible contaminant ATPases: 1 mM EGTA (Ca-ATPases), 500 µM ouabain (Na,K-ATPase), 1 µM oligomycin (mitochondrial ATPase), 10 nM bafilomycin (V type ATP-ases), and 100 nM thapsigargin (SERCa-ATPase)] in a total volume of 75 µl contained 1-3 µl of a membrane fraction, various concentrations of NH4Cl (0-40 mM), and 800 µM ATP. The reaction was terminated after 1 h at 37°C by adding 100 µl of stop solution containing 0.046% (wt/vol) malachite green, 1.43% (wt/vol) ammonium molybdate, 1.36 N HCl, and 1.26% (vol/vol) NP-10. After 1 min, 25 µl of 34% (wt/vol) sodium citrate were added. After 30 min, the absorbance at 680 nm was measured in a plate reader. Ion-stimulated ATPase activity was calculated as a difference between Pi release in the presence and in the absence of NH4Cl. The activity was expressed in µmol Pi · h-1 · mg total protein-1 and then converted to specific H,K-ATPase activity (µmol Pi · h-1 · mg H,K-ATPase-1) relative to a purified gastric vesicle preparation (G1) using the content of expressed H,K-ATPase calculated from the quantitative Western blot analysis (see above).
Determination of Vmax, Km,app for NH4+, and Ki for the SCH28080 inhibitor. Data for the specific H,K-ATPase activities at various NH4Cl concentrations (21-36 points) in the absence of inhibitor or at the fixed concentration of inhibitor were fitted by nonlinear regression to the Michaelis-Menten equation (Enzfitter, BIOSOFT, 1999), and the maximal velocity (Vmax) ± SE and the apparent Km (Km,app) ± SE for NH4+ were obtained. Each set of data points was plotted as an inverse plot to determine the mechanism of SCH28080 inhibition (competitive, noncompetitive, or mixed). The inhibitor constant (Ki) ± SE was obtained from linear regression of the Km,app/Vmax values plotted vs. inhibitor concentration. The intersection of the linear regression with x-axis gives the negative Ki.
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RESULTS |
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The microsomal membranes isolated from the cells stably expressing
YFP- were analyzed using monoclonal antibodies against GFP and against
the
-subunit (Fig. 1,
lanes 4-7). Both antibodies detected two bands, one at
80-100 kDa and another at
75 kDa (Fig.
1, lanes 4 and 5), presumably representing
complex- and core-glycosylated YFP-
fusion proteins, respectively. After
PNGase F treatment of the membrane fraction, bands at 80-100 and 75 kDa
disappeared, and a single band of
55 kDa was detected using the
monoclonal antibody against the
-subunit
(Fig. 1, lane 6). A
band of the same molecular weight was detected using anti-GFP antibodies (data
not shown). This band represents the nonglycosylated YFP-
fusion
protein. Treatment of the membranes with EndoH resulted in the
disappearance of only the lower band on the Western blot, whereas the band
correspondent to complex-glycosylated protein was retained
(Fig. 1, lane 7). A
small shift in the molecular weight of the EndoH cleavage product
compared with the PNGase F product reflects the difference in the cleavage
sites for these two enzymes in the glycoprotein. In
Fig. 1, lanes
8-10, the membranes isolated from the HEK-293 cells stably
expressing the gastric H,K-ATPase with and without treatment with PNGase F and
EndoH are shown. A pattern similar to the cleavage by glycosidases of
YFP-
fusion protein (Fig.
1, lanes 5-7) was observed. The difference in
molecular weights of nonglycosylated YFP-
fusion protein
(Fig. 1, lane 6) and
nonglycosylated
-subunit (Fig.
1, lane 9) corresponds to YFP portion of the fusion
protein.
The microsomal membranes isolated from HEK-293 cells transfected with
pECFP- alone did not possess ion-stimulated H,K-ATPase activity.
However, the microsomes isolated from HEK-293 cells cotransfected with
pECFP-
and pEYFP-
displayed ion-stimulated SCH28080-inhibited
ATPase activity (Fig. 2),
indicating that CFP and YFP tags of
- and
-subunits did not
disrupt the proper folding and association of the two subunits and only
moderately changed the Vmax (85 vs. 132 µmol ·
h-1 · mg-1) and
Km,app [NH4+] (1.0 vs.
2.4 mM) compared with the wild-type H,K-ATPase
(28).
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Characterization of glycosylation site mutants of the YFP-
fusion protein and the H,K-ATPase
-subunit. The
Western blot of the membrane fractions isolated from the cells expressing
wild-type and mutant YFP-
fusion proteins is shown in
Fig. 3. All the mutants,
similar to the wild type, were represented by both complex- and
core-glycosylated fractions of YFP-
fusion proteins, indicating that all
the mutants were able to leave the ER and reach the Golgi. A stepwise decrease
in molecular weights of mutants lacking one, two, or three glycosylation sites
was observed compared with the wild-type YFP-
fusion protein. PNGase F
cleavage of all the mutants resulted in a single product correspondent to the
size of nonglycosylated YFP-
fusion protein (only N1 mutant treated with
PNGase F is shown; Fig. 2,
lane 11). The ratio between complex-glycosylated and
core-glycosylated portions of the protein was decreased in most of the
mutants, especially in N3, N4, N7, and triple mutant N1/N2/N4.
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To determine whether removal of one or two glycosylation sites from the
-subunit affects the activity of the H,K-ATPase, three cell lines,
stably expressing the
-subunits lacking the fourth, the seventh, or the
first and the second glycosylation sites were obtained
(Table 1). These cell lines
were transfected with cDNA of the wild-type
-subunit, and stable lines
expressing
-subunit in a complex with each mutant
-subunit
(
+
N4,
+
N7, and
+
N1/N2) were
selected as described in METHODS. Microsomal membranes from the
cells were isolated and assayed for the H,K-ATPase activity and sensitivity to
SCH28080. All three mutants possessed ion-stimulated, SCH28080-inhibited
H,K-ATPase activity. The results of SCH28080 inhibition kinetics for
+
N1/N2 are presented in Fig.
4. Similar to that in the wild type, SCH28080 inhibition of the
mutant was purely competitive in respect to the stimulating ion. Mutants
+
N4,
+
N7 also displayed competitive kinetics of
SCH28080 inhibition (data not shown). Calculated kinetic parameters for all
three mutants were not significantly different from those in the wild type
(Fig. 4). These data indicate
that removing the fourth, the seventh, or first and the second glycosylation
sites from the
-subunit did not disrupt folding and proper association
of the two H,K-ATPase subunits.
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Plasma membrane expression of CFP- and YFP-
fusion proteins in HEK-293 cells. Transient expression of CFP-
fusion protein in HEK-293 cells resulted in the intracellular retention of the
protein (Fig. 5A),
whereas expression of the wild-type YFP-
fusion protein resulted in
plasma membrane localization of the protein
(Fig. 6A). When
HEK-293 cells stably expressing the H,K-ATPase
-subunit were transfected
with pECFP-
, the fusion protein was partly localized intracellularly
and partly on the plasma membrane (Fig.
5B). In HEK-293 cells cotransfected with pECFP-
and pEYFP-
, both CFP-
and YFP-
fusion proteins were
targeted to the plasma membrane with partial intracellular retention
(Fig. 5, C and
D).
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The effect of glycosylation site mutations on surface expression of
YFP- was studied using confocal microscopy
(Fig. 6). The confocal XY
images of the middle plane of the cells stably expressing the wild-type and
mutant YFP-
fusion proteins are shown on
Fig. 6. The wild-type
YFP-
predominantly was found on the plasma membrane
(Fig. 6A). A small
fraction of the protein pool was detected intracellularly in the perinuclear
region. The mutant lacking the second glycosylation site, N2, showed both
intracellular accumulation of the protein and plasma membrane localization
(Fig. 6C). Removing
each of the other six glycosylation sites from the exoplasmic loop of
YFP-
resulted in a loss of plasma membrane localization and
intracellular accumulation of the protein
(Fig. 6). Mostly, accumulation
of YFP-
was observed as bright areas near the nucleus, which appeared to
represent Golgi localization. A double mutant lacking the first and the second
glycosylation sites displayed intracellular accumulation of the protein.
In addition to confocal microscopy studies, the effect of mutating
glycosylation sites on trafficking and plasma membrane expression of
YFP- was studied by using Western blot analysis of biotinylated surface
proteins vs. total cell proteins. Biotinylated plasma membrane proteins and
total cell lysate obtained from the same cell line were run side by side on
SDS-PAGE and analyzed by Western blot with the anti-GFP antibody
(Fig. 7). Biotinylated plasma
membrane proteins of the wild-type and mutant YFP-
contained only the
fully glycosylated portion of YFP-
. By contrast, total cell lysates
contained both fully- and core-glycosylated fractions of the protein. In
agreement with the confocal microscopy studies, the amount of YFP-
detected in the plasma membrane was dramatically reduced in most of the
mutants, except N2, compared with the wild type. Normalization of the
YFP-
plasma membrane expression in mutants compared with the wild type
yielded a quantitative interpretation of the effect of glycosylation site
mutations on plasma membrane expression of YFP-
(Fig. 7). Mutation of the
second glycosylation site resulted in a 2.5-fold reduction of the relative
surface expression compared with the wild type. All other mutants showed a 13-
to 55-fold reduction of relative plasma membrane YFP-
content.
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DISCUSSION |
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Six of the seven N-glycosylation sites in the H,K-ATPase
-subunit are essential for its plasma membrane expression in HEK-293
cells. Removing each of the glycosylation sites, except the second one,
from the YFP-
results in a loss of plasma membrane localization of the
protein (Figs. 6 and
7). A double mutant lacking the
first and second glycosylation sites also shows intracellular retention. This
is consistent with earlier data showing the loss of surface delivery of
-and
-subunits in COS-7 cells as a result of removing three
glycosylation sites-the first, the third, and the seventh
(1).
One of the possible reasons for the loss of surface delivery of the mutants
could be impaired protein folding with subsequent retention of the protein in
the ER and the ER-dependent degradation cascade. However, the experimental
data show that N-glycosylation site defective mutants are properly
folded. The /
complexes containing the
-subunits lacking
the fourth, the seventh, or the first and the second glycosylation sites are
catalytically active and display kinetic properties, which are similar to the
wild-type enzyme (Fig. 4),
indicating that they retain the proper folding and assembly between
-
and
-subunits. These data are consistent with preservation of H,K-ATPase
activity after removal of any one of the seven N-glycosylation sites
from the
-subunit (1).
Serial removal of N-glycans from the
-subunit resulted in a
progressive loss of activity. Removing all the seven N-glycosylation
sites or inhibition of glycosylation resulted in prevention of
/
assembly and complete loss of H,K-ATPase enzymatic activity in HEK-293 cells
(1) or insect cells
(14).
N-glycosylation site defective mutants not only retain the native
structure of the enzyme heterodimer but also undergo normal processing and
reach the Golgi. The complex-glycosylated, EndoH
glycosidase-insensitive form of the protein is present in all the mutants,
indicating that the mutations do not prevent trafficking of the -subunit
from the ER to the Golgi. The ratio between complex-glycosylated and
core-glycosylated portions of the protein is, however, less in some of the
mutants compared with the wild-type
-subunit, indicating that mutations
increase the fraction of the protein pool that does not pass quality control
and is thus retained in the ER
(31). Nevertheless, all the
mutant fusion proteins undergo complex glycosylation, which is clear evidence
that they are able to reach the Golgi. As seen from
Fig. 6, accumulation of
YFP-
in Golgi occurs with all glycosylation site mutants.
The experimental observations described above allow the conclusion that the
trafficking step that is affected by the removal of N-glycosylation
sites is the route from the Golgi to the plasma membrane and not from ER to
Golgi. This implies that TGN-plasma membrane delivery depends on the presence
or absence of trafficking information encoded by particular glycosylation
sites within the extracellular domain of the -subunit. It is possible
that, similar to polarized cells, HEK-293 cells have machinery that recognizes
this trafficking signal and places the
-subunit into specific cargo
vesicles in TGN that deliver the protein to the plasma membrane. The
alternative explanation is that N-glycosylation sites are essential
not for the exit from TGN and surface delivery but for the stabilization of
the protein on the plasma membrane. However, recent data showing that
nonpolarized cells are capable of sorting basolateral and apical proteins to
the appropriate vesicles in TGN based on the sorting signals present within
the proteins (8,
12,
18,
19,
32) and that
N-glycosylation can mediate apical sorting for a number of secreted
and membrane proteins (8,
18,
24) favor the former
explanation. Recent data on the role of N-glycans in trafficking of
the homologous Na,K-ATP-ase
1-subunit, a basolateral protein,
also can be interpreted according to this hypothesis. In contrast to
H,K-ATPase
-subunit, removing N-glycans from the Na,K-ATPase
1-subunit does not prevent its ability to reach the plasma
membrane (17). Our
interpretation of this result suggests that the presence of a basolateral
signal within the Na,K-ATPase
1-subunit allows trafficking to
the plasma membrane in nonpolarized cells. Similar interpretation may explain
why a basolateral bile acid transporter retains plasma membrane targeting in
HEK-293 cells after complete removal of its two glycosylation sites
(9).
In summary, these results indicate that the fusion proteins of the gastric
H,K-ATPase - and
-subunits with CFP and YFP, respectively, are
appropriate tools for intracellular sorting and trafficking studies. Plasma
membrane delivery of the gastric H,K-ATPase
-subunit in HEK-293 cells
does not occur by a default pathway but is highly sensitive to structural
peculiarities of the normal or mutated proteins. Six of the seven
glycosylation sites in the gastric H,K-ATPase
-subunit are essential for
TGN to plasma membrane delivery, suggesting their role either in the exit of
the protein from TGN or in the stabilization on the plasma membrane. They are,
however, not required for movement into the TGN. The second glycosylation site
(Asn103), which is not conserved among the
-subunits from different
species, is not essential for sorting and trafficking of the gastric
H,K-ATPase.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() |
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2. Bamberg K, Mercier F, Reuben MA, Kobayashi Y, Munson KB, and Sachs G. cDNA cloning and membrane topology of the rabbit gastric H+/K+-ATPase alpha-subunit. Biochim Biophys Acta 1131: 69-77, 1992.[ISI][Medline]
3. Courtois-Coutry N, Roush D, Rajendran V, McCarthy JB, Geibel J, Kashgarian M, and Caplan MJ. A tyrosine-based signal targets H/K-ATPase to a regulated compartment and is required for the cessation of gastric acid secretion. Cell 90: 501-510, 1997.[ISI][Medline]
4. Duffield AS,
Brown AN, Folsch H, Mellman IC, and Caplan MJ. Differential sorting of the
H,K-ATPase -subunit in MDCK and LLC-PK1 cells is independent of µ1B
adaptin expression. In: 42nd American Society for Cell Biology
Annual Meeting, edited by Yamamoto KR. San Francisco, CA:
American Society for Cell Biology, 2002, p.
88a.
5. Dunbar LA,
Aronson P, and Caplan MJ. A transmembrane segment determines the
steady-state localization of an ion-transporting adenosine triphosphatase.
J Cell Biol 148:
769-778, 2000.
6. Gottardi CJ and Caplan MJ. An ion-transporting ATPase encodes multiple apical localization signals. J Cell Biol 121: 283-293, 1993.[Abstract]
7. Gottardi CJ,
Dunbar LA, and Caplan MJ. Biotinylation and assessment of membrane
polarity: caveats and methodological concerns. Am J Physiol Renal
Fluid Electrolyte Physiol 268:
F285-F295, 1995.
8. Gut A, Kappeler
F, Hyka N, Balda MS, Hauri HP, and Matter K. Carbohydrate-mediated Golgi
to cell surface transport and apical targeting of membrane proteins.
EMBO J 17:
1919-1929, 1998.
9. Hallen S, Mareninova O, Branden M, and Sachs G. Organization of the membrane domain of the human liver sodium/bile acid cotransporter. Biochemistry 41: 7253-7266, 2002.[ISI][Medline]
10. Ikonen E and Simons K. Protein and lipid sorting from the trans-Golgi network to the plasma membrane in polarized cells. Semin Cell Dev Biol 9: 503-509, 1998.[ISI][Medline]
11. Keller P and
Simons K. Post-Golgi biosynthetic trafficking. J Cell
Sci 110:
3001-3009, 1997.
12. Keller P, Toomre D, Diaz E, White J, and Simons K. Multicolour imaging of post-Golgi sorting and trafficking in live cells. Nat Cell Biol 3: 140-149, 2001.[ISI][Medline]
13. Kimura T,
Tabuchi Y, Takeguchi N, and Asano S. Mutational study on the roles of
disulfide bonds in the beta-subunit of gastric
H+,K+-ATPase. J Biol Chem
277: 20671-20677,
2002.
14. Klaassen CH, Fransen JA, Swarts HG, and De Pont JJ. Glycosylation is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells. Biochem J 321: 419-424, 1997.[ISI][Medline]
15. Kroepfl JF and Gardinier MV. Identification of a basolateral membrane targeting signal within the cytoplasmic domain of myelin/oligodendrocyte glycoprotein. J Neurochem 77: 1301-1309, 2001.[ISI][Medline]
16. Lambrecht N,
Munson K, Vagin O, and Sachs G. Comparison of covalent with reversible
inhibitor binding sites of the gastric H,K-ATPase by site-directed
mutagenesis. J Biol Chem 275:
4041-4048, 2000.
17. Laughery MD, Todd ML, Morgan LM, and Kaplan JH. Assembly and trafficking of the Na,K-ATPase (Abstract). Biophys J 84: 265a, 2003.
18. Matter K and Mellman I. Mechanisms of cell polarity: sorting and transport in epithelial cells. Curr Opin Cell Biol 6: 545-554, 1994.[ISI][Medline]
19. Musch A, Xu H, Shields D, and Rodriguez-Boulan E. Transport of vesicular stomatitis virus G protein to the cell surface is signal mediated in polarized and nonpolarized cells. J Cell Biol 133: 543-558, 1996.[Abstract]
20. Rabon EC, Bin Im W, and Sachs G. Preparation of gastric H+,K+-ATPase. Methods Enzymol 157: 649-654, 1988.[ISI][Medline]
21. Reuben MA, Lasater LS, and Sachs G. Characterization of a beta subunit of the gastric H+/K+-transporting ATPase. Proc Natl Acad Sci USA 87: 6767-6771, 1990.[Abstract]
22. Roush DL,
Gottardi CJ, Naim HY, Roth MG, and Caplan MJ. Tyrosine-based membrane
protein sorting signals are differentially interpreted by polarized
Madin-Darby canine kidney and LLC-PK1 epithelial cells. J Biol
Chem 273:
26862-26869, 1998.
23. Rustom A, Bajohrs M, Kaether C, Keller P, Toomre D, Corbeil D, and Gerdes HH. Selective delivery of secretory cargo in Golgi-derived carriers of nonepithelial cells. Traffic 3: 279-288, 2002.[ISI][Medline]
24. Scheiffele P, Peranen J, and Simons K. N-glycans as apical sorting signals in epithelial cells. Nature 378: 96-98, 1995.[ISI][Medline]
25. Smolka A,
Helander HF, and Sachs G. Monoclonal antibodies against gastric
H+-K+-ATPase. Am J Physiol Gastrointest Liver
Physiol 245:
G589-G596, 1983.
26. Traub LM and Kornfeld S. The trans-Golgi network: a late secretory sorting station. Curr Opin Cell Biol 9: 527-533, 1997.[ISI][Medline]
27. Urushidani T and Forte JG. Stimulation-associated redistribution of
H+-K+-ATPase activity in isolated gastric glands.
Am J Physiol Gastrointest Liver Physiol
252: G458-G465,
1987.
28. Vagin O, Denevich S, Munson K, and Sachs G. SCH28080, a K+-competitive inhibitor of the gastric H,K-ATPase, binds near the M5-6 luminal loop, preventing K+ access to the ion binding domain. Biochemistry 41: 12755-12762, 2002.[ISI][Medline]
29. Wieland FT, Gleason ML, Serafini TA, and Rothman JE. The rate of bulk flow from the endoplasmic reticulum to the cell surface. Cell 50: 289-300, 1987.[ISI][Medline]
30. Yeaman C,
Grindstaff KK, and Nelson WJ. New perspectives on mechanisms involved in
generating epithelial cell polarity. Physiol Rev
79: 73-98,
1999.
31. Yoshida Y, Chiba T, Tokunaga F, Kawasaki H, Iwai K, Suzuki T, Ito Y, Matsuoka K, Yoshida M, Tanaka K, and Tai T. E3 ubiquitin ligase that recognizes sugar chains. Nature 418: 438-442, 2002.[ISI][Medline]
32. Yoshimori T, Keller P, Roth MG, and Simons K. Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells. J Cell Biol 133: 247-256, 1996.[Abstract]
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