| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(Received for publication, March 22, 1995; and in revised form, July 21, 1995) From the
We compared the trafficking of the glycosylphosphatidylinositol
(GPI)-anchored placental alkaline phosphatase (PLAP) and two chimeric
transmembrane proteins containing the PLAP ectodomain in stably
transfected Madin-Darby canine kidney epithelial cells to determine
whether different mechanisms might be used in apical sorting of
GPI-anchored and transmembrane proteins. PLAP-G, which contained the
transmembrane and cytoplasmic domains of the vesicular stomatitis virus
glycoprotein, was delivered directly to the basolateral surface.
PLAP-HA contained the transmembrane and cytoplasmic domains of
influenza hemagglutinin. Both PLAP and PLAP-HA were delivered directly
to the apical membrane. PLAP becomes insoluble in Triton X-100 during
biosynthetic transport, as it associates with detergent-resistant
membranes. Neither hybrid protein was detergent insoluble, though the
small amount of PLAP that was missorted to the basolateral surface was
insoluble. We examined the effects of three drugs known to interfere
with membrane trafficking on sorting and delivery of PLAP and the
hybrid proteins. Monensin had no effect on sorting or surface
expression of any of the proteins. Nocodazole affected the sorting of
both PLAP and PLAP-HA but not of PLAP-G. Brefeldin A appeared to
disrupt the sorting of PLAP and PLAP-HA but not of PLAP-G. This
conclusion was tempered by the observation that this drug affected the
distribution of proteins at the cell surface. Thus, sorting and
transport of GPI-anchored and apical transmembrane proteins are similar
in a number of respects.
Polarized epithelial cells contain apical and basolateral plasma
membrane domains that are separated from each other by tight junctions
that maintain differences in the protein and lipid composition between
the two surfaces(1, 2, 3) . Compositional
differences in the two membrane domains are generated by the sorting of
proteins and lipids after intracellular synthesis. In the Madin-Darby
canine kidney (MDCK) ( Efforts
to understand the sorting process have focused on defining sorting
signals in transported proteins. Signals in the cytoplasmic domains of
several basolateral proteins are required for correct
targeting(6, 7, 8, 9, 10, 11, 12, 13) .
In some but not all cases, these regions overlap signals for
internalization of the proteins in clathrin-coated pits. Some
proteins are anchored in membranes by glycosylphosphatidylinositol
(GPI) instead of by conventional transmembrane peptides (reviewed in (14, 15, 16) ). GPI-anchored proteins are
apically polarized in epithelial cells in culture and in
tissues(17) . The role of the membrane anchor in specifying
this apical localization has been tested using hybrid
proteins(18, 19) . Hybrid proteins containing the
ectodomains of normally basolateral proteins, or soluble proteins
linked to GPI-anchors, are directed to the apical surface. Two lines
of investigation suggest that there may be differences between the
intracellular trafficking of GPI-anchored proteins and apical
transmembrane proteins. First, in two unusual cell lines, GPI-anchored
proteins are not restricted to the apical membrane. One of these is a
mutant MDCK cell line that is resistant to killing by concanavalin
A(20, 21) . GPI-anchored proteins are found in both
domains of these cells. The other is the FRT thyroid cell
line(22, 23) . Most GPI-anchored proteins in FRT cells
are present on the basolateral surface(24, 25) . By
contrast, many transmembrane proteins exhibit the same polarity in FRT
cells as in other epithelial cells (26, 27, 28) . Metabolic studies provide
another indication that the trafficking of GPI-anchored proteins and
transmembrane proteins may differ. Two groups showed that cholesterol
depletion specifically inhibits the cell-surface expression of
GPI-anchored proteins. Growth in low density lipoprotein-depleted serum
lowered the expression of a hybrid GPI-anchored protein, gD1-DAF, in
MDCK cells(64) . Cholesterol depletion had a similar effect on
the expression of the GPI-anchored protein CD14 on human
monocytes(29) . Thus, GPI-anchored proteins and apical
transmembrane proteins can show differential sorting and cell-surface
transport in polarized epithelial cells. To compare the trafficking of
GPI-anchored and transmembrane proteins in MDCK cells, we examined
three similar proteins, PLAP, PLAP-G, and PLAP-HA (collectively called
``PLAP proteins''). PLAP is an apical GPI-anchored protein. A
hybrid protein, PLAP-G, contains the ectodomain of PLAP and the
transmembrane and cytoplasmic domains of the vesicular stomatitis virus
glycoprotein (VSV G)(30) . We showed previously that PLAP-G is
present on the basolateral surface of MDCK cells(18) . Here, we
describe a second hybrid protein, PLAP-HA, that contains the ectodomain
of PLAP fused to the transmembrane and cytoplasmic domains of influenza
hemagglutinin (HA). To compare a GPI-anchored protein and two similar
chimeric proteins with the same ectodomain, we studied the delivery,
intracellular trafficking, and cell-surface localization of these three
proteins.
Figure 1:
A, plasmid pBC12/PLAP-HA. The fragment
of pSV-GH3A encoding the transmembrane and cytoplasmic domains of HA
were removed and inserted in pBC12/PLAP 489, generating pBC12/PLAP-HA. B, schematic diagram of PLAP, PLAP-G, and PLAP-HA in the
membrane. The same ectodomain in all three proteins (stripedoval) is linked to a GPI anchor in PLAP, to the VSV-G
transmembrane and cytoplasmic domains (darkstipples)
in PLAP-G, and the HA transmembrane and cytoplasmic domains (lightstipples) in PLAP-HA. C, the C-terminal amino
acid sequences of PLAP, PLAP-G, and PLAP-HA. The Asp residue of PLAP
that is linked to the GPI anchor is indicated(63) . Residues
beyond the site of GPI anchor attachment are not present in mature
PLAP. The transmembrane domains of PLAP-G and PLAP-HA are underlined.
As expected, only PLAP was released from cells by treatment
with 10 units/ml PI-PLC for 1 h at 37 °C (data not shown). The
fraction of PLAP that was released was variable, but generally did not
exceed about 50% and did not increase when more enzyme was used.
Incomplete cleavage by PI-PLC may result from structural heterogeneity
in the GPI anchor (37, 38) or from the membrane lipid
environment(39) .
Figure 2:
Delivery of PLAP, PLAP-HA, and PLAP-G to
the cell surface. Filter-grown cells expressing the indicated protein
were pulse labeled with [
Many transmembrane apical membrane proteins in
MDCK cells are solubilized by Triton X-100. However, a few
transmembrane cell-surface proteins are found in the
detergent-resistant complexes (42) . (
Figure 3:
Solubility of PLAP-HA and PLAP-G in
Triton X-100. Cells expressing PLAP-G (toppanel) or
PLAP-HA (bottompanel) were labeled for 5 min with
[
Figure 4:
Solubility of basolateral PLAP in Triton
X-100. Filter-grown cells expressing PLAP were labeled with
[
We studied the effect of monensin on transport and sorting of the
PLAP proteins. Cells treated with or without monensin were subjected to
the pulse-chase procedure described in Fig. 2and subjected to
domain-specific biotinylation. Biotinylated,
[
Figure 5:
Effect of monensin on polarized expression
of PLAP proteins. A, filter-grown cells expressing the
indicated protein were incubated with (+) or without(-)
monensin. Cells were labeled with [
Figure 6:
Effect of nocodazole on microtubules in
MDCK cells. Tubulin was detected by indirect immunofluorescence in
cells grown on coverslips treated with (A) or without (B) nocodazole.
Figure 7:
Effect of nocodazole on polarized
expression of PLAP proteins. A, filter-grown cells expressing
the indicated protein were incubated with (+) or without(-)
nocodazole. Cells were labeled with
[
Figure 8:
Effect of BFA on polarized expression of
PLAP proteins. Filter-grown cells expressing the indicated PLAP protein
were labeled with [
As the distribution of all three proteins
was affected, we were concerned about the specificity of the BFA
effect. We performed a control experiment to determine whether BFA
altered the distribution of proteins that were already on the plasma
membrane when drug treatment began. Cells were incubated with
[ We wondered whether a shorter exposure to
BFA might minimize this effect and allow us to detect any changes in
intracellular sorting. Proteins were pulse labeled and then incubated
with or without BFA for 1 h before domain-specific biotinylation. We
included an internal control to measure the effect of the drug on
cell-surface proteins. Cells on parallel filters were pulse labeled,
incubated for 2.5 h without BFA to allow newly synthesized proteins to
reach the cell surface, and then incubated for 1 h with BFA before
biotinylation. This allowed us to determine the effect of a 1-h
exposure to BFA on the distribution of cell-surface proteins. Results are shown in Fig. 8C and Table 1. When
BFA was included at the beginning of the chase, only 39% of PLAP and
15% of PLAP-HA were detected on the apical surface (Fig. 8C, +). However, when cells were treated
with BFA after labeled proteins had reached the plasma membrane, about
70-80% of each protein was found on the apical surface (Fig. 8C, +*). This was similar to the value for
the ``no-BFA'' control (Fig. 8C, -).
This result shows that 1 h of BFA treatment did not affect the polarity
of proteins already present on the apical surface. We conclude that BFA
affected the intracellular sorting of both proteins. The results
were different for PLAP-G. In the absence of BFA, 97% of the protein
was delivered to the basolateral surface (Fig. 8C,
-). When BFA was added at the beginning of the chase, only 70% of
PLAP-G was correctly localized to the basolateral surface (Fig. 8C, +). However, when BFA was added after
PLAP-G had reached the plasma membrane, a similar value of 78% of the
protein was detected on the basolateral membrane (Fig. 8C, +*). It appeared that the main effect of
BFA on PLAP-G was to alter the distribution of the protein after it
reached the cell surface. BFA seemed to have little effect on the
intracellular sorting of PLAP-G. Signals in the cytoplasmic domains of several proteins can
specify basolateral
targeting(6, 7, 8, 9, 10, 11) .
Casanova et al.(6) showed that a transmembrane form
of PLAP with a very short cytoplasmic domain was expressed apically but
could be redirected basolaterally by the addition of a sequence
containing a basolateral sorting signal. The cytoplasmic domain of VSV
G contains a basolateral sorting signal(53) . Thus, a sorting
signal in the cytoplasmic domain of PLAP-G is likely to be responsible
for its basolateral localization. If proper targeting of basolateral
proteins requires positive signals, does transport of apical proteins
occur by default, without the need for specific signals? If so, then
apical proteins should be correctly sorted or missorted coordinately.
The finding that GPI-anchored proteins are not apically polarized in a
concanavalin A-resistant MDCK cell line (21) and in FRT cells (24, 25) suggested that apical transmembrane and
GPI-anchored proteins may be sorted by different mechanisms. This
prompted us to characterize the trafficking of PLAP and PLAP-HA in
detail.
In previous work,
we used monensin in an attempt to block transport of PLAP out of the
Golgi apparatus in MDCK cells(34) . We found that the protein
was insoluble in Triton X-100 after monensin treatment and concluded
that it was insoluble while in the Golgi. Our current findings suggest
that transport was not actually blocked in the earlier experiment.
However, a separate result in the earlier paper also supported the same
conclusion ((34) , Fig. 2). As monensin was not used in
this experiment, the conclusion is still valid.
Several groups have also studied the effect of 3.5 µM BFA on basolateral proteins. Three groups found no effect on
basolateral sorting(58, 59, 61) . In
contrast, targeting of the low density lipoprotein receptor was
reported to be affected by BFA(60) . We found that treatment
with BFA for 3 h affected the polarity of proteins on the cell surface,
as measured by domain-specific biotinylation. The explanation for this
effect is unknown. An attractive possibility is that BFA may stimulate
transcytosis of the PLAP proteins. The drug is known to increase the
rate of basolateral to apical transcytosis of nonspecific
markers(62) , although specific transcytosis of the polymeric
immunoglobulin receptor is blocked(56) . This stimulation of
transcytosis probably reflects an effect of BFA on normal sorting in an
early endosomal compartment. An alternate explanation of the effect of
BFA that we observed, that the integrity of tight junctions is
disrupted, is unlikely(56, 62) . Regardless of the
mechanism, however, these data show that apparent effects of BFA on
sorting in the TGN may be complicated by changes in other membranes. Treatment of cells with BFA for 1 h had little effect on the
distribution of proteins that were already present on the apical
surface when drug treatment began. However, apical proteins were
missorted if exposed to BFA while they were in intracellular
compartments. Thus, we agree with others that BFA affects the sorting
of apical proteins. Qualitatively, BFA had the same effect on sorting
of a GPI-anchored protein (PLAP) as an apical transmembrane protein
(PLAP-HA). About 20-30% of a
[ We have shown that the
sorting and transport pathways of GPI-anchored proteins and a
closely-related transmembrane apical protein have several key features
in common. Further definition of these transport mechanisms and
determination of whether GPI-anchored and transmembrane proteins
inhabit the same transport vesicles remain challenges for the future.
Volume 270,
Number 40,
Issue of October 06, pp. 23641-23647, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)cell line, sorting occurs in the TGN
before delivery to the cell surface(4, 5) .
Materials, Cell Culture, and Plasmid
Construction
All chemicals were from Sigma unless otherwise
stated. MDCK strain II cells were grown as described(31) .
Stable transfection of these cells to generate independent lines
expressing PLAP, PLAP-G, or PLAP-HA was as described(32) .
Plasmids pBC12/PLAP 513 encoding PLAP and pBC12/PLAP 489G encoding
PLAP-G (30) were the gift of S. Udenfriend (Roche Institute,
Nutley, NJ). The construction of plasmid pBC12/PLAP-HA, encoding
PLAP-HA, was similar to that of pBC12/PLAP489G(30) . A 200-base
pair fragment encoding the transmembrane and cytoplasmic domains of HA
(H3 subtype) was excised from pSV2GH3A (33) by BamHI
digestion. The ends were filled in, and the fragment was ligated into
pBC12/PLAP 489 at the unique HpaI site, which was previously
modified by insertion of a filled-in HindIII linker to obtain
an in-frame fusion (Fig. 1).
Immunoprecipitation
MDCK cells expressing
the PLAP proteins were lysed in 1 ml of buffer D (50 mM Tris-Cl, pH 8.0, 6 mM EDTA, 0.4% deoxycholate, 1% Triton
X-100) containing 0.2 mM phenylmethylsulfonyl fluoride. Cell
lysates were warmed briefly to solubilize PLAP (34) and then
cleared by spinning for 1 min in the microfuge. All immunoprecipitation
reactions were performed with rabbit anti-PLAP antibodies (Dakopatts
A/S, Denmark). Immune complexes were recovered with heat-killed Staphylococcus aureus cells (Pansorbin, Calbiochem), washed,
and eluted with sample buffer (62.5 mM Tris-Cl, pH 6.8, 10%
(v/v) glycerol, 5% (v/v) 2-mercaptoethanol, 3% SDS). Electrophoresis
was performed using denaturing SDS-polyacrylamide gels (SDS-PAGE) (35) containing 10 or 11% acrylamide.Purification of PI-PLC
Plasmid pIC,
encoding Bacilluscereus PI-PLC, was the gift of Dr.
J. Volwerk. Purification of recombinant periplasmic PI-PLC from Escherichia coli was according to Koke et al.(36) with minor modifications.Detection of Proteins on the Cell
Surface
Domain-specific biotinylation was performed
according to Lisanti et al.(17) . Cells were grown to
confluency in plastic dishes or were seeded at confluency on
polycarbonate filters (Costar, Cambridge, MA) and grown for 1 week. For
pulse-chase experiments, cells were incubated for 20-30 min in
starvation media lacking methionine and containing 5% dialyzed serum,
pulse-labeled for 20 min with 1 mCi/ml
ExpreS
S (DuPont NEN, referred to as
[
S]methionine) in 50 µl total volume from
the basolateral surface, and then chased with media containing 5 mM unlabeled methionine. At various times, cells were placed on ice
in PBS or TEA buffer (10 mM triethanolamine HCl, pH 9.0, 2
mM CaCl
, 125 mM NaCl). Cell surface
proteins were biotinylated by addition of 0.5 mg/ml sulfo-NHS biotin
(Pierce) for two 15-min incubations. Cells were lysed with buffer D
with 0.2 mM phenylmethylsulfonyl fluoride. PLAP proteins were
immunoprecipitated from volumes of lysate containing equal amounts of
radioactivity, as determined by counting a small aliquot in a
scintillation counter. Immunoprecipitates were eluted by boiling for 5
min in 20 µl of 10% SDS. 10% of the eluate was reserved for
quantitation of the total labeled immunoprecipitated protein. The
remainder was diluted in 1 ml of immunoprecipitation buffer (20 mM phosphate buffer pH 7.5, 500 mM NaCl, 1% Triton X-100,
0.5% deoxycholate, 0.02% sodium azide). Biotinylated PLAP was recovered
by binding to streptavidin immobilized on agarose beads (Pierce)
overnight at 4 °C with rotating. Bound proteins were eluted in
sample buffer, analyzed by SDS-PAGE, incubated in 0.5 M salicylic acid in 30% methanol, dried, and visualized by
fluorography using pre-flashed x-ray film. Bands on fluorograms were
scanned using a Bio-Rad GS-670 imaging densitometer. When appropriate,
the relative amount of each protein on the apical and basolateral
surfaces was determined, correcting for the total labeled
immunoprecipitated protein in each sample. For monensin experiments,
starvation, pulse, and chase media contained 1.4 µM monensin, and the chase was for 3 h. For nocodazole experiments,
cells on filters or coverslips were preincubated for 30 min at 4 °C
in media containing 33 µM nocodazole and then for 2.5 h at
37 °C in the same media. Nocodazole was included in starvation,
pulse, and chase media. The chase was performed for 3 h. For brefeldin
A (BFA, Epicentre Technologies, Madison, WI) experiments, 3.5
µM BFA was included in the chase media. The chase was
performed for 2 h except as noted.Immunofluorescence
MDCK cells grown on
coverslips were fixed in PBS containing 3% paraformaldehyde for 30 min
at room temperature, permeabilized with PBS containing 1% Triton X-100,
and blocked with PBS containing 10 mM glycine and 1% bovine
serum albumin. Cells were incubated with monoclonal anti-tubulin
antibodies and then fluorescein-conjugated goat anti-mouse IgG, each
for 30 min at room temperature, before visualization by
immunofluorescence microscopy.
Expression of PLAP, PLAP-HA, and PLAP-G in MDCK
Cells
Although all three proteins were expressed at roughly
similar levels, expression of PLAP-G was often lower than that of PLAP
and PLAP-HA. Several independent stably transfected lines expressing
each protein were used in the course of these studies. These varied in
their expression level. In addition, expression of the transfected
proteins decreased with time in culture. Thus, a precise comparison of
the expression levels of the three proteins was difficult. None of the
results reported here varied as a function of expression level. All the
proteins were transported to a compartment in which their N-linked oligosaccharide chains became resistant to
endoglycosidase H with half-times of about 20-30 min (data not
shown). Most of each protein was delivered to the surface within 2 h of
synthesis, as shown by the following experiment. The ratio of each
[S]methionine-labeled PLAP protein that could be
recovered with immobilized streptavidin, to the amount of total labeled
PLAP protein in the lysate, was similar whether cell-surface
biotinylation was performed 2, 3, 6, or 10 h after pulse labeling. As
immunofluorescent observation showed no large intracellular pools of
any of the proteins at steady state, we concluded that most of each
protein reached the plasma membrane within 2 h of synthesis (data not
shown).
PLAP and PLAP-HA Are Delivered Directly to the Apical
Plasma Membrane Domain, While PLAP-G Is Delivered to the Basolateral
Membrane
Most proteins in MDCK cells are transported
directly from the TGN to the plasma membrane domain in which they will
accumulate. However, some specialized proteins are initially delivered
to one domain and are then internalized and transported to the other
surface by transcytosis(40) . To determine the behavior of the
PLAP proteins, cells grown on semi-permeable filters were incubated
with [S]methionine for 20 min and then
transferred to media containing excess unlabeled methionine in a
pulse-chase protocol. At various times, proteins on the apical or
basolateral surface were subjected to domain-specific biotinylation,
and the cells were lysed. PLAP proteins were recovered by
immunoprecipitation. Biotinylated proteins were recovered from eluted
immunoprecipitates on streptavidin-agarose beads, separated by
SDS-PAGE, and analyzed by fluorography as shown in Fig. 2. PLAP
and PLAP-HA appeared first on the apical surface and were always more
abundant there, while PLAP-G was only detected on the basolateral
surface. PLAP-HA was expressed with apical polarity at steady state, as
determined by surface biotinylation of filter-grown cells (data not
shown).
S]methionine for 20 min
and then chased in unlabeled methionine for the indicated times (in
min). Domain-specific biotinylation was then performed from the apical (Api) or basolateral (Baso) surface of the monolayer.
Biotinylated PLAP proteins were recovered by sequential
immunoprecipitation and absorption onto streptavidin agarose, separated
by SDS-PAGE, and detected by fluorography.
PLAP-HA and PLAP-G Are Always Soluble in Triton
X-100
GPI-anchored proteins are insoluble in non-ionic
detergents at low temperatures. Insolubility results from association
of the proteins with detergent-resistant membranes(34) , at
least partially through interactions with lipid acyl
chains(41) .)To determine
whether PLAP-HA or PLAP-G associated with detergent-resistant
membranes, transfected cells were subjected to a pulse-chase protocol
as described in Fig. 2. After lysis on ice, Triton-soluble and
insoluble fractions were separated by centrifugation, and the pellets
were solubilized in SDS(43) . The hybrid proteins were
recovered from both fractions by immunoprecipitation and analyzed by
SDS-PAGE and fluorography. This experiment was previously performed on
cells expressing PLAP(34) . PLAP shifted from the
Triton-soluble to the Triton-insoluble fraction with a half-time of
about 30 min. After 3 h of chase, approximately 95% of the protein was
insoluble in Triton ( Fig. 1in (34) ). By contrast, both
PLAP-G and PLAP-HA were recovered exclusively from the Triton-soluble
fraction at all times of chase, showing that neither protein associated
with the detergent-resistant membranes (Fig. 3).
S]methionine and then incubated with unlabeled
methionine for the indicated times (in min). After lysis, Triton
X-100-soluble and -insoluble fractions were separated by
centrifugation, and the pellets were solubilized in SDS. The PLAP
proteins were immunoprecipitated from Triton-soluble (S) and
Triton-insoluble (P) fractions, separated by SDS-PAGE, and
analyzed by fluorography.
Basolateral PLAP Is Insoluble in Triton
X-100
A small amount of PLAP is missorted to the
basolateral surface. To determine whether this protein were insoluble
in Triton X-100, a pulse-chase experiment was performed on cells
expressing PLAP as described in Fig. 2, using a 5-min pulse and
a 3-h chase. After domain-specific biotinylation and lysis, the
detergent-soluble and -insoluble fractions were separated.
[S]Methionine-labeled, biotinylated PLAP was
recovered and analyzed as described in Fig. 2. A fluorograph is
shown in Fig. 4. The small amount of PLAP that was present on
the basolateral surface was largely insoluble in Triton X-100.
S]methionine for 20 min and then transferred to
media containing unlabeled methionine for 2 h. Filters were
biotinylated on the apical (A) or basolateral (B)
surface, and cells were lysed in buffer containing Triton X-100. Triton
X-100-soluble and -insoluble fractions were separated by
centrifugation, and the pellets were solubilized in SDS. PLAP was
immunoprecipitated from Triton-soluble (S) and
Triton-insoluble (P) fractions, separated by SDS-PAGE, and
analyzed by fluorography.
Monensin, Nocodazole, Brefeldin A, and
Transport
Three drugs have been reported to affect sorting
or biosynthetic transport of transmembrane proteins. These drugs,
monensin, nocodazole, and BFA, are thus useful tools for characterizing
the trafficking of a GPI-anchored protein. Examining two closely
related chimeric proteins also provides a unique opportunity to compare
the effects of the drugs on apical and basolateral transport.Monensin Does Not Block Surface Delivery of PLAP,
PLAP-HA, or PLAP-G
The monovalent cation ionophore monensin
blocks transport along the secretory pathway in the cis or medial Golgi
in most mammalian cells(44) . In MDCK cells, however, the
effect is different. Early studies using virally infected cells showed
that apical transport of influenza HA was unaffected, while basolateral
transport of VSV G was blocked(45, 46, 47) . S]methionine-labeled PLAP proteins were
recovered and detected by fluorography (Fig. 5). In contrast to
the earlier results cited above, we found that monensin had little
effect on the sorting or cell-surface transport of any of these
proteins. However, the electrophoretic mobility of all three proteins
was altered (Fig. 5). The proteins migrated faster after
monensin treatment, consistent with the previously described effects of
this drug on glycosylation(46) .
S]methionine
for 20 min and then incubated with unlabeled methionine for 3 h before
biotinylation from the apical (A) or basolateral (B)
surface. Biotinylated PLAP proteins were recovered and analyzed as in Fig. 4. A fluorograph is shown. B, bands on fluorograms
from three experiments similar to the one shown in panelA were quantitated by scanning densitometry. The average values are
shown. The percent of each protein localized to the apical surface with
(+) or without(-) monensin was calculated, using the
arbitrary units of the densitometer, as [amount apical/(amount
apical + amount basolateral)]
100. Errorbars indicate standard
deviation.
Nocodazole Affects the Sorting of PLAP and PLAP-HA
but Not of PLAP-G
Treatment of epithelial cells with
microtubule-disrupting drugs specifically affects polarized delivery to
the apical
membrane(48, 49, 50, 51, 52) .
To ensure that nocodazole disrupted microtubules, MDCK cells grown on
coverslips were treated with (Fig. 6A) or without (Fig. 6B) nocodazole and examined by immunofluorescence
using anti-tubulin antibodies. Only a diffuse cytoplasmic staining was
observed after drug treatment. We then determined the effect of
nocodazole on expression of the PLAP proteins using a pulse-chase
protocol. Results are shown in Fig. 7. Only 50% of PLAP and 60%
of PLAP-HA was targeted to the apical membrane after microtubule
disruption, while 94% of PLAP-G was expressed on the basolateral
surface. Thus, in accord with previous findings, sorting of a
basolateral protein was not altered by the drug. Recent results suggest
that this may result from incomplete microtubule disruption (See
``Discussion''). Despite this possibility, our results
clearly demonstrate that polarized delivery of a GPI-anchored protein,
like that of apical transmembrane proteins, requires intact
microtubules.
S]methionine for 20 min and then incubated with
unlabeled methionine for 3 h before biotinylation from the apical (A) or basolateral (B) surface. Biotinylated PLAP
proteins were recovered and analyzed as in Fig. 2and detected
by fluorography. B, bands on fluorographs from three
experiments similar to the one shown in panelA were
quantitated by scanning densitometry. The average values are shown. The
percent of each protein localized to the apical surface with (+)
or without(-) nocodazole was calculated as in Fig. 5. P-HA, PLAP-HA; P-G,
PLAP-G.
Effect of BFA on Sorting of PLAP Proteins and on Cell
Polarity
Low concentrations of the fungal metabolite BFA do
not affect secretion in MDCK cells but have been reported to alter
membrane trafficking in these cells (see ``Discussion''). To
examine the effect of BFA on sorting of the PLAP proteins, cells
expressing them were pulse labeled with
[S]methionine for 5 min and then incubated for 3
h with unlabeled methionine with or without BFA. BFA had a dramatic
effect on the localization of all three proteins (Fig. 8A and Table 1).
S]methionine for 5 min. A, cells were then incubated with unlabeled methionine with
(+) or without(-) BFA for 3 h before biotinylation from the
apical (A) or basolateral (B) surface. B,
after labeling, cells were incubated with unlabeled methionine for 3 h.
Cells were then treated with (+*) or without(-) BFA for 2 h
(PLAP-G) or 3 h (PLAP and PLAP-HA). C, after labeling, cells
were incubated with (+) or without(-) BFA for 1 h or for 2.5
h without BFA and then for 1 h with BFA (+*). A-C,
domain-specific biotinylation was performed from the apical (A) or basolateral (B) side, and biotinylated PLAP
proteins were recovered and processed as in Fig. 2and detected
by fluorography.
S]methionine and then chased for 2-3 h to
allow newly synthesized plasma membrane proteins to reach the cell
surface. Cells were then treated for 3 h with or without 3.5 µM BFA. Monolayers were subjected to domain-specific biotinylation,
and the [
S]methionine-labeled, biotinylated
proteins were recovered and detected as described in Fig. 2.
Results are shown in Fig. 8B and Table 1. The
distribution of all three proteins was affected by this treatment. This
effect did not appear to result from disruption of the tight junctions,
as we detected little effect of BFA on the leakage of fluorescein
isothiocyanate-dextran (M
3860) across the
monolayer in 3 h (data not shown). BFA thus affects a step in sorting,
trafficking, or distribution of surface proteins that is not related to
biosynthetic sorting in the TGN. A likely explanation of this behavior
is the recently described effect of BFA on transcytosis (see
``Discussion'').
Solubility of the PLAP Proteins in Triton
X-100
PLAP-HA and PLAP-G were fully soluble in Triton
X-100, while both apical and basolateral pools of PLAP were insoluble.
Simons and van Meer (2) suggested that apical proteins
associate with glycolipid patches or rafts in the TGN and that this
association is important in apical sorting. Based on this model, we
suggested that association of PLAP with glycolipid-rich
detergent-resistant membrane domains in the TGN might play a role in
sorting. The fact that basolateral PLAP is also detergent resistant
shows that glycolipid-rich domains also exist in the basolateral
membrane. Such domains are thus more widespread than was originally
postulated. However, our finding does not directly contradict the
sorting model. Extensive transcytosis in both directions occurs in MDCK
cells(54) . Thus, the distribution of lipids between the apical
and basolateral membrane does not necessarily reflect the lipid
composition of apical and basolateral transport vesicles. The small
fraction of PLAP that is missorted basolaterally could easily associate
with glycolipid-rich microdomains after delivery to that surface.Monensin
Treatment of virally infected
MDCK cells with monensin inhibited the surface expression of the
basolateral VSV G protein but did not affect the polarity of influenza
HA(45, 47) . These results suggested that monensin
affected transport of apical and basolateral proteins differently. We
found that monensin did not alter the expression or polarity of PLAP,
PLAP-HA, or PLAP-G, and we conclude that the drug has no general effect
on apical or basolateral transport in MDCK cells.Nocodazole
Most previous studies have
indicated that treatment with microtubule-disrupting drugs disrupts the
polarized delivery of apical but not basolateral transmembrane
proteins(48, 49, 50, 51, 52) .
It is likely that microtubule disruption affects targeting of apical
transport vesicles rather than sorting of proteins in the TGN. We found
that nocodazole affected the polarized expression of PLAP and PLAP-HA
but not PLAP-G. While this work was in progress, Lafont et al.(55) reported that nocodazole affected transport of both
apical and basolateral proteins in permeabilized MDCK cells. They
suggested that the drug does not fully disrupt microtubules in intact
cells. This may explain why delivery of PLAP-G, and other basolateral
proteins studied earlier, was not affected by nocodazole. Our most
significant result, however, was that both PLAP and PLAP-HA were
affected to the same extent. We conclude that microtubules are
important in the polarized delivery of both GPI-anchored and apical
transmembrane proteins.Brefeldin A
Low concentrations (1-3
µM) of BFA have little effect on the efficiency of
secretion in MDCK cells(56, 57, 58) .
However, apically directed proteins are missorted in the presence of
the drug(57, 59, 60, 61) . S]methionine-labeled basolateral protein
(PLAP-G) was present on the apical surface after 1 h of incubation in
BFA, whether the drug was applied during biosynthetic sorting or after
the protein had reached the plasma membrane. Thus, there appears to be
little additional effect of BFA on the intracellular sorting of PLAP-G
beyond the effect on the protein already present at the cell surface.
This phenomenon may have been responsible for the apparent effect of
BFA on biosynthetic sorting of the low density lipoprotein receptor (60) and may explain the discrepancy between the fact that TGN
sorting of this protein appeared to be affected by BFA, while that of
other basolateral proteins did
not(58, 59, 61) .
))
We thank J. Rose, in whose laboratory PLAP-HA was
constructed, S. Udenfriend and J. Volwerk for plasmids, B. Theurkauf
for anti-tubulin antibodies, and N. Dean, B. Haltiwanger, and J. Crosby
for critically reading the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Paladino, T. Pocard, M. A. Catino, and C. Zurzolo GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells J. Cell Biol., March 27, 2006; 172(7): 1023 - 1034. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Paladino, D. Sarnataro, R. Pillich, S. Tivodar, L. Nitsch, and C. Zurzolo Protein oligomerization modulates raft partitioning and apical sorting of GPI-anchored proteins J. Cell Biol., November 22, 2004; 167(4): 699 - 709. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Ren, A. G. Ostermeyer, L. T. Ramcharan, Y. Zeng, D. M. Lublin, and D. A. Brown Conformational Defects Slow Golgi Exit, Block Oligomerization, and Reduce Raft Affinity of Caveolin-1 Mutant Proteins Mol. Biol. Cell, October 1, 2004; 15(10): 4556 - 4567. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Ji, C. Lee, M. Jeoung, Y. Koo, G. A. Sievert, and T. H. Ji Trans-Activation of Mutant Follicle-Stimulating Hormone Receptors Selectively Generates Only One of Two Hormone Signals Mol. Endocrinol., April 1, 2004; 18(4): 968 - 978. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. G. Ostermeyer, L. T. Ramcharan, Y. Zeng, D. M. Lublin, and D. A. Brown Role of the hydrophobic domain in targeting caveolin-1 to lipid droplets J. Cell Biol., January 5, 2004; 164(1): 69 - 78. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. J. McIntosh, A. Vidal, and S. A. Simon Sorting of Lipids and Transmembrane Peptides Between Detergent-Soluble Bilayers and Detergent-Resistant Rafts Biophys. J., September 1, 2003; 85(3): 1656 - 1666. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.-Q. Sun, R. Salkar, Sachchidanand, S. Xu, L. Zeng, M.-M. Zhou, and F. J. Suchy A 14-Amino Acid Sequence with a beta -Turn Structure Is Required for Apical Membrane Sorting of the Rat Ileal Bile Acid Transporter J. Biol. Chem., January 31, 2003; 278(6): 4000 - 4009. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. T. Dunphy, W. K. Greentree, and M. E. Linder Enrichment of G-protein Palmitoyltransferase Activity in Low Density Membranes. IN VITRO RECONSTITUTION OF Galpha i TO THESE DOMAINS REQUIRES PALMITOYLTRANSFERASE ACTIVITY J. Biol. Chem., November 9, 2001; 276(46): 43300 - 43304. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Leitner, R. Szlauer, I. Ellinger, A. Ellinger, K.-P. Zimmer, and R. Fuchs Placental Alkaline Phosphatase Expression at the Apical and Basal Plasma Membrane in Term Villous Trophoblasts J. Histochem. Cytochem., September 1, 2001; 49(9): 1155 - 1164. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. F. Pickl, F. X. Pimentel-Muinos, and B. Seed Lipid Rafts and Pseudotyping J. Virol., August 1, 2001; 75(15): 7175 - 7183. [Abstract] [Full Text]
|
|
|
|
 |
|
 A.-Q. Sun, I'K. Swaby, S. Xu, and F. J. Suchy Cell-specific basolateral membrane sorting of the human liver Na+-dependent bile acid cotransporter Am J Physiol Gastrointest Liver Physiol, June 1, 2001; 280(6): G1305 - G1313. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V Gouyer, E Leteurtre, P Delmotte, W. Steelant, M. Krzewinski-Recchi, J. Zanetta, T Lesuffleur, G Trugnan, P Delannoy, and G Huet Differential effect of GalNAc(&agr;)-O-bn on intracellular trafficking in enterocytic HT-29 and Caco-2 cells: correlation with the glycosyltransferase expression pattern J. Cell Sci., January 4, 2001; 114(8): 1455 - 1471. [Abstract] [PDF]
|
|
|
|
|
|
C. Lipardi, L. Nitsch, and C. Zurzolo Detergent-insoluble GPI-anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting Mol. Biol. Cell, February 1, 2000; 11(2): 531 - 542. [Abstract] [Full Text]
|
|
|
|
|
|
S. Moffett, D. A. Brown, and M. E. Linder Lipid-dependent Targeting of G Proteins into Rafts J. Biol. Chem., January 21, 2000; 275(3): 2191 - 2198. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Saunders and L. E. Limbird Microtubule-Dependent Regulation of alpha 2B Adrenergic Receptors in Polarized MDCKII Cells Requires the Third Intracellular Loop but Not G Protein Coupling Mol. Pharmacol., January 1, 2000; 57(1): 44 - 52. [Abstract] [Full Text]
|
|
|
|
|
|
A. G. Ostermeyer, B. T. Beckrich, K. A. Ivarson, K. E. Grove, and D. A. Brown Glycosphingolipids Are Not Essential for Formation of Detergent-resistant Membrane Rafts in Melanoma Cells. METHYL-beta -CYCLODEXTRIN DOES NOT AFFECT CELL SURFACE TRANSPORT OF A GPI-ANCHORED PROTEIN J. Biol. Chem., November 26, 1999; 274(48): 34459 - 34466. [Abstract] [Full Text] [PDF]
|
|
