(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) ()cell line, sorting occurs in the TGN
before delivery to the cell surface(4, 5) .
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 [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.
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) . ()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).
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
[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.
Figure 4:
Solubility of basolateral PLAP in Triton
X-100. Filter-grown cells expressing PLAP were labeled with
[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.
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,
[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) .
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 [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.
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
[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.
Figure 8:
Effect of BFA on polarized expression of
PLAP proteins. Filter-grown cells expressing the indicated PLAP protein
were labeled with [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.
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
[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'').
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
[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 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.