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INTRODUCTION |
Polarized cells such as neurons and epithelial cells maintain
separate plasma membrane domains, each with a distinct protein and
lipid composition, through intracellular sorting mechanisms that
recognize classes of proteins and deliver them into separate vesicles
for transport to the correct surface domain (1, 2). Sorting to the
correct membrane is essential for the proteins to exhibit their
biological functions, whereas missorting often results in pathological
conditions (3, 4). The recognition event responsible for sorting has
been under intense investigation for two decades, and a number of
peptide sequences capable of specifying transport to the basolateral
surface of epithelial cells (5-11), or cell body of neurons (12-16),
have been characterized. All of these signals are located in the
cytoplasmic domains of transmembrane glycoproteins. In addition to
basolateral signals, three types of signals for sorting proteins to the
apical surface of epithelial cells, or axon of neurons, are known.
Glycolipid anchors direct proteins to the apical surface of several
types of epithelial cells (17, 18), apparently by associating in the
trans-Golgi network (19, 20) with detergent-insoluble membrane domains enriched in glycosphingolipids and cholesterol (21).
Oligosaccharides on some secreted proteins appear to specify apical
transport (22), although this mechanism does not apply to all secreted
proteins (23-26).
For many transmembrane glycoproteins, deletion of cytoplasmic sequences
containing a basolateral sorting signal results in efficient transport
of the protein to the apical surface, rather than the random transport
expected for the deletion of specific sorting information (10, 27-30).
For other proteins, deletion of cytoplasmic sequences caused randomized
transport, proving that transport to the apical surface does not occur
by default (9, 31). In the reverse approach, introducing basolateral sorting signals into the cytoplasmic domain of the influenza
hemagglutinin (HA)1 was shown
to have a dominant effect over apical sorting information (8, 31, 32)
that has been recently localized to the transmembrane domain (8). These
observations implied that some proteins carry apical sorting
information that is recessive to cytoplasmic basolateral sorting
signals. Basolateral signals could dominate over apical signals simply
by being recognized earlier in the biosynthetic pathway, or sorting
could occur in a common compartment where basolateral signals might
bind tighter to the sorting machinery than apical signals. To
investigate these questions, we attached a series of basolateral
sorting signals to the strictly polarized membrane protein of small
intestinal epithelial cells, lactase-phlorizin hydrolase (LPH, EC
3.2.1.23-3.2.1.62), and determined their effect on the sorting of
LPH.
LPH, an integral type I membrane glycoprotein, is 1927 amino acids long
containing a membrane anchor of 19 contiguous hydrophobic amino acids
and a cytoplasmic domain of 26 amino acids. It is synthesized as a
precursor with apparent molecular masses of 215 and 230 kDa,
representing the mannose-rich (pro-LPHh) and complex (pro-LPHc) glycosylated forms. Maturation of LPH involves
proteolytic cleavage after complex glycosylation of the precursor to
yield the brush-border form of 160 kDa (33-37). LPH is targeted
strictly to the apical membrane of intestinal epithelial cells and
Madin-Darby canine kidney (MDCK) cells (38). To investigate the
position and relative strength of the apical sorting signal of LPH,
sorting of a tailless LPH mutant (LPH
ct) (39) and
chimeric proteins made by fusing LPH external and transmembrane
sequences to the short, 12-amino acid-long cytoplasmic domain of
several HA mutants was studied in MDCK cells. Wild type HA lacks
basolateral sorting signals and is transported to the apical surface
(40), but point mutations in the cytoplasmic domain of HA were
identified that created both internalization signals and dominant
basolateral sorting signals (8, 32, 41-43). In contrast to their
function in HA, these basolateral sorting signals did not affect strict apical delivery of LPH. However, the chimeric LPH proteins gained the
internalization capacity similar to those of the HA counterparts.
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EXPERIMENTAL PROCEDURES |
Construction of cDNA Encoding Mutant LPH
ct and
Chimeras of Intestinal LPH and Influenza Virus HA--
Standard
recombinant DNA techniques were employed according to Sambrook et
al. (44). The LPH mutant lacking the cytoplasmic tail,
LPH
ct, has been reported previously (39). cDNAs encoding the external and transmembrane domain of LPH (amino acids 1-1901) (45) and the short, 12-amino acid-long, cytoplasmic domain of
HA (amino acids 536-547) (36) (denoted as LPH-HA) were generated by
polymerase chain reaction SOEing (46). The polymerase chain reaction
product encoding LPH-HAwt was cloned into the expression
vector pJB20 (32). A similar strategy was utilized to construct the
LPH-HA chimeras which contain a single mutation
(LPH-HAY543) or a double mutation
(LPH-HAY543/Y546 and LPH-HAY543/R546) in the HA
cytoplasmic domain. The sequence of LPH
ct and each LPH-HA
chimera was determined by sequencing with a Sequenase kit according to
the instructions of the manufacturer (U. S. Biochemical Corp.).
Transfection and Generation of Stable Cell Lines--
MDCK cells
and COS-1 cells were maintained in Dulbecco's modified Eagle's medium
(DMEM, Life Technologies, Inc., Eggenstein/Germany) supplemented with
10% fetal bovine serum, 2 mM glutamine, 50 units/ml penicillin, and streptomycin at 37 °C in a 5% CO2
atmosphere. Cells were transfected with 5 µg of the appropriate
recombinant DNA using DEAE-dextran (for COS-1 cells) as described (47)
or Polybrene (for MDCK cells) (38). Stably transfected MDCK cells were
selected in the presence of 0.25 mg/ml active G418 (Life Technologies,
Inc.), and after 18-23 days, surviving colonies were isolated with
cloning rings. Stable transformants expressing LPH
ct or
LPH-HA chimeras were screened by immunoprecipitation and by
immunofluorescence staining. Expression of LPH-HA chimeras in
intestinal Caco-2 cells was preformed transiently on membrane filters
using the calcium phosphate procedure (48). Here, the cells were grown
to confluency and the corresponding DNA was added at 2 µg/ml. For
higher transfection efficiency on filters, the cells were treated prior
to transfection with trypsin to dissociate the cells and to achieve an
optimal exposure of cells to DNA. Three days post-transfection the
cells were processed for cell surface immunoprecipitation with mAb
anti-LPH (see below) after biosynthetic labeling for 18 h. We
found that a 3-day period was sufficient for the cell layer to achieve
complete polarity. This was biochemically assessed by cell surface
immunoprecipitation of sucrase-isomaltase, which is targeted in Caco-2
cells to the apical membrane. Infection of Caco-2 cells with HA
cDNA was performed as described by Naim and Roth (42) for MDCK cells.
Biosynthetic Labeling of Cells, Immunoprecipitation, and
SDS-PAGE--
Metabolic labeling of MDCK cells grown on filters or
plated in six-well culture dishes was performed as described previously (38). MDCK clones expressing LPH
ct or LPH-HA chimeras were labeled for 1 h with 100 µCi of
[35S]methionine (10 mCi/ml
L-[35S]RedivueTM
Pro-mixTM, Amersham, Braunschweig/Germany) and chased for
different times with unlabeled methionine. Caco-2 cells expressing
transiently transfected LPH-HAwt or
LPH-HAY543/F546 were labeled continuously for 18 h to
ensure a maximum labeling of the expressed recombinant proteins. Caco-2
cells infected with HAwt or HAY543/F546 were continuously labeled for 2 h. Cell lysates were immunoprecipitated with mouse mAb anti-LPH (from hybridoma HBB 1/900/34/74) (34) as
described by Naim et al. (47), and cell surface antigens were immunoprecipitated from intact cells on filters by addition of
anti-LPH or anti-HA (42) antibody to either the apical or basolateral
compartments. The immunoprecipitates were analyzed by SDS-PAGE
according to the method of Laemmli (49). After electrophoresis the gels
were fixed, soaked in 16% salicylic acid for signal amplification, and
subjected to fluorography.
Detergent Extractability of LPH and
Sucrase-Isomaltase--
MDCK-ML cells expressing LPH were
biosynthetically labeled for 1 h with
[35S]methionine and chased over several time points. The
cells were solubilized in the cold for 2 h with 1% Triton X-100
in 25 mM Tris-HCl, pH 8.0, 50 mM NaCl. The
detergent extracts were centrifuged, and the supernatant was
immunoprecipitated with mAb anti-LPH. The pellet was dissolved by
boiling in 1% SDS for 10 min. Thereafter 10-fold volume of buffer
containing 1% Triton X-100 was added. These extracts were centrifuged
and the supernatant was immunoprecipitated with a mixture of two
monoclonal antibodies, MLac 6 and MLac 10, that recognize denatured and
native forms of LPH (39). A similar experimental procedure was followed
to assess the detergent solubility of intestinal brush-border
sucrase-isomaltase by using the colon carcinoma Caco-2 cells.
Immunoprecipitation of native and denatured forms of sucrase-isomaltase
was performed with a mixture of monoclonal antibodies (34). The
immunoprecipitates were analyzed by SDS-PAGE on 5% or 6% gels.
Internalization Assays--
COS-1 cells transiently transfected
with pJB20 encoding LPH
ct or the LPH-HA chimeras were
grown in duplicate on coverslips. 48 h post-transfection, cells
were rinsed in ice-cold DMEM and placed on ice. The cells were
incubated for 2 h with mAb anti-LPH diluted 1:200 in 5% bovine
serum albumin/PBS. Unbound antibody was removed by three washes with
DMEM containing 10% fetal calf serum. Control samples were retained at
4 °C and for a second set of samples the temperature was raised to
37 °C with DMEM for 10 min in a circulating water bath. The cells
were chilled on ice, rinsed in ice-cold PBS, and then fixed with 2%
paraformaldehyde for 20 min at room temperature. After two washes with
PBS, the cells were incubated with fluorescein
isothiocyanate-conjugated goat anti-mouse antibody (Boehringer
Mannheim, Mannheim, Germany), diluted 1:100 in 5% bovine serum
albumin/PBS for 30 min at room temperature. Intracellular localization
of proteins was assessed in transfected cells that were permeabilized
with 0.1% Triton X-100 after fixing. The cells were examined with an
Axioplan fluorescence microscope (Zeiss, Oberkochen, Germany) equipped
with a 100× immersion objective.
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RESULTS |
The Cytoplasmic Tail of LPH Is Not Required for Its Sorting to the
Apical Membrane--
To evaluate the role of the cytoplasmic tail of
LPH in its transport and sorting, we constructed a mutant cDNA
lacking the entire sequence encoding the cytoplasmic tail of LPH
(denoted pro-LPH
ct, Table
I) and found that in COS-1 cells this deletion did not affect the transport-competence of the molecule (39).
Biosynthetic processing of LPH
ct in a MDCK cell line
continuously expressing the protein was similar to that of wild type
pro-LPH in intestinal and MDCK cells (35, 38). The first detectable
biosynthetic form, the 215-kDa mannose-rich pro-LPH
ct species, chased into the complex glycosylated 230-kDa
pro-LPH-ct polypeptide after 4-6 h, and a cleaved form of
LPH
ct (approximate apparent molecular mass 160 kDa)
appeared (Fig. 1). The cleaved 160-kDa
form is the tailless analogue of LPH
previously characterized in
intestinal biopsy specimens (35), in transfected MDCK and CHO cells
(38, 50), and will be therefore denoted LPH
ct.
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Table I
Transmembrane and carboxyl-terminal sequences of wild type LPH,
LPH-ct, and LPH-HA chimeras
All sequences are shown in single-letter code. The cytoplasmic domains
are illustrated in bold letters. The first depicted sequence is that of
the transmembrane domain (19 amino acids) and the cytoplasmic domain
(26 amino acids) of wild type LPH (45). LPH-ct is a mutant form
of LPH lacking the cytoplasmic domain (39). Chimeras containing the
external and transmembrane domain of LPH and the cytoplasmic domain of
HA are indicated as LPH-HA. The cytoplasmic domain of HA contains 12 amino acids. This sequence is predicted to be the maximum length of the
A/Japan HA cytoplasmic domain (36). Single or double mutations in the
cytoplasmic tail of HA are indicated by underlined letters.
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Fig. 1.
Transport kinetics of LPH ct in
MDCK cells. MDCK cells stably expressing LPH ct were
pulse-labeled for 1 h with [35S]methionine and
chased for the indicated times with 2.5 mM unlabeled
methionine. LPH ct was purified by immunoprecipitation and
analyzed by electrophoresis on a 6% SDS gel and fluorography.
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The kinetics of appearance of pro-LPH
ct in the apical or
basolateral domains were investigated by cell surface
immunoprecipitation of pro-LPH
ct and its derivative
LPH
ct from cells grown on transparent polyester
membrane filters as described previously for wild type LPH (38). Fig.
2 shows that the complex glycosylated 230-kDa pro-LPH
ct precursor and LPH
ct
appeared after 4 h of chase at the apical surface. The intensity
of the bands isolated from the apical domain became stronger at the 6-h chase point. No significant bands corresponding to these two LPH species were detected at the basolateral surface. Together, the pulse-chase and sorting analyses indicate that intracellular processing and targeting of pro-LPH
ct in MDCK cells is similar to its wild type pro-LPH counterpart and that the cytosolic portion of
pro-LPH is devoid of apical sorting signals.

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Fig. 2.
Polarized delivery of newly synthesized
LPH ct to the surface of MDCK cells. MDCK cells
stably transfected with LPH ct were grown on filters,
pulse-labeled with [35S]methionine for 1 h, and
chased in medium with 2.5 mM unlabeled methionine for the
indicated times. LPH ct was immunoprecipitated either from
the apical (a) or the basolateral (b) surface and
analyzed as in Fig. 1.
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Apical Transport of LPH Is Independent of Its Association with
Sphingolipid-Cholesterol Rafts--
A number of apically sorted
proteins, such as influenza virus neuraminidase, HA, and some
intestinal proteins, have been shown to be selectively associated with
sphingolipid-cholesterol rafts (21, 51, 52). One of the characteristics
of these protein-membrane structures is their insolubility in
detergents such as Triton X-100 at 4 °C. We therefore examined
whether pro-LPH is associated with rafts in the polarized cell line
MDCK-ML that expresses pro-LPH (38). Cells were pulse-labeled with
[35S]methionine for 1 h and chased for various time
points. The cells were extracted with Triton X-100, and the detergent
solubility of pro-LPH biosynthetic forms versus insolubility
was examined. Fig. 3 shows two
representative chase time points. The 215-kDa mannose-rich pro-LPH
appeared in the supernatant fraction (denoted S) at the earliest chase
time point (1 h pulse, 0 h chase), and the pellet (P) was devoid
of this form. The complex glycosylated 230-kDa pro-LPH was also found
exclusively in the supernatant after 4 h of chase together with
the mannose-rich 215-kDa species. Similar results were obtained with
chase points earlier and later than 4 h. The absence of pro-LPH in
the detergent insoluble fraction (P) indicates that pro-LPH is not
associated with sphingolipid-cholesterol rafts. These data agree with
previous observations that pro-LPH was only found in the
detergent-soluble form in biosynthetically labeled explants from the
pig small intestine (52). By contrast to pro-LPH, the 245-kDa complex
glycosylated mature form of another brush-border protein,
sucrase-isomaltase (34, 53), could be found in the Triton X-100
insoluble pellet (P) after 3 h of chase in biosynthetically
labeled intestinal Caco-2 cells (Fig. 3). The mannose-rich form
(210-kDa), on the other hand, was found only in the supernatant (Fig.
3). This result indicates that the 245-kDa mature form of
sucrase-isomaltase is associated with sphingolipid-cholesterol rafts
and suggests a role of these structures in the targeting of this
glycoprotein to the apical membrane, but not in the sorting of LPH.

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Fig. 3.
LPH is not associated with
sphingolipid-cholesterol rafts. MDCK-ML and intestinal Caco-2
cells expressing LPH and sucrase-isomaltase (SI),
respectively, were biosynthetically labeled for 1 h with
[35S]methionine and chased over several time points.
Triton X-100 detergent extracts were centrifuged, and the supernatants
were immunoprecipitated with mAb anti-LPH or mAb
anti-sucrase-isomaltase. The cellular pellets were extracted by boiling
in 1% SDS followed by dilution with 10-fold volume of buffer
containing 1% Triton X-100. These extracts were centrifuged, and the
supernatants were immunoprecipitated with antibodies that recognize
denatured and native forms of LPH or sucrase-isomaltase. The
immunoprecipitates were analyzed by SDS-PAGE on 6% (LPH) or 5%
(sucrase-isomaltase) gels and fluorography. Representative chase time
points are shown.
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The Chimeric LPH-HA Mutants--
The high fidelity of sorting of
LPH (more than 90% was apically targeted) and its cleaved product
(exclusively apically located) strongly suggest that this protein
contains strong apical sorting signals. The strength and efficiency of
the basolateral sorting machinery has lead to the notion that
basolateral signals are dominant over apical signals in MDCK cells.
Consistent with this concept, HA mutants constructed to contain
basolateral signals were sorted to the basolateral rather than the
apical membrane (8). To examine the strength of the potential apical
sorting signal within the LPH molecule, the same cytosolic sequences
that completely reversed the polarity of HA were fused to the
ectodomain and transmembrane domain of pro-LPH (the chimeras are
indicated LPH-HA). The cytosolic tails of the HA mutants used in this
study were derived from mutant HAY543,
HAY543/F546, and HAY543/R546. The corresponding
chimeras are referred to as LPH-HAY543,
LPH-HAY543/F546, and LPH-HAY543/R546. As a
control, the pro-LPH tail was replaced by the cytosolic tail of wild
type HA (LPH-HAwt). These chimeric proteins were stably
expressed in MDCK cells, and the biosynthesis, processing,
transport, and sorting of the chimeric proteins were investigated.
LPH-HA Chimeras Containing Basolateral Sorting Signals Are Sorted
to the Apical Membrane--
Exchanging the cytosolic tails of pro-LPH
with mutants of the HA tail had no significant effects on the
biosynthesis, processing, and transport rate of pro-LPH. In a fashion
similar to wild type pro-LPH (38), the mutants were processed from the
mannose-rich 215-kDa species to the complex glycosylated mature 230-kDa
and proteolytically cleaved to the 160-kDa LPH
analogues (Fig.
4A). To examine whether the
transplanted cytosolic tails in pro-LPH had affected its sorting to the
apical in a fashion similar to the effects observed with mutant HA
molecules, monolayers of MDCK cells expressing LPH-HAY543,
LPH-HAY543/F546, LPH-HAY543/R546, or
LPH-HAwt were grown on filters and were pulse-labeled with [35S]methionine for 1 h and chased for several
intervals. Cell surface immunoprecipitation with mAb anti-LPH was
performed followed by SDS-PAGE. Fig. 4B demonstrates that
all the biosynthetic forms of the LPH chimeras, i.e. the
uncleaved complex glycosylated pro-LPH and the corresponding cleaved
LPH
analogues, appeared exclusively in the apical membrane. Very
little, if any, was found at the basolateral membrane. Even the chimera
containing the double mutation (Tyr543/Phe546)
in the cytosolic domain was not effective in the context of the pro-LPH
species. By contrast, this mutation completely reversed the sorting of
HA from an apically to a basolaterally sorted molecule (8). Since the
proportion of the labeled species located in the basolateral membrane
was minor throughout the chase periods and because this proportion did
not change in relation to the apical proportion, we conclude that the
transport of the chimeras to the apical surface was direct, as it was
the case with wild type pro-LPH (38). We demonstrate that not even a
minor effect on the polarized sorting of pro-LPH to the apical membrane
could be discerned when basolateral signals in the cytosolic tail of HA
were examined in the pro-LPH species.

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Fig. 4.
A, transport kinetics of LPH-HA
chimeras. MDCK cells expressing the chimeras LPH-HAwt,
LPH-HAY543, LPH-HAY543/F546, or
LPH-HAY543/R546 were biosynthetically labeled for 1 h
with [35S]methionine and were chased over a period of
6 h. Samples were analyzed by SDS-PAGE on 6% slab gels and
fluorography. B, polarized expression of LPH chimeras in
MDCK cells. Monolayers of MDCK cells expressing LPH-HAwt,
LPH-HAY543, LPH-HAY543/F546, or
LPH-HAY543/R546 were grown on filters. Six days after
confluence, the cells were labeled with [35S]methionine
for 1 h and chased for the indicated times. Chimeras were
immunoprecipitated either from the apical (a) or basolateral
(b) surface with mAb anti-LPH and analyzed by SDS-PAGE on
6% gels and fluorography. C, polarized expression of LPH
chimeras in Caco-2 cells. LPH-HAwt and
LPH-HAY543/F546 were transiently expressed in Caco-2 cells.
Three days after transfection and confluence, the cells were
continuously labeled with [35S]methionine for 18 h.
Chimeras were immunoprecipitated either from the apical (a)
or basolateral (b) surface with mAb anti-LPH and analyzed by
SDS-PAGE on 6% gels and fluorography. D, polarized
expression of wild type and mutant HA forms in Caco-2 cells.
HAwt and HAY543/F546 were expressed by
infection of Caco-2 cells grown on membrane filters 6 day after
confluence. The cells were continuously labeled with
[35S]methionine for 2 h. HAwt and
HAY543/F546 were immunoprecipitated either from the apical
(a) or basolateral (b) surface with anti-HA and
analyzed by SDS-PAGE on 12.5% gels and fluorography.
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To examine whether a similar or a different sorting behavior of these
mutants occurs in an enterocytic cell line, we used colon carcinoma
Caco-2 cells. In these cells endogenous or recombinant LPH is processed
in a similar fashion to its counterparts in the intestinal mucosa and
is targeted to the apical membrane (54). For this purpose the
LPH-HAwt and LPH-HAY543/F546 chimeras as well
as the full-length HA cDNA analogues, i.e.
HAwt and HAY543/F546, were expressed in Caco-2
cells. Fig. 4C demonstrates that the LPH-HA chimeras
examined, LPH-HAwt and LPH-HAY543/F546, behaved in a fashion similar to their analogues in MDCK cells. Here, pro-LPH (230-kDa) as well as the cleaved 160-kDa LPH
species were sorted to
the apical membrane. Likewise, the biosynthesis and sorting of
HAwt and HAY543/Y546 Caco-2 cells were
essentially similar to their counterparts in MDCK cells (Fig.
5D). Thus, HAwt
was predominantly found at the apical and HAY543/F546 at
the basolateral membrane. In essence, the data demonstrate that the
sorting pathways for the LPH-HA chimeras and also for the HA molecule
are similar in MDCK and Caco-2 cells.

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Fig. 5.
Internalization of LPH ct and
LPH-HA chimeras. Duplicates of COS-1 cells expressing
LPH ct (A), LPH-HAwt
(B), LPH-HAY543 (C),
LPH-HAY543/F546 (D), and
LPH-HAY543/F546 (E) were treated with mAb
anti-LPH antibody at 0 °C. One set of transfected cells was kept on
ice as controls (indicated as 0 min), whereas the other set of cells
was chased for 10 min at 37 °C to induce internalization (indicated
as 10 min). All cells were fixed with 2% paraformaldehyde and either
permeabilized with Triton X-100 (denoted as internal, b and
d) or non-permeabilized (denoted as surface, a
and c). The cells were stained with a second fluorescein
isothiocyanate-conjugated antibody to determine the location of
anti-LPH antibodies bound to the expressed proteins.
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Internalization of LPH-HA Chimeras Is Dependent on Mutations in the
Cytoplasmic Domain--
Two possibilities could explain the apical
sorting of the LPH-HA chimeras. The apical sorting signal of pro-LPH
might be dominant over the basolateral signals or the introduced
signals might not function in the context of pro-LPH and were not
accessible to the sorting machinery. One way to examine the latter
possibility is to determine whether these sequences function as
internalization signals in the context of the pro-LPH sequences, as
they do in the context of HA (55). Normally, LPH does not contain an
internalization signal; it escapes coated pits and remains exclusively
at the cell surface. To determine whether the LPH-HA mutants were
internalized, expression plasmids encoding each protein were
transfected into COS-1 cells. Wild type pro-LPH and the tailless
pro-LPH
ct mutant were included as controls, since neither
should be internalized. Transfected COS-1 cells were grown on
coverslips for 48 h post-transfection and were assayed for
endocytosis by indirect immunofluorescence procedures (Fig. 5). Cells
expressing either tailless pro-LPH
ct or
LPH-HAwt that were incubated with monoclonal anti-LPH
antibody at 0 °C (Fig. 5A, a) or at 37 °C
to allow endocytosis to proceed, had predominantly bright staining at
the cell surface. By contrast, all three chimeric proteins containing
internalization signals were internalized. Most notably, cells
expressing the LPH chimeras with HA tails containing the double
mutations, Tyr543/Phe546 and
Tyr543/Arg546 revealed punctate fluorescence
staining after incubation at 37 °C (Fig. 5, D and
E, panel d in each case) but fluorescence that was restricted to the cell surface at 0 °C (Fig. 5, D and
E, panel a in each case). LPH-HAY543
revealed a punctate pattern to a lesser extent (Fig. 5C), as
would be expected from its slower rate of internalization (43, 55).
These data demonstrate that the cytoplasmic sequences of the LPH-HA
chimeras were available to be bound by components of the
internalization apparatus and therefore must be exposed to the cytosol.
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DISCUSSION |
In MDCK cells basolateral signals have been observed to dominate
over apical signals when both are present in the same protein. Many
basolateral proteins become apical proteins when the cytoplasmic basolateral sorting signal is mutated or removed. Basolateral sorting
signals, when transferred to an apical protein, direct the chimeric
protein to the basolateral surface. A single exception to this
generality has been reported recently. When a portion of the ectodomain
of the normally apically expressed neurotropin receptor is removed, the
mutated protein is routed to the basolateral surface (56). However, it
is not yet clear whether the neurotrophin receptor contains a recessive
basolateral signal or whether the deletion in the ectodomain causes
conformational changes in the protein that expose a cryptic basolateral
signal that is dominant when available to interact with the sorting
machinery. To address the question of whether basolateral signals are
always dominant over apical signals, we made a series of mutants of LPH
that contain overlapping basolateral and internalization signals that
differed in the efficiency with which each specified either
internalization or basolateral sorting (8, 42). Because these signals
had dual function, we could determine whether the signals were
available to interact with cellular sorting machinery by monitoring the capacity of the internalization signals to allow endocytosis of LPH,
which normally lacks that capacity. Our results show clearly that a
series of basolateral sorting signals capable of directing the
influenza virus HA to the basolateral surface could not redirect the
LPH, although the internalization signals in those sequences did cause
LPH to be internalized. These results demonstrate that basolateral
sorting signals are not always dominant over apical signals. This
eliminates the possibility that basolateral signals are simply
recognized earlier in the biosynthetic pathway and suggests that
sorting is determined by the relative affinity of various sorting
signals for the sorting machinery.
The nature of the signal or signals in LPH that allow it to be sorted
so efficiently to the apical surface of epithelial cells remains to be
determined. In contrast to basolateral signals, which all appear to be
short amino acid motifs located in the cytosolic domain, three quite
different features have been proposed to be important in apical
targeting of transmembrane and secreted proteins in polarized
epithelial cells (18, 21, 57). Recent data indicate that
oligosaccharides can mediate protein sorting to the apical membrane.
For instance, engineering two N-linked glycosylation sites
into the normally unglycosylated growth hormone leads to a polarized
targeting to the apical membrane of the otherwise unsorted protein
(22). Another type of glycosylation, O-glycosylation, may
constitute a targeting signal to the apical membrane (56). As yet
unidentified apical sorting signals have been proposed to reside in the
ectodomain of a number of proteins, such as intestinal brush-border
proteins (58), but whether these signals depend upon glycosylation or
not is currently unclear. Finally, protein association with
sphingolipid-cholesterol rafts has been proposed as a potential
mechanism in targeting proteins to the apical surface (22). This
association occurs in the trans-Golgi network and results in
detergent-insoluble membranes. Three examples are known of apical
signals residing in transmembrane segments of proteins that associate
with detergent insoluble membranes (59-61), although detergent
insolubility has been shown not to be sufficient for apical sorting in
one of these cases (60).
For LPH, a number of observations strongly support the idea that apical
sorting signals are located in a specific portion of the ectodomain
extending from Ala869 to Ile1646. The LPH
precursor, pro-LPH, is cleaved intracellularly to LPH
(Ser20-Arg868) and LPH
, which is targeted
to the brush-border membrane. Cleavage is not required for sorting,
since the uncleaved pro-LPH precursor is also sorted almost exclusively
to the apical membrane (38). The LPH
is apparently not necessary for
sorting pro-LPH since LPH
expressed individually in MDCK cells is
correctly sorted in a fashion similar to wild type pro-LPH (50).
Deletion of 236 amino acids in the homologous region IV (45) of the
ectodomain that is juxtaposed to the membrane generates a
transport-competent and correctly sorted mutant protein (62).
Importantly, the deletion of this stretch of 236 amino acids eliminates
four potential N-glycosylation sites and the entire
O-glycosylated domain of pro-LPH. Since these substantial
changes in the glycosylation pattern of this mutant do not affect its
sorting behavior, it is likely that neither N- nor
O-linked glycosylation is directly responsible for the sorting of LPH. The cytosolic portion of LPH does not contain targeting
signals, since a tailless LPH mutant is sorted to the apical membrane
with similar fidelity to wild type LPH. Finally, the membrane anchoring
domain of LPH does not have the characteristics of other transmembrane
domains that contain apical sorting signals. LPH is completely soluble
in Triton X-100 at 4 °C and is therefore not associated with lipid
rafts (this study and Ref. 52). As there are other apically sorted
proteins that could not be demonstrated to associate with
detergent-insoluble membrane domains (52, 63), a separate mechanism for
sorting these proteins into a parallel pathway to the apical surface is
possible, or these proteins might associate with detergent-insoluble
membranes with an affinity too weak to be observed experimentally.
Currently, we do not understand why the LPH has stronger apical sorting
signals than does the HA. At least 90% of wild type LPH expressed in
MDCK cells is delivered to the apical membrane compared with 75%
apical sorting of HA expressed in continuous MDCK cell lines (11, 60).
Since there are a number of different ways in which proteins can
interact with apical sorting machinery, it is possible that apical
sorting of LPH is regulated by several signals, which then act
cooperatively to ensure strong association with the apical sorting machinery.