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J. Biol. Chem., Vol. 276, Issue 49, 46597-46604, December 7, 2001
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From the Department of Physiological Chemistry, School of
Veterinary Medicine Hanover, Bünteweg 17, Hanover D-30559,
Germany
Received for publication, August 24, 2001, and in revised form, September 25, 2001
The apical sorting of the small intestinal
membrane glycoprotein sucrase-isomaltase (SI) depends on the presence
of O-linked glycans and the transmembrane domain. Here, we
investigate the role of O-glycans carried by the
Ser/Thr-rich stalk region of SI as an apical sorting signal and
evaluate the spatial requirements for an efficient recognition of this
signal. Several hybrid proteins are generated comprising the unsorted
and unglycosylated protein, the rat growth hormone (rGH), fused to
either the transmembrane domain of SI (GH-SITM), or the
transmembrane and the stalk domains (GH-SISR/TM). Both
constructs are randomly distributed over the apical and basolateral
membranes of MDCK cells indicating that neither the transmembrane
domain nor the O-glycans are sufficient per se
for an apical delivery. Only when a polyglycine spacer is inserted
between the stalk region of SI and the luminal part of rGH in the
GH-SIGly/SR/TM fusion protein does efficient apical sorting
of an O-glycosylated protein as well as a
time-dependent association with detergent-insoluble lipid
microdomains occur. Obviously, the polyglycine spacer facilitates the
accessibility of the O-glycans in
GH-SIGly/SR/TM to a putative sorting receptor, whereas
these glycans are inadequately recognized in GH-SISR/TM. We
conclude that the O-glycans in the stalk region of SI act
as an apical sorting signal within a sorting machinery that comprises at least a carbohydrate-binding protein and fulfills specific spatial
requirements provided, for example by a polyglycine spacer in the
context of rGH or the P-domain within the SI enzyme complex.
The polarity of epithelial cells is characterized by two
functionally and structurally different plasma membrane domains, the
apical and the basolateral. Separated by tight junctions, the two
surfaces contain distinct compositions of proteins and lipids. The
maintenance of polarity requires sorting as well as domain-specific
retention of newly synthesized and recycling proteins (1, 2). Protein
constituents are transported along the secretory pathway to the
trans-Golgi network
(TGN)1 where they are sorted
to either one of these two domains (3-5). Basolateral targeting
generally depends upon the existence of a tyrosine-based cryptic signal
or a di-leucine motif in the cytoplasmic tail of the sorted protein (1,
6). The apical delivery of membrane and secretory proteins is more
complex and utilizes several types of signals suggesting the existence
of multiple binding sites for apical signals in the sorting machinery.
Glycolipid anchors direct proteins to the apical surface of several
types of epithelial cells (7), apparently by associating in the TGN with detergent-insoluble membrane domains enriched in
glycosphingolipids and cholesterol (8, 9). The transmembrane segments
of influenza virus neuraminidase (NA) (10, 11) and hemagglutinin (HA)
specify apical transport, and in the case of HA, several residues
critical for this function have been identified (12).
N-Linked oligosaccharides on some secreted proteins appear
to specify apical transport (13), although this mechanism does not
apply to all secreted proteins (14, 15) and has not been conclusively
demonstrated for membrane-bound proteins (16, 17).
O-Glycosylation is also critically important in the sorting
event of some membrane glycoproteins (18). Specific inhibition of
O-glycosylation of intestinal sucrase-isomaltase with
benzyl-GalNAc
(benzyl-N-acetyl- It is not obvious, however, from these observations whether
O-glycans constitute the sorting signal per se,
or they impose a particular folding determinant in the context of the
actual sorting signal. Analysis of various deletion mutants of SI
demonstrated that O-glycosylation and membrane anchoring of
SI are required for the association of the enzyme with cholesterol and
glycosphingolipid-rich lipid microdomains and subsequent apical
sorting. Moreover, a possible role for an O-glycosylated
Ser/Thr-rich stalk domain that immediately follows the membrane domain
and belongs to the isomaltase subunit in the sorting of SI has emerged.
However, SI comprises two strongly homologous subunits that are both
O-glycosylated and a possible contribution of
O-glycans in the sucrase subunit to the structure of a
putative sorting signal cannot be excluded. The basic aim of this paper
is to assess the requirements needed for an efficient sorting of apical
proteins using the high polarized protein sucrase-isomaltase as a
model. In particular, the role of O-glycans located in the
stalk region of SI and the spatial constraints that influence an
efficient recognition of these structures through putative sorting
elements have been investigated. Using chimeras of the non-polarized
protein model, the rat GH, fused to the stalk transmembrane domains of
SI we could show that O-glycosylation is absolutely required
for apical sorting. However, specific spatial requirements should be
fulfilled for an efficient recognition of these glycans by a putative
carbohydrate-binding protein.
Materials--
Restriction enzymes, Taq DNA
polymerase, ligase, endo- Expression of Chimeras of the Rat Growth Hormone Fused to Various
Domains of SI in MDCK Cells--
Hydrophilic rat GH is an
unglycosylated polypeptide that is randomly secreted in polarized MDCK
cells from the apical as well as basolateral membranes (13). It could
be therefore conveniently used as a reporter gene to analyze the role
of specific putative sorting signals. Previous studies on a possible
role of N-glycosylation in apical sorting have utilized
mutants of this protein, which contained potential
N-glycosylation sites (13). As such the trafficking
randomness of the protein could be abolished and polarized secretion of
the protein through the apical membrane has occurred. Obviously,
N-glycans are implicated in the sorting event either directly being the sorting signal itself or indirectly by generating particular structural determinants in rat GH that constitute the sorting signal. Previous data from our own work have provided ample
evidence for a strong implication of O-glycans in the
sorting of intestinal sucrase-isomaltase (18-20). We examined the role of O-glycosylation in the apical sorting event by making
chimeras comprising unglycosylated rat GH as a reporter gene and the
Ser/Thr-rich stalk domain and the transmembrane domain of human
sucrase-isomaltase. In the wild type SI protein the Ser/Thr-stalk
domain is heavily O-glycosylated and the transmembrane
domain is required for SI to enter into cholesterol/sphingolipid-rich
membrane microdomains (19). Using wild type rat GH in these chimeras
rather than a mutagenized form of rat GH in which potential
N- or O-glycosylation sites are introduced should
provide direct evidence regarding the location of the apical sorting
signal in the stalk region. This is because no alteration in the
structural features of rat GH per se would be expected, and
therefore any influence on the trafficking behavior of this reporter
gene would be directly related to the introduced domain. We first
generated two chimeras. In the first one the cleavable signal sequence
of the type I protein rat GH was eliminated and replaced by the
N-terminal transmembrane domain (TM) of the type II protein SI, which
contains an uncleavable signal sequence (23). The generated construct
comprised the sequences Met1 -Ala32 of SI and
Leu27-Phe216 of rat GH and is denoted
GH-SITM (Fig. 1). The second
construct was designed to directly assess the role of the stalk domain
of SI in sorting and comprised the transmembrane domain and the stalk region of SI (Met1 -Ser60) fused to the
Leu27-Phe216 sequences of rat GH (denoted
GH-SISR/TM) (Fig. 1). These constructs were expressed in
MDCK cells and their transport kinetics and sorting were analyzed. We
excluded wild type rat GH from these studies, because the biosynthesis
and trafficking of this protein are well established (24, 25) and will
be only referred to where appropriate. The expression constructs were
generated as follows. The cDNA of rat GH lacking its own signal
sequence and encoding amino acid residues 27-190 was fused to the
cDNA coding for the cytoplasmic tail (amino acid residues 1-12)
and the membrane anchoring domain of the type II protein human SI
(amino acid residues 13-32), which contains the signal sequence for ER
translocation. The construct was generated by polymerase chain reaction
using the plasmids pSG8-hSI (26) and pSVGH (24) as templates and the
following oligonucleotides: SI 5'HindIII,
5'-GGAAGCTTGCTATGAAATAAGATGG-3'; crGH,
5'-GGGCGGCCGCTAGAAAGCACAGCTGCTTTC-3'; SItmGH,
5'-GTTGTTTTAGCATTACCTGCCATGCCCTTGTCCA-3'; cSItmGH,
5'-CATGGCAGGTAATGCTAAAACAACAATTAAGGCA-3'.
The resulting product was cloned as a
HindIII-NotI fragment into pCDNA3
(Invitrogen, Groningen, The Netherlands) to generate pCDNA3-GHSITM. Similarly, the
pCDNA3-GHSISR/TM encoding the SI cytoplasmic tail,
transmembrane region, and stalk region of SI (amino acids 1-60) fused
to rat growth hormone without the signal sequence (amino acids 27-190)
was generated using the oligonucleotides: SI5'HindIII,
5'-AAGCTTGCTATGAAATAAGATGG-3'; cSIstalkGH,
5'-CATGGCAGGTAATGAATCAGAAGGATTTGTAGTC-3'; SIstalkGH,
5'-CCTTCTGATTCATTACCTGCCATGCCCTTGTCCA-3'; and crGH3'NotI, 5'-GGGCGGCCGCTAGAAAGCACAGCTGCTTTC-3'.
For generation of the plasmid pCDNA3-GHSIGly/SR/TM
encoding eight consecutive glycines inserted between the stalk domain
of SI and the rat GH, an AccIII site was introduced into
pCDNA3-GHSISR/TM by site-directed mutagenesis according
to the QuikChange protocol (Stratagene, Amsterdam, The Netherlands)
using the following oligonucleotides: ACCIII,
5'-CAAATCCTTCTGATTCCGGACCTGCCATGCCCTTG-3', and cACCIII, 5'-CAAGGGCATGGCAGGTCCGGAATCAGAAGGATTTG-3'. In addition to the introduction of the AccIII restriction site, this mutation
resulted in an amino acid substitution of leucine by a glycine at amino acid residue 27 of rat GH. The resulting plasmid was opened with AccIII and dephosphorylated, and a doublestrand linker of
8Gly, 5'-CCGGAGGTGGCGGGGGAGGCGGAGGCG-3', and c8Gly,
5'-CCGGCGCCTCCGCCTCCCCCGCCACCT-3' containing 5'-phosphates were ligated
into the plasmid. The inserted sequence encodes eight glycines and an
alanine located between the final serine belonging to the stalk region
of SI at residue 60 and the first glycine of rat GH at amino acid
residue 27. All the hybrid cDNA sequences were verified by complete sequencing.
Cell Culture and Transfection--
Madin-Darby canine kidney
(MDCK) cells (strain II) were maintained subconfluent in DMEM (Life
Technologies, Inc., Eggenstein, Germany) supplemented with 10% fetal
bovine serum, penicillin (100 units/ml), and streptomycin (100 µg/ml)
at 37 °C in a humidified 5% CO2 incubator.
Transfections were performed using a calcium phosphate-DNA
precipitation procedure (27). To establish stably expressing cell
lines, cells were selected with 0.4 mg/ml G418 (Life Technologies,
Inc.) for 2 weeks after which individual clones were isolated and
screened for expression of the chimeric protein with a polyclonal
monkey anti-mouse GH antibody which cross-reacts with rat GH (22). For
analysis of cell surface polarity, cells were grown on Transwell
filters (24 mm, 0.4 µm) (Becton Dickinson GmbH, Heidelberg, Germany)
for 1 week after confluence. For all other experiments, cells were
grown in 100-mm culture dishes (Greiner GmbH, Frickenhausen, Germany).
Biosynthetic Labeling of Cells, Immunoprecipitation, and
SDS-PAGE--
Stably transfected MDCK cells were biosynthetically
labeled with 80 µCi of L-[35S]Redivue
PRO-MIX (Amersham Pharmacia Biotech, Freiburg, Germany) as described by
Naim et al. (28) either continuously or by employing a
pulse-chase protocol. Here, cells were pulse-labeled for 30 min and
chased for different periods of time with cold methionine. The cells
were solubilized for 20 min at 4 °C with a Nonidet P-40 lysis buffer
(1% Nonidet P-40, 0.1% SDS, 50 mM Tris-HCl, pH 8.0) containing a mixture of protease inhibitors 1 µg/ml aprotinin, 17.4 µg/ml benzamidine, 5 µg/ml leupeptin, and 1 µg/ml pepstatin. The
cell lysates and the media after the biosynthetic labeling were
immunoprecipitated with the polyclonal monkey anti-mouse GH antibody
used at 1:1000 concentration, and the antigen-antibody complex was
captured by protein A-Sepharose essentially as described by Naim
et al. (28). The Immunobeads were washed according to the
procedure described by Thomas et al. (29). Cell surface immunoisolation of rat GH from MDCK cells expressing rat GH and grown
on filters was performed essentially as described by Jacob et
al. (30) for intestinal lactase-phlorizin hydrolase. Here, MDCK
cells were labeled for 4 h with 100 µCi of
[35S]methionine. The protein chimeras were
immunoprecipitated from intact cells by addition of anti-mouse GH to
either the apical or basolateral compartments for 2 h at 4 °C.
After extensive washing to remove the excessive antibody, the cells
were solubilized on the filters by the addition of TX-100 extracts of
non-labeled transfected MDCK cells and the antigen-antibody complex was
captured by protein A-Sepharose. The immunoprecipitates were analyzed
by SDS-PAGE according to Laemmli (31). After electrophoresis the gels
were fixed, soaked in 16% salicylic acid for signal amplification, and
subjected to fluorography, or quantified using a phosphorimaging device (Bio-Rad, Munich, Germany).
Analysis of the Glycosylation Pattern of the Chimeras--
The
presence of N-linked glycans in the various protein chimeras
was analyzed by treatment of the immunoprecipitated biosynthetically labeled proteins with endo H or endo F followed by SDS-PAGE on essentially according to Naim et al. (18). Following
electrophoresis, the radioactive bands were visualized using a
phosphorimaging device (Bio-Rad) or autoradiography. The
presence of O-linked glycans in the protein chimeras was
examined by biosynthetic labeling MDCK cells expressing the various
chimeras in the presence or absence of 6 mM benzyl-GalNAc
(Sigma, Deisenhofen, Germany), an inhibitor of
O-glycosylation (32) as described by Alfalah et al. (20).
Association of the rGH-SI Chimeras with Membrane
Microdomains--
The association of the various protein chimeras with
sphingolipid/cholesterol-rich microdomains was assessed in detergent extractability assays using TX-100 essentially as described before (20). Here, the cells were subjected to a pulse-chase protocol and
solubilized for 2 h at 4 °C in a lysis buffer containing 1% TX-100, 25 mM Tris-HCl, pH 8.0, 50 mM NaCl. The
detergent extracts were centrifuged at 100,000 × g for
1 h at 4 °C, and the supernatant or the detergent-soluble
fraction was retained for immunoprecipitation. The detergent-insoluble
proteins recovered in the pellet were dissolved by boiling in 1% SDS
for 10 min. Thereafter, 10-fold of a buffer containing 1% TX-100 was
added. These and the TX-100-soluble fraction were immunoprecipitated
with the polyclonal anti-mouse GH that recognizes native and denatured
forms of rat GH (22).
Biosynthesis and Processing of GH-SITM and
GH-SISR/TM--
MDCK cells expressing chimeric forms of
rat GH and SI were subjected to a pulse-chase protocol followed by
immunoprecipitation of detergent extracts of the labeled cells with
anti-GH antibodies. Fig. 2A
compares the various GH forms. GH-STTM was found in the cell lysates as a 25-kDa polypeptide, and its molecular form did not
alter during the chase. The culture medium was devoid of a comparable
polypeptide confirming the membrane association of this chimera (not
shown). The chimera comprising GH and the stalk and the transmembrane
domains of SI, GH-SISR/TM, on the other hand revealed two
biosynthetic forms, 28- and 34-kDa species, which displayed a
precursor-product relationship. The 28-kDa polypeptide was revealed at
early chase points and gradually disappeared with a concomitant
appearance of the 34-kDa protein. The 28-kDa protein represents,
therefore, the precursor form of GH-SISR/TM
(pGH-SISR/TM) and the 34-kDa protein its final mature form
(mGH-SISR/TM). The faint but definite
mGH-SISR/TM band appearing already at an early chase point
is reminiscent of rapid kinetics of processing of pGH-SISR/TM to mGH-SISR/TM. Wild type GH is
neither N- nor O-glycosylated; the shift to a
larger size of 34-kDa during chase can be, therefore, assigned
exclusively to the presence of the Ser/Thr-rich stalk domain of SI and
possible O-glycosylation of the chimeric protein. We
therefore examined the glycosylation features of GH-SISR/TM using enzymatic treatments. As expected, neither endo H nor endo F,
which have specificities in cleaving N-linked glycans,
altered the electrophoretic pattern of pGH-SISR/TM or
mGH-SISR/TM (Fig. 2B). We further corroborated
these results by treating cells with benzyl-GalNAc, which specifically
inhibits O-glycosylation. While the precursor
pGH-SISR/TM was not affected upon benzyl-GalNAc treatment,
a significant shift in the size of mGH-SISR/TM could be
observed (Fig. 2C). Together, the results unequivocally
demonstrate that the 34-kDa mGH-SISR/TM polypeptide is
O-glycosylated whereas pGH-SISR/TM is not. By
virtue of the fact that O-glycosylation occurs in the Golgi
apparatus, it is obvious that the chimera GH-SISR/TM has at
least egressed the ER to the Golgi. By contrast to
GH-SISR/TM, the GH-SITM chimera did not undergo
post-translational processing, because the earliest detectable form did
not change throughout the pulse and chase periods. The amino acid
sequence of GH-SITM does not reveal potential N-
or O-glycosylation, and it is unlikely that this chimera is
glycosylated. We confirmed this by employing deglycosylation analyses
with endo H and endo F (Fig. 2B) and by inhibition of
O-glycosylation by treatment of the MDCK cells with
benzyl-GalNAc as described for GH-SISR/TM (Fig.
2C). These treatments did not show any shift in the
apparent molecular weight of GH-SITM, therefore, excluding
that this polypeptide is glycosylated.
Cell Surface Expression of GH-SITM and
GH-SISR/TM--
The biosynthetic labeling experiments have
provided unequivocal evidence for transport competence of
GH-SISR/TM from the ER to the Golgi and an efficient
processing of the pGH-SISR/TM form to
mGH-SISR/TM. We investigated further the fate of
GH-SISR/TM and its transport to the cell surface
particularly emphasizing the polarized sorting of this chimera in MDCK
cells and the possible role played by the stalk and transmembrane
domains of SI in this event. With this in mind, MDCK cells expressing
the chimera GH-SISR/TM were grown on transparent polyester
membranes in multiwell tissue culture plates to allow separate
isolation of the apical and basolateral surface antigens by cell
surface immunoprecipitation. Postconfluent cells were biosynthetically
labeled, and the antigenic material expressed on either surface was
immunoprecipitated by adding anti-GH antibody to the apical or
basolateral compartments. The control employed the chimera, containing
the transmembrane domain and lacking the stalk region
GH-SITM. Fig. 2D shows that GH-SITM
was almost equally distributed over the apical and basolateral
membranes (55-45%) indicative of a default targeting of this chimera
to either membrane. The presence of the stalk region in the chimera GH-SISR/TM increased the targeting of this chimera to the
apical membrane to about 60%. The apical versus basolateral
distribution of this chimera (60-40%) is comparable to the
distribution of a number of proteins described as being targeted by
default or are not sorted in MDCK cells (29, 33). Previous studies have demonstrated that O-glycosylation of pro-SI, particularly
that of its stalk domain, is required for sorting of pro-SI to the apical membrane with high fidelity, with almost 90% of
surface-expressed pro-SI being found at the apical surface (19). It
appears at first glance that the O-glycosylated stalk domain
in the GH-SISR/TM chimera is not sufficient for its
efficient apical sorting. Presumably, O-glycans in the
context of this chimera are not sufficiently accessible or exposed to
putative sorting elements.
The sorting mechanism of pro-SI occurs through its association with
glycosphingolipid- and cholesterol-rich membrane microdomains, and this
association is mediated by O-glycans. GH is a soluble protein and is not associated with lipid microdomains during its entire
life cycle. We examined whether inducing O-glycosylation in
the GH-SISR/TM chimera results in its
association with lipid microdomains and analyzed the detergent
extractability of GH-SISR/TM with TX-100 in pulse-chase
experiments. Fig. 2E shows that both forms of
GH-SISR/TM, pGH-SISR/TM as well as
mGH-SISR/TM, were detergent-soluble and appeared in the
supernatant fraction. The detergent-insoluble pellet that contains
sphingolipids- and cholesterol-rich microdomains was devoid of either
protein form at all chase time points. Therefore, in a sharp contrast
with pro-SI, it appeared at this stage of investigation that, in the
context of GH-SISR/TM, O-glycans neither
constitute a strong apical sorting signal nor do they mediate
association of this chimera with detergent-insoluble membrane
microdomains. One explanation is that a recognition element, a
lectin-like protein for example, may be implicated in the sorting event
and that O-glycans in the chimera are not sufficiently
accessible to this molecule due to steric hindrance imposed by the
globular part of GH.
Biosynthesis, Processing, and Cell Surface Expression of a Chimeric
Protein Containing a Glycine Spacer Inserted between the Stalk Domain
of SI and GH--
Based on the hypothesis above, we inserted a spacer
region comprising eight glycines between the SR of SI and GH,
GH-SIGly/SR/TM, to examine whether this additional space
would improve the access of O-glycans to a putative receptor
and increase thus the apical sorting fidelity. The glycine linker is
expected to be a flexible structure that does not assume a particular
folding determinant within the overall context of GH as predicted from
algorithmic analyses (34). Moreover, a sequence of eight glycines has
not been described in a cell surface protein rendering a possible function of this spacer as a sorting signal very unlikely.
Pulse-chase experiments with cell lines expressing
GH-SIGly/SR/TM revealed after 30 min of pulse one band
corresponding to the expected 28.6 kDa of the unmodified earliest
detectable precursor (denoted pGH-SIGly/SR/TM) (Fig.
3A). This protein was rapidly chased into a 34-kDa polypeptide, the intensity of which reached a
maximum after 2 h of chase and persisted at comparable levels throughout the remaining chase periods (this form will be referred to
as mature or mGH-SIGly/SR/TM). Here again and as described above for the chimeras, GH-SITM and GH-SISR/TM,
the glycosylation pattern of GH-SIGly/SR/TM was analyzed.
As expected, endo H and endo F did not alter the electrophoretic
pattern of either form of GH-SIGly/SR/TM indicating that
the chimera is not N-glycosylated (Fig. 3B). This
result was corroborated by treatment of cells expressing
GH-SIGly/SR/TM with the inhibitor of
O-glycosylation benzyl-GalNAc, which generated a shift in
the size of mGH-SIGly/SR/TM to ~31-32 kDa pointing to
the presence of O-glycans in this fusion protein (Fig.
3C). O-Glycosylation of
GH-SIGly/SR/TM indicates also that this chimera has at
least reached the Golgi apparatus (35). To examine the cell surface
expression and polarized sorting of GH-SIGly/SR/TM, the
MDCK cell line expressing this chimera was grown on membrane filters
and cell surface immunoprecipitation was performed after biosynthetic
labeling. Fig. 3D demonstrates that
mGH-SIGly/SR/TM was found at the cell surface and, more
importantly, that its targeting reached high fidelity levels, because
almost 85% of this chimera was localized at the apical surface.
pGH-SIGly/SR/TM was exclusively recovered intracellularly.
The sorting pattern of mGH-SIGly/SR/TM is comparable to
that of wild type SI in Caco-2 cells (36) or when expressed in MDCK
cells (20). Obviously, the substantial increase in the apical transport
of mGH-SIGly/SR/TM could be attributed to the presence of
the glycine spacer.
As shown above and in sharp contrast to pro-SI,
O-glycosylated mGH-SISR/TM, i.e. the
mutant lacking the glycine spacer, neither enters into
detergent-insoluble membrane microdomains nor is it sorted with high
fidelity to the apical membrane. We asked whether the high sorting
fidelity revealed after introduction of the glycine spacer in the
GH-SIGly/SR/TM chimera is associated with an alteration in
the detergent solubility of GH-SIGly/SR/TM with TX-100.
Pulse-chase analysis revealed that mGH-SIGly/SR/TM, the
O-glycosylated form of GH-SIGly/SR/TM, could be
identified in the TX-100-insoluble fractions at all time points (Fig.
3E), whereas its earliest detectable form, the
non-glycosylated pGH-SIGly/SR/TM precursor, was not. The
presence of the glycine spacer has therefore not only generated a
highly sorted chimera GH-SIGly/SR/TM but has concomitantly
lead to the acquisition of this mutant to detergent insolubility as compared with the spacer-free GH-SISR/TM fusion protein. We
wanted therefore to determine whether the glycine spacer per
se directly alters the detergent insolubility of
mGH-SIGly/SR/TM or, more reasonably, its effect is indirect
in providing sufficient spatial requirements for the
O-glycans in the SISR to interact with putative cellular components implicated in the biosynthesis of the lipid microdomains.
One approach is to inhibit or to influence the
O-glycosylation event and determine whether an alteration in
the detergent extractability has occurred.
GH-SIGly/SR/TM-expressing MDCK cells were biosynthetically
labeled in the presence or absence of benzyl-GalNAc, and the chimeric
protein was assayed for its detergent solubility toward TX-100. Fig.
4A demonstrates that treatment
of cells with benzyl-GalNAc generated a substantial shift in the size
of mature GH-SIGly/SR/TM due to decreased
O-glycosylation. Concomitantly, this form became entirely
soluble in TX-100 and was exclusively retained in supernatant fraction.
The internal control, the non-glycosylated pGH-SIGly/SR/TM, was as expected not affected by
benzyl-GalNAc, and its solubility with TX-100 remained also unchanged
(Fig. 4A). Because the glycine spacer is not glycosylated,
the observed effect could be exclusively attributed to the reduced
O-glycosylation of GH-SIGly/SR/TM, which was
ensued by benzyl-GalNAc. The results demonstrate that the lipid
raft-associated sorting of SI requires the presence of the
O-linked glycans of the stalk. Furthermore, the glycans need
to be in an accessible context for sorting to take place, because
GH-SIGly/SR/TM, but not GH-SISR/TM, was
predominantly found at the apical membrane.
Cell Surface Expression of GH-SIGly/SR/TM in
Benzyl-GalNAc-treated MDCK Cells--
Finally, we wanted to determine
whether the reduction in O-glycosylation and the resulting
failure to associate with membrane microdomains had implications on the
sorting behavior of GH-SIGly/SR/TM. To achieve this goal,
MDCK cells expressing GH-SIGly/SR/TM were treated with
benzyl-GalNAc on membrane filters and the sorting pattern of the fusion
protein was analyzed by cell surface immunoprecipitation. Here a
default targeting to the apical and basolateral membranes of
this glycoform (55%:45%) was discerned when
O-glycosylation was affected in the presence of
benzyl-GalNAc, whereas the normally O-glycosylated species
(control) was targeted predominantly to the apical membrane as shown
above (Fig. 4B).
Recent observations have implicated O-linked glycans as
possible apical sorting signals (14, 18-20). The selective delivery of
intestinal SI to the apical membrane in polarized Caco-2 or MDCK cells
could be attributed to O-glycosidically linked carbohydrates that function in concert with the transmembrane domain of SI in driving
SI to associate with detergent-insoluble lipid microdomains (18-20).
In particular, the heavily O-glycosylated stalk domain of SI
appears to constitute the main apical targeting signal. These
observations, however, could not exclude a possible function of other
heavily O-glycosylated domains in the apical sorting event
of SI. The major focus of this report is therefore to determine whether
the O-glycosylated stalk domain and the transmembrane region
are sufficient to target SI to the apical membrane and possess
therefore the exclusive function as a sorting signal per se.
One approach is to fuse this stalk and the transmembrane domain to an
unglycosylated and unsorted secretory protein. The rat GH fulfills
adequately these criteria, and a chimeric protein containing rat GH
fused to the stalk and transmembrane domains of SI
(GH-SISR/TM) has been therefore used to delineate the
function of these two domains in the sorting process. The
GH-SISR/TM is a transport-competent protein that traverses
the secretory pathway rapidly. The two structural components TM and SR
in GH-SISR/TM endow this protein with characteristics
reminiscent of a membrane-bound and O-glycosylated protein,
indicating a correct integration and processing of these two domains, respectively.
The transport and secretion of rat GH into the external milieu in
polarized MDCK cells occurs randomly, indicating that rat GH does not
harbor specific sorting signals. The conversion of rat GH into a
membrane-bound protein by fusing the TM of SI to the globular domain
(GH-SITM) also does not alter the sorting behavior, thus
indicating that the TM of SI is not equipped with polarized sorting
elements. On the other hand, the TM of SI is a critical component in
the overall sorting event of SI, and its presence constitutes an
absolute requirement for SI to associate with lipid rafts and to be
ultimately sorted to the apical membrane (19). Nevertheless and unlike
the TM of certain integral viral protein, such as the influenza HA (12,
37), neuraminidase (38), and the envelope glycoprotein of simian virus
(39), the TM of SI alone is not sufficient to mediate apical targeting. In fact, an SI deletion mutant containing the TM domain, but lacking another structurally critical domain, the O-glycosylated
stalk region, is sorted randomly in epithelial cells and does not
acquire TX-100 detergent insolubility during its passage through the
TGN (19). The information harbored in the TM of the HA and
neuraminidase, for example, is presumably indirectly utilized for the
apical sorting event. It is most likely essential for the acquisition of these proteins to a particular quaternary structure, such as trimers
or tetramers, and these structures facilitate the association of these
proteins with lipid rafts that function as vehicles to the apical
membrane. In fact, certain mutations in the TM domain of HA strongly
affect its folding properties, including trimerization and
susceptibility to trypsin, and inhibit its association with lipid rafts
and subsequent apical targeting (12). A similar potential role for the
TM of SI cannot therefore be hypothesized, because SI maintains a
monomeric structure throughout its life cycle. Within the SI protein,
the TM domain functions in concert with another critical and essential
region in mediating association of SI with lipid rafts and apical
sorting. Like the TM domain, the heavily O-glycosylated
stalk region (SR) is a necessary but not a sufficient component of the
sorting mechanism. Given the primordial importance of the TM and
SR domains in the context of SI apical sorting, it is surprising at
first glance that these domains in the GH-SISR/TM chimera
are not capable of routing this protein to the apical membrane and
mediating its interaction with lipid rafts. The possibility of altered
structural features in the SR and TM within the context of rat GH is
unlikely to explain the altered sorting behavior. Heavy
O-glycosylation of the stalk region in the
GH-SISR/TM occurs and is expected to generate a rigid
unfolded structure that would behave autonomously in SI as well as in
GH-SISR/TM. Likewise, the successful anchorage of GH-SISR/TM in the membrane through the foreign TM of SI is
reminiscent of a fulfillment of the structural requirements of a type
II protein. Defective or incorrectly processed fused domains can
therefore be excluded as an argument to explain the default sorting of
GH-SISR/TM. The association of SI with lipid microdomains
is known to directly implicate O-glycans of the SR (19, 20)
and occurs probably through an interaction with a specific lectin-type
protein (20) that recognizes these O-linked sugar chains.
One likely explanation for the failure of GH-SISR/TM to
associate with lipid microdomains or rafts lies in an inadequate
recognition of the O-glycans in GH-SISR/TM by a
putative lectin-receptor due to geometrical restrictions upstream of
the SR in GH-SISR/TM. Such restrictions are presumably not
found in the SI molecule. This idea is strongly supported by the data
presented here on the processing and sorting pathway of the
GH-SIGly/SR/TM mutant. In this chimera SR is extended
by eight aliphatic glycine residues fused between the SR and the globular rat GH domain to possibly eliminate a hypothesized geometrical restriction and to ultimately provide a better accessibility of the
O-glycan residues to a putative lectin-like receptor.
The inclusion of the glycine spacer in GH-SISR/TM remains
without effects on the biosynthesis, processing,
O-glycosylation, and transport kinetics of the chimera but
generates dramatic effects on its interaction with lipid microdomains
and subsequent polarized sorting. The glycine-containing
GH-SIGly/SR/TM construct associates with lipid
microdomains and is sorted to the apical membrane with high fidelity
similar to that of the SI molecule (19, 20). The association of
GH-SIGly/SR/TM and many apically sorted proteins with
detergent-insoluble lipid microdomains is transient and is reduced or
terminated upon delivery of the protein to the apical membrane. Also, a
shift from random transport, as in the case of GH-SISR/TM
(60% apical versus 40% basolateral), to a high sorting fidelity of the GH-SIGly/SR/TM chimera (85%) is not
exclusively due to an association of only an additional proportion (in
this case 25%) of the molecules with detergent-insoluble lipid microdomains.
The data presented here could therefore unequivocally underscore the
necessity of a spatial environment such as that endowed by the glycine
spacer for a sufficient exposure and subsequent recognition of
O-glycans in the GH-SIGly/SR/TM. It is
worthwhile to note that a possible role for the glycine spacer in the
sorting and membrane association of the GH-SIGly/SR/TM
chimera can be fully excluded, because inhibition of
O-glycosylation causes randomness in the sorting behavior
and complete detergent solubility of GH-SIGly/SR/TM, despite the presence of the glycine spacer. Furthermore, a set of eight
glycines does not impose a particular structural motif as deduced from
algorithmic predictions (34) and, finally, a multiglycine structural
motif has never been demonstrated to be implicated in the trafficking
events of a membrane glycoprotein. Obviously, structural constraints
upstream of the SR domain in the SI molecule itself do not exist, which
perhaps mask the O-linked glycans or reduce their access to
a putative interacting protein. One major structural domain immediately
upstream of the SR in SI is a 46-amino acid P-domain or trefoil
structural motif that endows the SI molecule with a particular
conformational flexibility near the rigid SR and may play an essential
role in protein-protein or lectin-like interactions. A similar
structure is not present in the immediate vicinity of the SR in the rat
GH construct and in the globular domain of rat GH itself, and this is
perhaps why a spacer is required to fulfill spatial requirements for
binding to a putative lectin-like receptor.
In conclusion, the data demonstrate that O-glycans of the SR
of SI are essential components of the apical sorting event of a
normally unsorted secretory protein. These residues can be considered to directly act as sorting signal and to constitute the
recognition site required for the sorting event to ensue.
It can be excluded that protein-folding determinants endowed by the SR
are responsible for the apical sorting behavior, otherwise the
GH-SISR/TM, which contains the stalk, would have been
correctly sorted; however, this is not the case. Furthermore,
O-linked glycans in the stalk regions of proteins are rigid
autonomous structures that are not expected to influence other
proteinaceous domains, and in fact, inhibition of
O-glycosylation in SI by benzyl-GalNAc does not affect its
folding pattern as judged by its unchanged reactivity toward
trypsin.2
Despite the primordial importance of the O-glycans, they are
not sufficient per se to warrant a correct sorting of
proteins to the apical membrane. It is rather the adequate recognition of the O-glycans that constitutes a critical criterion for
sorting, and this requires auxiliary elements that ultimately ensure
the high fidelity of this event. Along these lines, our data are
reminiscent of the existence of a recognition machinery that at least
comprises a carbohydrate-binding protein and requires specific spatial
requirements provided by the P-domain in SI or a glycine spacer in the
context of rat GH.
A direct interaction of O-glycans with glycolipid membrane
components in immediate proximity can be excluded as a mechanism underlying apical sorting of O-glycosylated
GH-SISR/TM or the glycine-spacer containing
GH-SIGly/SR/TM construct or their association with lipid
rafts, otherwise the O-glycosylated GH-SISR/TM
would have also been interacting with lipid rafts and sorted correctly. It should be noted that the common feature of proteins that utilize O-glycans for their apical sorting is the organization and
clustering of these chains into rigid stalks. Perhaps this is an
important criterion for the strength and functionality of
O-glycans as a signal and may explain why
O-glycosylation does not play a role in sorting when it is
not extensive or sporadically distributed over several protein domains.
The nature, identity, and structure of a putative cellular lectin-like
protein specific for binding residues comprising O-linked chains, such as galactose or N-acetylgalactosamine,
is far from being unraveled. For another type of glycosylation,
N-linked glycosylation, two lectin-like proteins have been
described in TGN and post-Golgi compartments that may recognize these
chains as apical sorting signals on proteins and mediate thus their
incorporation into apical carrier vesicles. One of these is VIP 36, which possesses striking sequence homologies to proteins of the family
of leguminous lectins (40), and the other is the thyroglobulin receptor
(41), a protein with strong affinity toward binding
N-acetylglucosamine in the presence of calcium and at low
pH, i.e. at conditions that prevail in the TGN. Although
thyroglobulin can be excluded as a possible lectin sorter for
O-linked glycans, which do not contain N-acetylglucosamine, VIP 36 is an interesting protein by
virtue of its binding specificities. It binds galactose and
N-acetylgalactosamine residues and can therefore
function as a receptor for both N- and O-linked
glycans. Nevertheless, VIP 36 has been detected not only in apical
transport vesicles but also in basolateral carriers (42, 43). Moreover,
elimination of galactose in a particular mutant MDCK cell line or
through inhibition of N-glycan processing by mannosidase I
and II remains without marked alteration of the sorting pattern of many
N-linked glycosylated membrane-bound as well as secretory
proteins. These observations together do not underscore a
definite role of VIP 36 in the sorting machinery of
N-linked, presumably also, in that of O-linked
glycoproteins. The absence of N-linked carbohydrates from
our rat GH constructs, which may interfere with the folding pattern and
the exclusive presence of O-linked glycans in the SR, should
be helpful in directly analyzing the binding of these constructs to
lectin-like cellular components.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, September 27, 2001, DOI 10.1074/jbc.M108187200
2
N. Spodsberg, M. Alfalah, and H. Y. Naim,
unpublished data.
The abbreviations used are:
TGN, trans-Golgi network;
SI, sucrase isomaltase;
GH, growth
hormone;
rGH, rat growth hormone;
PAGE, polyacrylamide gel
electrophoresis;
benzyl-GalNAc, benzyl-N-acetyl-
Characteristics and Structural Requirements of Apical Sorting of
the Rat Growth Hormone through the O-Glycosylated Stalk
Region of Intestinal Sucrase-isomaltase*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactosaminide) as well as deletion of the potentially O-glycosylated Ser/Thr-rich
stalk domain abolished the high fidelity of apical sorting of SI and resulted in a random transport of the protein to both membranes (19,
20). Likewise, a dominantly O-glycosylated domain juxtapose the membrane of the neurotrophin receptor is presumably implicated in
its apical sorting (14, 21).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-N-acetylglucosaminidase H (endo
H), endo--N-acetylglucosaminidase F (containing
N-glycosidase F) (endo F/GF), and phenylmethanesulfonyl fluoride were purchased from Roche Molecular Biochemicals (Mannheim, Germany). Streptomycin, penicillin, Dulbecco's modified Eagle's medium (DMEM), minimum essential medium, methionine-free DMEM, and
fetal calf serum were purchased from Life Technologies, Inc. (Eggenstein, Germany). L-[35S]Redivue PRO-MIX
(>800 Ci/mmol) and protein A-Sepharose was purchased from Amersham
Pharmacia Biotech (Freiburg, Germany). Acrylamide, ammonium persulfate,
dithiothreitol, 2-mercaptoethanol,
N,N'-methylenebisacrylamide, SDS, TEMED,
and Triton X-100 were acquired from Carl Roth GmbH & Co. (Karlsruhe,
Germany). DEAE-dextran, benzamidine, aprotinin, leupeptin, pepstatin,
molecular weight standards for SDS-PAGE, and trypsin were purchased
from Sigma (Deisenhofen, Germany). Polyclonal anti-mouse GH antibody,
which cross-reacts with rat GH, was a generous gift of Dr. Sinha
(22).
View larger version (14K):
[in a new window]
Fig. 1.
Schematic representation of SI-GH fusion
protein constructs. Structural features of SI are shown according
to previous studies (23, 44). The constructs are type II membrane
proteins (Nin/Cout) containing the uncleavable
signal sequence/membrane anchor of SI. The light-shaded
boxes (CT) represent the 12-amino acid cytoplasmic tail
of SI (amino acid residues Met1-Ser12). The
black boxes (TM) represent the 20-amino acid
transmembrane domain of SI (Leu13-Ala32) SI,
which also functions as signal for translocation into the endoplasmic
reticulum. The white boxes (SR) represent the
O-glycosylated 28-amino acid serine/threonine-rich stalk
region (Thr33-Ser60). The stippled
box (Gly) represents an eight-glycine stretch. The
dark-shaded boxes (rGH) represent the mature rat growth
hormone lacking the cleavable signal sequence
(Leu27-Phe216).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (23K):
[in a new window]
Fig. 2.
Biosynthesis of GH-SITM or
GH-SISR/TM in MDCK cells. A, MDCK cells
stably expressing either GH-SITM or GH-SISR/TM
were biosynthetically labeled for 30 min at 37 °C with
[35S]methionine and chased with 2.5 mM
unlabeled methionine for the indicated times. The cells were
solubilized with 1% TX-100, and the fusion proteins were
immunoprecipitated from the detergent extracts with a polyclonal
anti-mouse GH antibody followed by SDS-PAGE on 12% slab gels and
fluorography. B, MDCK cells stably expressing
GH-SITM or GH-SISR/TM were biosynthetically
labeled for 4 h at 37 °C with [35S]methionine and
processed for immunoprecipitation as in A.
Immunoprecipitates were divided into three parts, which were treated or
not treated with endo H or endo F/GF followed by SDS-PAGE.
C, MDCK cells stably expressing GH-SITM or
GH-SISR/TM were biosynthetically labeled for 4 h at
37 °C with [35S]methionine in the absence or presence
of 6 mM benzyl-GalNAc and further processed for
immunoprecipitation and SDS-PAGE as in A. D, MDCK
cells stably expressing GH-SITM or GH-SISR/TM
were grown on filters for 7 days after confluence and labeled with
[35S]methionine at 37 °C for 4 h. The fusion
GH-SI proteins located at the cell surface were immunoprecipitated from
either the apical (A) or basolateral (B)
membrane. The apical/basolateral distribution was quantified
by scanning of the fluorograms using a phosphorimaging device
(Bio-Rad). n = 3. E, MDCK cells stably
expressing GH-SITM or GH-SISR/TM were labeled
at 37 °C with [35S]methionine for 30 min and chased
with 2.5 mM unlabeled methionine for the indicated times.
The cells were solubilized with 1% TX-100, and the SI-GH fusion
proteins were immunoprecipitated from the detergent-soluble
(S) and detergent-insoluble pellet (P) fractions.
The immunoprecipitates were analyzed by SDS-PAGE on 12% slab
gels.
View larger version (27K):
[in a new window]
Fig. 3.
Biosynthesis of
GH-SIGly/SR/TM in MDCK cells. A, MDCK cells
stably expressing GH-SIGly/SR/TM were biosynthetically
labeled for 30 min at 37 °C with [35S]methionine and
chased with 2.5 mM unlabeled methionine for the indicated
times. The cells were solubilized with 1% TX-100, and the detergent
extracts were immunoprecipitated with a polyclonal anti-mouse GH
antibody followed by SDS-PAGE on 12% slab gels and fluorography.
B, MDCK cells stably expressing GH-SIGly/SR/TM
were biosynthetically labeled for 4 h at 37 °C with
[35S]methionine and further processed for
immunoprecipitation as in A. The immunoprecipitates were
divided into three parts, which were treated or not treated with endo H
or endo F/GF. C, MDCK cells stably expressing
GH-SIGly/SR/TM were biosynthetically labeled for 4 h at 37 °C with [35S]methionine in the absence or
presence of 6 mM benzyl-GalNAc. D, MDCK cells
stably expressing GH-SIGly/SR/TM were grown on filters for
7 days after confluence and labeled with [35S]methionine
at 37 °C for 4 h. GH-SIGly/SR/TM expressed at the
cell surface was immunoprecipitated from either the apical
(A) or basolateral (B) membrane. The
apical/basolateral distribution was quantified by scanning of the
fluorograms using a phosphorimaging device (Bio-Rad). n = 3. E, MDCK cells stably expressing
GH-SIGly/SR/TM were labeled at 37 °C with
[35S]methionine for 30 min and chased with 2.5 mM unlabeled methionine for the indicated times. The cells
were solubilized with 1% TX-100, and GH-SIGly/SR/TM was
immunoprecipitated from the detergent-soluble (S) and
detergent-insoluble pellet (P) fractions. The
immunoprecipitates were analyzed by SDS-PAGE on 12% slab gels.
View larger version (31K):
[in a new window]
Fig. 4.
Effect of benzyl-GalNAc on the association of
GH-SIGly/SR/TM with lipid microdomains and polarized
sorting. A, MDCK cells stably expressing
GH-SIGly/SR/TM were labeled at 37 °C with
[35S]methionine for 4 h in the absence or presence
of 6 mM benzyl-GalNAc. The cells were solubilized with 1%
TX-100, and the detergent-soluble (S) and
detergent-insoluble (P) extracts were immunoprecipitated
with anti-mouse GH followed by SDS-PAGE on 12% slab gels and
fluorography. B, MDCK cells stably expressing
GH-SIGly/SR/TM were grown on filters for 7 days after
confluence and labeled with [35S]methionine at
37 °C for 4 h in the absence or presence of 6 mM
benzyl-GalNAc. The cell surface antigens were immunoprecipitated from
either the apical (A) or basolateral (B)
membrane, and the immunoprecipitates were subjected SDS-PAGE on 12%
slab gels followed by fluorography. The apical/basolateral
distribution was quantified by scanning of the fluorograms using a
phosphorimaging device (Bio-Rad). n = 3.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FOOTNOTES
To whom correspondence should be addressed: Tel.: 49-511-953-8780;
Fax: 49-511-953-8585; E-mail: hassan.naim@tiho-hannover.de.
ABBREVIATIONS
-D-galactosaminide;
endo H, endoglycosidase H;
endo F, endoglycosidase F;
DMEM, Dulbecco's
modified Eagle's medium;
MDCK, Madin-Darby canine kidney cells;
TM, transmembrane;
SR, stalk region;
TX-100, Triton X-100;
NA, neuraminidase;
HA, hemagglutinin;
ER, endoplasmic reticulum;
TEMED, N,N,N',N'-tetramethylethylenediamine.
REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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  R. S. Chmelar and N. M. Nathanson Identification of a Novel Apical Sorting Motif and Mechanism of Targeting of the M2 Muscarinic Acetylcholine Receptor J. Biol. Chem., November 17, 2006; 281(46): 35381 - 35396. [Abstract] [Full Text] [PDF]
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K. Kato, C. Jeanneau, M. A. Tarp, A. Benet-Pages, B. Lorenz-Depiereux, E. P. Bennett, U. Mandel, T. M. Strom, and H. Clausen Polypeptide GalNAc-transferase T3 and Familial Tumoral Calcinosis: SECRETION OF FIBROBLAST GROWTH FACTOR 23 REQUIRES O-GLYCOSYLATION J. Biol. Chem., July 7, 2006; 281(27): 18370 - 18377. [Abstract] [Full Text] [PDF]
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D. Castelletti, G. Fracasso, M. Alfalah, S. Cingarlini, M. Colombatti, and H. Y. Naim Apical Transport and Folding of Prostate-specific Membrane Antigen Occurs Independent of Glycan Processing J. Biol. Chem., February 10, 2006; 281(6): 3505 - 3512. [Abstract] [Full Text] [PDF]
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 B. A. Potter, R. P. Hughey, and O. A. Weisz Role of N- and O-glycans in polarized biosynthetic sorting Am J Physiol Cell Physiol, January 1, 2006; 290(1): C1 - C10. [Abstract] [Full Text] [PDF]
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M. Alfalah, G. Wetzel, I. Fischer, R. Busche, E. E. Sterchi, K.-P. Zimmer, H.-P. Sallmann, and H. Y. Naim A Novel Type of Detergent-resistant Membranes May Contribute to an Early Protein Sorting Event in Epithelial Cells J. Biol. Chem., December 30, 2005; 280(52): 42636 - 42643. [Abstract] [Full Text] [PDF]
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 S. Schuck and K. Simons Polarized sorting in epithelial cells: raft clustering and the biogenesis of the apical membrane J. Cell Sci., December 1, 2004; 117(25): 5955 - 5964. [Abstract] [Full Text] [PDF]
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 T. J. Proszynski, K. Simons, and M. Bagnat O-Glycosylation as a Sorting Determinant for Cell Surface Delivery in Yeast Mol. Biol. Cell, April 1, 2004; 15(4): 1533 - 1543. [Abstract] [Full Text] [PDF]
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