1 Consiglio Nazionale delle Ricerche Cellular and Molecular Pharmacology Center
and Department of Medical Pharmacology, University of Milan, Milan,
Italy
2 Faculty of Pharmacy, University of Catanzaro `Magna Graecia', Catanzaro,
Italy
* Present address: Centre for Study and Research on Obesity, Department of
Preclinical Sciences, L. Sacco Hospital, University of Milan, Italy
Author for correspondence (e-mail:
Nica{at}csfic.mi.cnr.it
)
Accepted 14 January 2002
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Summary |
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Key words: Apical and basolateral sorting, Cytochrome b(5), Madin Darby Canine Kidney cells, N-glycosylation, Syntaxins, Transmembrane domain
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Introduction |
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Because multiple, hierarchically acting sorting determinants are often
present on the same protein (for a review, see
Mostov et al., 2000), their
identification in multidomain polypeptides or oligomers has proved to be
difficult. As simple models to study the role of the TMD in trafficking, we
have been using tail-anchored (TA) proteins. Proteins of this class, which are
defined by a cytosolic N-terminal domain anchored to the bilayer by a TMD
close to the C-terminus (Borgese et al.,
1993
; Kutay et al.,
1993
), provide convenient models for investigating trafficking of
membrane proteins in the absence of a lumenal domain. Using cytochrome b(5)
(b(5)) as a representative TA protein, we were able to uncover for the first
time a role for the TMD in determining the localisation of an ER-resident
protein. Like other ER-restricted TA proteins, b(5) has a relatively short
TMD, a feature that is absolutely required for its ER residency. This was
demonstrated by a b(5) mutant with a lengthened TMD, which escapes from the ER
and reaches the plasma membrane
(Pedrazzini et al., 1996
). The
TMD-dependent sorting mechanism first described for b(5) is shared also by
other TA proteins (Rayner and Pelham,
1997
; Yang et al.,
1997
).
Here, we have pursued our studies of the role of the TMD in sorting of TA proteins. To exclude any effect of the catalytic domain of b(5), we have replaced it with GFP and then systematically investigated the effect of TMDs of different length, hydrophobicity and sequence on the intracellular distribution of the corresponding fusion proteins. Moreover, we have extended our observations to polarised epithelial cells and asked which plasma membrane domain is reached by TA proteins with extended TMDs. We find that GFP fused to the extended tail of b(5) is localised to the basolateral surface of MDCK cells; however it can be partially relocated to the apical domain by addition of a short lumenal sequence containing an N-glycosylation consensus. A similar effect is obtained by replacing the extended tail of b(5) with the TMD of the apically targeted TA protein, syntaxin 3.
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Materials and Methods |
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Constructs with altered TMD length and hydrophobicity (GFP-14, GFP-19,
GFP-22, GFP-25 or GFP-17-HH) (Fig.
1C) were obtained by inserting paired synthetic oligonucleotides,
coding for the desired sequences and designed to have Xho1 and
Age1 compatible extremities, into the basic GFP-17 plasmid cut with
Xho1 and Age1. Similarly, GFP-22-Nglyc and GFP-22-MutNglyc
(Fig. 1C), which bear a
C-terminal tag corresponding respectively to the N-terminal region of bovine
opsin or to the same region mutated in its Nglycosylation consensus sequences,
were constructed by substituting the appropriate oligonucleotide pairs within
the Age1-Xba1 sites of GFP-22. The GFP-ST, GFP-Syn3 and
GFP-Syn4 (Fig. 1C) constructs
were obtained by substituting the entire Xho1-Xba1 region of
GFP-17 with oligonucleotide pairs coding respectively: (i) for the TMD of rat
-2,6-sialyl transferase flanked at the N-terminus by its three Lys
residues, and at the C-terminal end by the downstream polar region of b(5);
(ii) for the 29 C-terminal residues of rat syntaxin 3; (iii) for the 29
C-terminal residues of rat syntaxin 4. The GFP-17-
UPS construct (not
listed in Fig. 1), in which
b(5)'s UPS was deleted, was obtained by substituting the
BamH1-Xho1 fragment of GFP-17 with an oligonucleotide
cassette to obtain the modified linker sequence (
UPS linker;
Fig. 1B) directly attached to
the TMD region.
We also produced two constructs, GFP-22-myc and
GFP-22-MutNglyc-
myc (not listed in
Fig. 1), in which the
corresponding constructs (GFP-22 and GFP-22-MutNglyc) were modified to contain
a linker lacking the myc epitope (
myc linker
Fig. 1B). To this purpose, the
sequence coding for enhanced GFP in pEGFP-N1 (Clontech) was amplified with an
upper primer (5'-ACC-CACCCAAGCTTGCCACCATGGTGAGCAAGGG-3'), covering
nucleotides 673-692 of the plasmid and preceded by an extra sequence
containing a Hind3 site, and a lower primer
(5'-GCGGATCCGCCACCTCCAGATCCACCTCCTCCGGAACCTCCTCCACCCTTGTACAGCTCGTCCATGC-3')
complementary to a sequence comprising nucleotides 1376-1395 of the plasmid
followed by a stretch coding for [GGGS]3 and containing a
BamH1 site The amplified fragment, cut with Hind3 and
BamH1, was used to replace the Hind3-BamH1 fragment
of similarly digested GFP-22 or GFP-22-MutNglyc, to create the corresponding
myc plasmids.
Cell culture, transient transfections and selection of stable
transformants
CV-1 cells were grown and transiently transfected by the
Ca2PO4 method as previously described
(De Silvestris et al.,
1995).
MDCK strain II cells were cultured as previously described
(Borgese et al., 1996). To
obtain clones stably expressing our constructs, cells were cotransfected by
the Ca2PO4 method with GFP-reporter constructs in pCDNAI
and with a plasmid conferring resistance to G-418 (pCB6) in a 20:1 ratio.
After 10 days of selection with G-418 (GIBCO BRL, Gaithersburg, MD) at 0.9
mg/ml, resistant colonies were visually inspected with an inverted microscope
equipped for epifluorescence (Zeiss IM35), and GFP-expressing clones were
picked and expanded in complete medium containing 0.4 mg/ml G-418. For each
construct, all experiments were performed on at least two separate clones,
with identical results.
To obtain confluent monolayers of polarised stably transfected cells with easily detectable levels of our constructs, cells were seeded onto 24 or 6.5 mm diameter polycarbonate Transwell filters with 0.4 µm pores (Corning Costar Corp. Cambridge, MA) at a density of 250,000 cells/cm2 and cultured for 4 days, with daily changes of the medium. At the end of the fourth day, the monolayers were exposed to medium containing 10 mM Na+ butyrate for 8-12 hours. This medium was then replaced with butyrate-free medium, and the cells were returned to the incubator for a further 4 hours before fluorescence or biotinylation analysis (see below).
In some experiments, we used electroporation to transiently transfect MDCK
cells, as described by Rowe et al. (Rowe
et al., 2001). After elimination of dead cells by centrifugation
over a cushion of Ficoll (Ficoll Plus from Pharmacia Biotech Italia, Cologno
Monzese, Milan, Italy), live cells were plated at confluent density onto
Transwell filters and analysed after 4 days of culture.
Antibodies
A monoclonal antibody (mAb R2-15) against the N-terminal peptide of bovine
opsin was a gift of Paul Hargrave (University of Florida, Gainesville, FL). A
mAb (6.23.3) against a 58 kDa basolateral protein of MDCK cells was donated by
Kai Simons (Max Planck Institute of Molecular Cell Biology and Genetics,
Dresden, Germany). Antigiantin antiserum was provided by M. Renz (Institute of
Immunology and Molecular Genetics, Karlsruhe, Germany). Other antibodies were
from the indicated commercial sources: polyclonal anti-GFP, Medical and
Biological Laboratories, Naka-ku Nagoya, Japan; monoclonal anti-Protein
Disulfide Isomerase (PDI), StressGen Biotechnologies, Victoria BC, Canada; rat
monoclonal anti-mouse E-cadherin (clone no. DECMA-1), Sigma Italia, Milano,
Italy; a polyclonal antibody against the N-terminal peptide of human caveolin
I (N-20 antibody), Santa Cruz Biotechnology.
Secondary fluorescent antibodies were from Jackson Immunoresearch Laboratories (West Grove, PA). Biotinylated or peroxidase-conjugated secondary antibodies and fluorochrome-conjugated streptavidin were from Amersham Pharmacia Biotech Italia.
Immunofluorescence and lectin labelling
Paraformaldehyde-fixed cells were permeabilised with Triton X-100 and
processed for immunofluorescence as previously described, with the use either
of secondary fluorescent antibodies or of an amplification protocol based on
biotinylated secondary antibodies and fluorochrome-conjugated streptavidin
(De Silvestris et al., 1995).
Cells were examined either under a Zeiss Axioplan microscope equipped for
epifluorescence or with a Bio-Rad MRC 1024 ES laser confocal microscope. In
double- or triple-labelling experiments, the signal from each fluorophore was
acquired separately, and merged images were created subsequently using Adobe
Photoshop software.
For in vivo immunolabelling of non-permeabilised, GFP-expressing, MDCK cells, the Transwell filter chambers were transferred to the cold room, medium was removed from both chambers and the cells were washed with ice-cold PBS, containing 1 mM MgCl2 and 0.5 mM CaCl2 (Ca2+Mg2+-PBS). The cells were then exposed to anti-opsin mAb at 10 µg/ml in Ca2+Mg2+-PBS, in both the bottom and top chambers, for 1 hour at 4°C to label the extracellular epitope of the Nglyc and MutNglyc constructs. After washing, the monolayers were incubated on both sides with Cy5-conjugated secondary antibodies, diluted in Ca2+Mg2+-PBS, for 30 minutes at 4°C, then washed, fixed, and permeabilised. At this point they were immunostained for E-cadherin using Cy3-conjugated anti-rat secondary antibodies.
To label apical glycoproteins in non-permeabilised MDCK cells with Concanavalin A (Con A), we proceeded as described above, but the cells were incubated for 30 minutes with biotinylated Con A (Pierce, Rockford, ILL) at 2.5 µg/ml in 0.15 M NaCl, 10 mM Hepes-Na+, pH 7.2, 0.1 mM MnSO4, 0.1 mM CaCl2, administered only in the upper chamber, whereas the lower chamber contained the same solution, lacking the lectin. Surface glycoproteins of CV-1 cells grown on coverslips were labelled in the same way. After washing, bound Con A was revealed with Texas-Red-conjugated streptavidin.
Quantitative analysis of confocal Z series
To estimate the percentage of GFP construct localising to the Golgi
complex, transfected CV-1 cells were fixed and stained with anti-giantin
antibodies. Z series of confocal sections at 1 µm intervals were acquired
separately with the FITC and Texas Red filters from 25 randomly chosen cells.
The laser power and gain were adjusted to maintain the signal below saturation
levels. To avoid possible errors owing to bleaching, 50% of the Z series were
acquired from the apical to the basal side and 50% were acquired in the
opposite direction. The Z series were imported as stacks into the Metamorph
program. The mean pixel intensity and area of each cell was measured
throughout the stack. Within the same stacks, the amount of GFP fluorescence
in giantin-positive areas as well as the background pixel intensity in areas
devoid of cells was determined.
To compare the surface distribution of GFP reporter constructs with that of E-cadherin in polarised MDCK cells, Z series of filter grown cells labelled with anti-opsin and anti-cadherin antibodies were acquired as described above. The mean pixel intensity of areas comprising 10-15 GFP expressing cells was measured throughout the stack. After background subtraction, the distribution of label, as a percentage of the total, was calculated. For the anti-opsin antibodies, background for each section was obtained from areas devoid of expressing cells. For E-cadherin, areas inside of the cells, with borders removed by 1 µm from the lateral membrane, were taken as background. Data from eight separate determinations were averaged and standard errors were calculated.
Biotinylation assay
Biotinylation from the apical or basolateral side of filter grown cells was
carried out as described by Zurzolo et al.
(Zurzolo et al., 1994) using
EZ-Link Sulfo-NHS-LC Biotin from Pierce. After quenching of excess biotin with
50 mM NH4Cl, the filters were excised from the Transwell chambers
and incubated for 1 hour at 4°C in 35 mm wells with 1 ml of TNE (150 mM
NaCl, 5 mM EDTA, 50 mM Tris-Cl, pH 7.4 plus protease inhibitors, as in
Pedrazzini et al. (Pedrazzini et al.,
2000
), containing 1% Triton X-100. After complete detachment of
the cells by scraping with a policeman, the lysate was clarified by
centrifugation (700 g for 10 minutes) and then supplemented with 200
µl of a 1:2 slurry of streptavidin agarose beads (Immunopure Immobilised
Streptavidin Gel from Pierce). Attached proteins were solubilised with Laemmli
denaturation buffer and analysed by western blotting, as described in previous
publications (Pedrazzini et al.,
2000
). Bound antibodies were revealed by enhanced
chemiluminescence with reagents from Pierce (Pico Supersignal).
Treatment with Peptide N-glycanase F (PNGase F) and Endoglycosidase H
(Endo H)
MDCK transfectants expressing GFP-22-Nglyc were grown to subconfluence on
10 cm Petri dishes and treated with Na+butyrate as described above.
The cells from one dish were lysed with 0.5 ml TNE + 1% Triton X-100. After
clarification by centrifugation, the Triton soluble proteins were precipitated
with -20°C acid acetone. The pellets, deriving from 1/4 of the initial
lysate, were washed with acetone, resuspended in 40 µl of H2O
and then subjected to digestion with Endo H or PNGase F from New England
Biolabs (Beverly, MA) using the reagents and the protocol supplied by the
manufacturer. After 3 hours of incubation, the samples were supplemented with
Laemmli denaturation buffer and analysed by SDS-PAGE/western blotting.
Sucrose flotation gradients of detergent lysed cells
Subconfluent MDCK transfectants, grown on 10 cm Petri dishes and treated
with 10 mM Na+ butyrate as described above, were scraped with 1 ml
of ice-cold TNE buffer containing 1% Triton X-100 and homogenised with 10
strokes of a Dounce homogeniser. The lysate obtained from two Petri dishes was
immediately diluted with an equal volume of 80% w/v sucrose in TNE and loaded
under a step gradient, consisting of two 4 ml layers of 30% and 5% sucrose in
TNE. After centrifugation at 39,000 g for 19 hours at 4°C in the
SW 41 rotor (Beckman Instruments, Palo Alto, CA), 12 1 ml fractions were
collected from the top. These were supplemented with 2 ml of PBS containing 50
µg/ml bovine Haemoglobin (Sigma) as carrier, and 3 ml 20% trichloracetic
acid. Precipitates were washed with -20°C acetone and then solubilised
with Laemmli denaturing buffer. Equal aliquots of the fractions were analysed
by SDS-PAGE western blotting followed by enhanced chemiluminescence.
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Results |
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Since elongation of the TMD results in an increase in total hydrophobicity,
we considered the possibility that hydrophobicity rather than length was the
important sorting determinant. The hydrophobicity of the TMD region of the
constructs, calculated by adding the value of the scale of Engelman et al.
(Engelman et al., 1986) for
each residue in the sequence between the last upstream charged residue and the
Arg residue in the DPS, is indicated in italics next to the name of each
construct in Fig. 1. To test
whether the effect on sorting was caused by the increased length or
hydrophobicity, we produced a construct, GFP-17-HH, in which no amino acids
were added to the b(5) tail, but four residues were substituted for more
hydrophobic ones, as shown in Fig.
1. This construct, like the ones with lengthened TMD, exited the
ER and was localised to the Golgi region as well as at the cell surface
(Fig. 2).
Rayner and Pelham (Rayner and Pelham,
1997) reported that in the TMD of the yeast ER-resident TA protein
Ufe 1p, the order of the residues, in addition to length, is important for
sorting. The cytosolic half of Ufe 1p's TMD is richer in hydrophilic residues
than the exoplasmic half. Inversion or randomisation of the sequence resulted
in the escape of the mutated Ufe 1p to the Golgi complex
(Rayner and Pelham, 1997
).
b(5)'s TMD has a similar asymmetry; however, a TMD with reversed amino acid
sequence retained the capacity to keep the GFP construct in the ER (not
shown).
The result with the GFP-17-HH construct was unexpected, as its TMD is
similar in length and hydrophobicity to those of resident Golgi type II
transmembrane proteins. These short (17 residue) but hydrophobic TMDs are
both necessary to retain Golgi residents and sufficient to determine Golgi
localisation of a type II reporter protein
(Munro, 1998
). As the TMD of
GFP-17-HH was not completely retained in the Golgi, we investigated whether
the TMD of a bona fide Golgi-resident protein,
-2,6-sialyl transferase,
could cause retention in the context of a TA protein. To this end, the TMD of
b(5) was replaced with the one of sialyl transferase, including a short basic
region on the cytosolic side of the membrane, to obtain GFP-ST
(Fig. 1C). As shown in
Fig. 2, this construct,
although concentrated in the Golgi region, was also capable of reaching the
cell surface. Plasma membrane staining was visible also in cells with low
levels of expression of the construct (arrows). The double Golgi/surface
localisation of GFP-ST is better seen in the confocal images of
Fig. 3A, in which
colocalisation with the Golgi marker giantin (panels d-f) and with surface
glycoproteins labelled with Con A (panels a-c) is apparent.
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To investigate whether the failure of GFP-ST to be completely retained in
the Golgi was because of its overexpression, we determined the percentage of
GFP-ST colocalising with giantin in randomly selected cells showing different
levels of expression of the transfected cDNA. As shown in
Fig. 3B (grey bars), over a
wide range (30 fold) of average GFP fluorescence, cells showed a similar
proportion of Golgi staining, indicating that overexpression was not the cause
of transport of the constructs to the cell surface. Similar results were
obtained with the GFP-17-HH and GFP-22 constructs, although the proportion of
Golgi staining with these constructs was lower, especially for GFP-22 (A.B.
and N.B., unpublished). To see whether Golgi-localised GFP-ST could be
subsequently transported to the cell surface we analysed cells treated with
cycloheximide for 4 hours (Fig.
3B; pink bars). Although Golgi staining remained clearly
detectable, the treatment caused a statistically highly significant decrease
in its relative intensity (for the 25 cells examined 4.74%±0.66
(s.e.m.) versus 8.09%±0.63 for untreated cells, P<0.001 by
Student's t test), suggesting a slow transport of GFP-ST to the cell
surface.
The results of Figs 2 and 3 indicate that low TMD hydrophobicity is the critical factor determining ER residence of TA proteins. Moreover, the TA constructs that escaped from the ER were all capable of travelling to the cell surface, regardless of the length of their TMD, although constructs with short and hydrophobic TMDs (GFP-17-HH and GFP-ST) were also partially retained in the Golgi.
A plasma membrane delivered GFP TA protein (GFP-22) is preferentially
localised to the basolateral surface in MDCK cells
At least two routes for transport from the Golgi complex to the cell
surface exist and are thought to correspond, respectively, to the apical and
basolateral pathways in polarised epithelial cells
(Muesch et al., 1996;
Yoshimori et al., 1996
). To
investigate whether the TMD length of our GFP constructs influenced their
post-Golgi sorting, we turned to polarised MDCK cells and produced clones
stably expressing the plasma-membrane-directed constructs GFP-22 or GFP-25 as
well as the ER-retained construct GFP-17.
A comparison of the localisation of GFP-17 and GFP-22 in MDCK cells is
shown in Fig. 4. As in CV-1
cells, GFP-17 (panel A) appeared confined to intracellular compartments and
showed a distribution similar to that of PDI
(Fig. 4B,C) as expected for an
ER protein. In contrast, GFP-22, although partly intracellular, was also
present at the cell surface (Fig.
4D), together with a 58 kDa basolateral protein
(Keller and Simons, 1998)
(Fig. 4E,F). Confocal x-z
sections indicated that GFP-22 was preferentially localised to the basolateral
membrane of the transfected cells (Fig.
4G) and was not detectable at the apical surface, revealed by
bound lectin administered in the upper compartment of a Transwell filter
chamber (Fig. 4H,I). Identical
behaviour was observed for the GFP-25 construct (not shown).
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N-glycosylation of a C-terminal lumenal tag causes partial relocation
of GFP-22 to the apical surface
Basolateral sorting of membrane proteins is generally mediated by specific
signals that are located in the cytosolic tails
(Matter and Mellman, 1994).
These signals usually dominate over apical determinants, with which they often
coexist within the same polypeptide (for a review, see
Mostov et al., 2000
). On the
other hand, there is also evidence that some transmembrane proteins can reach
the basolateral surface without a signal, simply by exclusion from apical
carriers (Lin et al., 1998
).
We initially considered the possibility that the myc epitope in our
constructs, because of the di-hydrophobic couple Leu-Ile that it contains,
could be responsible for basolateral localisation and produced constructs
without this epitope (
myc constructs)
(Fig. 1), but this deletion did
not have any obvious consequence on sorting
(Fig. 6C). We then investigated
whether GFP-22 carries a dominant basolateral signal in any part of its
sequence by providing it with an apical determinant. This was achieved by
tagging its C-terminus with a peptide corresponding to the N-terminus of
bovine opsin and containing an N-glycosylation site (GFP-22-Nglyc)
(Fig. 1C). N-linked
oligosaccharides are known to favour apical targeting of some secretory
(Scheiffele et al., 1995
) and
membrane (Gut et al., 1998
)
proteins.
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First, we investigated whether GFP-22-Nglyc was glycosylated as expected.
Lysates obtained from stably expressing transfectants were digested either
with PNGase F, with Endo H or mock digested and then analysed by western
blotting with anti-opsin mAbs. As shown in
Fig. 5, two bands were present
in the undigested samples, which were both converted to a lower Mr
(40 kDa) species by PNGase F (lanes 1 and 2). However, the higher
Mr band was resistant to Endo H, whereas the more rapidly migrating
one (arrow in lanes 1 and 4) was sensitive. Thus, GFP-22-Nglyc is efficiently
glycosylated, and most of the N-linked oligosaccharde undergoes Golgi
processing. The Endo-H-sensitive polypeptide probably represents the fraction
of molecules that are in the ER at steady state. It should be mentioned that a
small amount of unglycosylated GFP-22-Nglyc was also present; this species is
detected more efficiently by the anti-GFP antibodies used in the blots of Figs
7 and
8.
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We then analysed the distribution of GFP-22-Nglyc in polarised cells. As
shown in Fig. 6A, this
construct produced a strong apical signal, although its presence on the
basolateral membrane was also detectable. The N-linked glycan, and not the
lumenal amino acid sequence, was responsible for the altered targeting,
because a construct containing a modified opsin tag, in which the
glycosylation consensus site was eliminated by conversion of its Asn residue
to Gln (GFP-22-MutNglyc) (Fig.
1C) reverted to a basolateral localisation
(Fig. 6B). Fig. 6 also shows a vertical
section of a clone expressing a myc construct
(GFP-22-MutNglyc-
myc) (Fig.
6C) whose distribution was similar to that of the corresponding
myc-containing protein (Fig.
6B).
Because GFP-22Nglyc and GFP-22-MutNglyc have an extracellular epitope, it became possible to analyse their surface distribution quantitatively. We used a method based on immunolabeling of the extracellular opsin sequence in non-fixed, non-permeabilised cells, as well as a biochemical biotinylation assay (Fig. 7). Fig. 7A shows confocal sections taken from an apical or lateral plane of a field of cells expressing GFP-22-Nglyc and stained for extracellular opsin, as well as for the adhesion protein E-cadherin. Total GFP fluorescence is shown in panels a and e, and merged images of all three fluorochromes are shown in d and h. The opsin epitope in non-fixed cells was accessible to antibodies both from the apical and from the basolateral surface. The intracellular GFP construct was not labelled by the anti-opsin mAbs (compare e with f and with the merge in h). Fluorescence intensity of the extracellular opsin tag was quantified throughout Z series of confocal sections of monolayers of GFP-22-Nglyc- or GFP-22-MutNglyc-expressing cells, and the distribution was compared with that of E-cadherin. As shown in Fig. 7B, surface GFP-22-Nglyc displayed a prominent peak in the apical region (left panel), which was not apparent in the case of GFP-22-MutNglyc (right panel). With both constructs a relatively low degree of labelling was obtained in the junctional region, probably because of the difficulty of penetration of the antibody in these non-fixed preparations.
The results of the confocal analysis were fully substantiated by the biotinylation assay (Fig. 7C). GFP-22-MutNglyc (lanes 5 and 6), like E-cadherin, was biotinylated only from the basolateral chamber, whereas the glycosylated construct (lanes 3 and 4) was biotinylated also at the apical surface (45% apical and 55% basolateral by densitometry of the blots). Interestingly, within the GFP-22-Nglyc-expressing cells, only the mature glycosylated form was accessible to apical biotin (square bracket), whereas the non-glycosylated form (arrowhead) was biotinylated exclusively from the basolateral side.
Sucrose gradient analysis of TA construct solubility in cold
detergent solution
In order to gain insight into the mechanism underlying the targeting of
GFP-22-Nglyc to the apical surface, we analysed whether it partitions into
cold detergent-insoluble sphingolipid and cholesterol-enriched complexes
(low-density detergent-insoluble
glycosphingolipid-enriched membrane domains DIGs or
rafts), which are thought to be involved in apical sorting
(Simons and Ikonen, 1997).
Sucrose gradient flotation was used to analyse a possible association of
GFP-22-Nglyc and GFP-22-MutNglyc with DIGs. The distribution of the GFP
constructs was compared with that of caveolin I, which partitions into DIGs
(Lisanti et al., 1994
), and
with that of a non-raft protein, E-cadherin
(Fig. 8A).
As expected, cadherin remained in the load zone and was not detectable in
the upper fractions of the gradient even after prolonged exposures of the
blots; in contrast, a sizeable proportion of caveolin floated into the
low-density fractions (21 and 38% of total caveolin in fractions 1-7, as
determined by densitometry of the GFP-22-Nglyc and GFP-22-MutNglyc blots,
respectively). An extremely minor portion of both GFP constructs floated into
the raft-containing fractions. However, close inspection revealed that the
mature glycosylated form of GFP-22-Nglyc was recruited preferentially with
respect to the non-glycosylated form of the same protein and to
GFP-22-MutNglyc. This can be seen more clearly in
Fig. 8B, where the western blot
pattern of an aliquot of fraction four, corresponding to the 30%/5% sucrose
interface, is compared with the one obtained with 1/10th the amount of a
fraction from the load zone (fraction 11). The mature glycosylated form of
GFP-22Nglyc (square bracket) was enriched in fraction four relative to the
Endo-H-sensitive and non-glycosylated polypeptide (arrow and arrowhead,
respectively), and a smaller proportion of GFP-22-MutNglyc than of mature
glycosylated GFP-22-Nglyc floated to the 30%/5% sucrose interface. After
reducing the amounts of load zone fractions (8-12) to 1/10th the amount loaded
for the low density fractions (1-7) (as exemplified in
Fig. 8B) we carried out
densitometric analyses of the blots, by which the percentage of floating
mature glycosylated GFP-22-Nglyc was estimated at 2.8%, whereas the
corresponding value for the Endo-H-sensitive and for the unglycosylated form,
as well as for GFP-22-MutNglyc, was
1%.
Reporter constructs bearing the tails of syntaxin 3 and 4 are sorted
differently in MDCK cells
From the results described above, no differences in post-Golgi sorting
between the 22- and 25-residue TMD TA constructs were detected, and apical
delivery was induced only by addition of an artificial glycosylated lumenal
tag. However, some TA proteins, without lumenal domains, do have a polarised
surface distribution in epithelial cells
(Low et al., 1996;
Delgrossi et al., 1997
). To
investigate whether the TMDs of these endogenous TA proteins are involved in
apical/basolateral sorting, we produced fusion constructs in which GFP was
attached to the tail region of syntaxin 3 or 4, two TA target-Soluble
N-ethylmaleimide sensitive factor Attachment protein Receptors (t-SNAREs),
which are sorted respectively to the apical and basolateral domain in MDCK
cells (Low et al., 1996
). The
distribution of the two constructs, GFP-Syn3 and GFP-Syn 4
(Fig. 1C) was analysed in
polarised monolayers by confocal microscopy. As shown in
Fig. 9, neither of the
constructs had a completely polarised surface distribution; however, GFP-Syn3
produced a pronounced apical signal (Fig.
9A-C), whereas GFP-Syn4 appeared to be more concentrated in the
basolateral domain (Fig. 9D)
where it colocalised with E-cadherin (Fig.
9E,F). These results suggest that the TMDs of syntaxins play a
role in sorting in polarised epithelial cells.
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Discussion |
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TMD-dependent sorting of GFP TA constructs
For our investigation, we chose GFP as a neutral and easily detectable
reporter for the TMD-dependent sorting of TA proteins. As found for other
proteins with this topology, the tendency to escape from the ER of our GFP
constructs correlated with TMD length. Two novel observations in this study
are, however, worth underlining: first, the degree of hydrophilicity rather
than length alone appears to determine the capacity of a TMD to prevent ER
exit; second, all of the constructs that could exit the ER, with TMDs
characterised by different combinations of length and hydrophobicity, were
able to reach the plasma membrane at least to some extent. Even the construct
bearing the TMD of sialyl transferase, previously shown to be the key
determinant for Golgi localisation of type II proteins, in addition to being
concentrated in the Golgi complex as expected, showed a clear surface
localisation. Watson and Pessin (Watson
and Pessin, 2001) reported a Golgi localisation for GFP linked to
the TMD of the Golgi TA protein syntaxin 5. Yang et al.
(Yang et al., 1997
) also
reported that the ER TA enzyme UBC6 was relocated to the Golgi after moderate
lengthening of its TMD, whereas further elongation resulted in transport to
the cell surface. However, in line with our results, a recent paper reports
that a group of Golgi-resident coiled-coil TA proteins requires a sequence of
the cytosolic domain adjacent to the TMD to be retained in the Golgi
(Misumi et al., 2001
).
It has been suggested that short TMDs cause retention of Golgi-resident
proteins because their length in the -helical conformation more closely
matches the width of the Golgi bilayer than that of the plasma membrane
(Bretscher and Munro, 1993
).
The latter, presumably because of its high cholesterol content, is
characterised by increased thickness compared with intracellular membranes.
Although the fundamental role of the length of the TMD in Golgi protein
localisation (Munro, 1998
) is
not questioned, and although we do not know at present why sialyl
transferase's TMD failed to be completely retained in the Golgi in the context
of our TA construct, it is intriguing that the short TMD of a Golgi protein
can reside on the cell surface, notwithstanding the mismatch between
its expected length and the thickness of the plasma membrane. One might
speculate that at the plasma membrane it is confined to microdomains of
reduced thickness, depleted in cholesterol and sphingolipids.
In summary, it appears that for TA proteins containing a neutral reporter as the cytosolic domain, the crucial sorting decision is whether or not to exit the ER. Sorting at subsequent stations of the secretory pathway appears to be looser, as all constructs that exit the ER attain at least some surface localisation. The choice whether or not to remain in the ER depends on the hydrophobicity of the TMD: short, moderately hydrophilic ones determine ER residence, whereas short or long ones with increased hydrophobicity favour ER exit.
In the present study, we have not addressed the mechanism by which polar
TMDs effect ER residence, whether by real retention (exclusion from
anterograde transport vesicles) or retrieval from a downstream compartment by
retrograde traffic. However, in a previous investigation
(Pedrazzini et al., 2000) we
showed that both mechanisms operate to keep b(5) in the ER. Thus, the
moderately hydrophobic TMD of ER-resident TA proteins could be involved in
both retention and retrieval. Moderate hydrophobicity is indeed a requirement
for interaction of TMDs with rer1p
(Letourneur and Cosson, 1998
),
a recycling receptor operating at the ER-Golgi interface
(Sato et al., 2001
). On the
other hand, the membrane of the ER is enriched in short chain, unsaturated
fatty acyl containing phospholipids and is poor in cholesterol and
sphingolipids (for a review, see Sprong et
al., 2001
). This lipid composition, resulting in a particularly
fluid bilayer, might provide a favourable environment for moderately polar
TMDs and contribute to their retention in the ER.
Sorting of GFP TA proteins with extended TMD in polarised epithelial
cells. Effect of an N-linked glycan
Transport from the Golgi complex to the cell surface occurs by at least two
different routes, which are thought to correspond to the apical and
basolateral pathways in polarised epithelial cells
(Muesch et al., 1996;
Yoshimori et al., 1996
). It is
difficult to study these different pathways in non-polarised cells, which lack
clearly demarcated surface domains. To investigate the post-Golgi sorting of
our plasma-membrane-directed TA constructs, we therefore turned to MDCK cells
and found that both GFP-22 and GFP-25 had a polarised distribution on the
basolateral surface.
Basolateral transport of membrane proteins is usually mediated by signals
in the cytosolic tails of these proteins. These signals are generally dominant
over apical ones, which are of more variable nature, and can be in any region
of a transmembrane protein (for reviews, see
Matter and Mellman, 1994;
Mostov et al., 2000
). We
investigated the possible presence of a basolateral signal in our constructs
by adding an apical determinant an N-linked glycan
(Scheiffele et al., 1995
)
to the C-terminus of GFP-22. Because of the recessive nature of apical
sorting determinants, we expected this sorting determinant to be without
effect in the case of a signal-mediated transport of GFP-22 to the basolateral
membrane. Instead, we found that the N-linked glycan fulfilled its targeting
potential quite efficiently, relocating roughly 50% of the construct to the
apical domain. This result is consistent with the idea that GFP-22 and GFP-25
are targeted to the basolateral domain without a signal, simply by exclusion
from apically directed transport carriers. Support for the existence of this
type of exclusion mechanism has been provided from studies on Influenza virus
Hemagglutinin mutants (Lin et al.,
1998
). We cannot, of course, rule out the possibility that our
constructs have a weak basolateral targeting signal, recessive to the N-linked
glycan. This would not be present in the myc epitope, as its deletion
had little effect on basolateral sorting, but could conceivably be present in
the UPS of b(5)'s tail or in GFP itself.
The effect of adding the opsin sequence to GFP-22 was quite striking and
deserves some further discussion. First, the N-glycosylation consensus was
used efficiently, and the N-linked glycan underwent Golgi processing,
confirming that TA proteins reach the surface after translocation of their
C-terminus across the ER membrane and transport through the secretory pathway
(Jäntti et al., 1994;
Kutay et al., 1995
;
Pedrazzini et al., 1996
).
Second, the apical targeting it caused was dependent on glycosylation, as
point mutation of the consensus resulted in a protein that was again targeted
to the basolateral surface. Third, the addition of this extracellular opsin
epitope made it possible to quantitatively evaluate the surface distribution
of GFP-22-Nglyc and GFP-22-MutNglyc by morphology and biochemistry. By both
techniques, the glycosylated version was distributed approximately equally
between the two surface domains. Interestingly, in the same cells, the
fraction of GFP-22-Nglyc that escaped glycosylation was detected only on the
basolateral membrane by biotinylation, indicating that the glycosylated and
non-glycosylated forms of the construct are sorted from each other within the
same cells and ruling out the possibility that apical targeting was an
artefact caused by different expression levels of GFP-22-Nglyc and
GFP-22-MutNglyc.
The role of N-linked glycans in apical targeting has been the subject of
debate (for a review, see Rodriguez-Boulan
and Gonzales, 1999). On the one hand, it has been suggested that
oligosaccharides may be recognised by a lectin receptor involved in apical
transport from the trans Golgi network
(Fiedler and Simons, 1995
;
Scheiffele et al., 1995
). On
the other hand, an alternative proposal
(Rodriguez-Boulan and Gonzales,
1999
) suggests that oligosaccharides play an indirect role in
apical sorting by stabilising proteinaceous apical sorting signals. The role
of N-linked glycans in apical sorting was first unequivocally demonstrated for
a secretory protein (Scheiffele et al.,
1995
) and subsequently reported for three membrane protein
chimeras, which in the absence of glycosylation are trapped in the Golgi
complex (Gut et al., 1998
),
and for a glycosyl-phosphatidylinositol-anchored growth hormone chimera
(Benting et al., 1999
). The
GFP-22-Nglyc construct used in the present study differs from the above models
because the bulk of the polypeptide mass is cytosolic and the lumenal domain
is small (29 residues), barely long enough to allow placement of the
glycosylation consensus at the minimal distance from the bilayer required for
it to be functional (Nilsson and von
Heijne, 1993
). Thus, it seems unlikely that the apical relocation
we observed was an indirect effect of glycosylation caused by either altered
folding of the cytosolic domain or unmasking or stabilisation of a cryptic
apical targeting determinant contained in the opsin tag. Therefore, we believe
that our observations indirectly support the hypothesis of a direct role of
N-linked glycans in apical sorting
(Fiedler and Simons, 1995
) and
that the GFP-22-Nglyc construct could be a useful tool in the hunt for
putative lectins involved in apical sorting.
Like the function of glycans in polarised sorting, the role of
sphingolipid-cholesterol rafts within the exoplasmic leaflet of the Golgi
membrane as platforms for apical targeting
(Simons and Ikonen, 1997) is
also controversial. Partitioning of proteins within these lipid domains is
generally evaluated indirectly by assessing their association with cold
detergent-insoluble low-density material. Such an association has been shown
for glyco-phosphatidylinositol-linked proteins
(Brown and Rose, 1992
), which
are targeted apically in MDCK cells
(Lisanti et al., 1989
), and
also for apical transmembrane proteins (for a review, see
Harder and Simons, 1997
;
Mostov et al., 2000
).
Recently, however, exceptions to this relationship between apical targeting
and detergent insolubility have been reported
(Lin et al., 1998
;
Zheng et al., 1999
;
Benting et al., 1999
;
Lipardi et al., 2000
). In our
system, neither GFP-22-Nglyc nor its non-glycosylated counterpart were
significantly associated with DIG-containing fractions after flotation on
sucrose gradients, although more of the mature glycosylated polypeptide than
the other forms was Triton insoluble. As the proportion of glycosylated
protein recovered in raft-containing fractions was small (<3%), the
relevance of this observation to the apical sorting of GFP-22-Nglyc remains to
be investigated.
Sorting of GFP-syntaxin tail fusion proteins in polarised epithelial
cells
Syntaxins are t-SNAREs that play a central role in recognition and fusion
of vesicles and target membrane (for a review, see
Chen and Scheller, 2001). In
polarised epithelial cells, syntaxins 3 and 4 are enriched at the apical and
basolateral membrane, respectively (Low et
al., 1996
; Fujita et al.,
1998
; Delgrossi et al.,
1997
); this different localisation is thought to contribute to the
specificity of vesicle fusion with these surface domains
(Galli et al., 1998
). However,
the mechanism of specific targeting of the two syntaxin isoforms themselves is
at present poorly understood.
Although the extended TMDs deriving from b(5) in GFP-22 and 25 were incapable of determining apical localisation, we considered the possibility that the syntaxin TMDs might contain information for polarised sorting. Therefore, we expressed two fusion proteins, which resulted from the replacement in our constructs of b(5)'s TMD and DPS with the C-terminal tails of syntaxin 3 or 4, and we observed a clear difference in their localisation in MDCK cells, with the tail of syntaxin 3 determining a more pronounced apical distribution of GFP.
It is now known that the TMDs of SNAREs play an important role in
determining specificity in vesicle-SNARE/t-SNARE interactions
(McNew et al., 2000), as well
as in SNARE localisation along the secretory pathway
(Banfield et al., 1994
;
Rayner and Pelham, 1997
;
Lewis et al., 2000
;
Watson and Pessin, 2001
). The
result reported here demonstrates yet another role for syntaxin TMDs. Although
it is likely that the cytosolic domains of syntaxin 3 and 4 also contain
targeting information, as reported for other SNAREs
(Banfield et al., 1994
;
Grote et al., 1995
;
Watson and Pessin, 2000
), our
results indicate that the TMDs contribute to the final polarised distribution
of the two syntaxins.
At present, we do not know what feature of the two syntaxin TMDs determine their differential targeting. The two sequences are of similar length and differ only slightly in hydrophobicity; both are considerably more hydrophobic than the TMD of GFP-25 (total hydrophobicity 62.6 and 57.7 for syntaxin 3 and 4, respectively, calculated as explained in the legend to Fig. 1). It is possible that the slightly higher hydrophobicity of syntaxins 3's TMD or the abundant hydroxylated residues in the one of syntaxin 4 (Fig. 1C) are important elements in sorting. The GFP TA constructs we have described here should offer a good starting point to investigate the features that define the sorting role of the TMDs of these two syntaxin isoforms.
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