From the Department of Physiology II, University of Freiburg, Hermann-Herder-Strasse 7, 79104 Freiburg, Germany
Received for publication, December 2, 2002, and in revised form, February 10, 2003
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ABSTRACT |
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Traffic of integral membrane proteins
along the secretory pathway is not simply a default process but can be
selective. Such selectivity is achieved by sequence information within
the cargo protein that recruits coat protein complexes to drive the
formation of transport vesicles. A number of sequence motifs have been
identified in the cytoplasmic domains of ion channels that regulate
early trafficking events between the endoplasmic reticulum and the
Golgi complex. Here, we demonstrate that the following trafficking step from the Golgi compartment to the plasma membrane can also be selective. The N-terminal domain of the inward rectifier potassium channel Kir2.1 contains specific sequence information that is necessary
for its efficient export from the Golgi complex. Lack of this
information results in accumulation of the protein within the Golgi and
a significant decrease in cell surface expression. As similar results
were obtained for the N terminus of another Kir channel subfamily
member, Kir4.1, which could functionally substitute for the Kir2.1 N
terminus, we propose a more general role of the identified N-terminal
domains for post-Golgi trafficking of Kir channels.
The type and number of ion channels expressed in the plasma
membrane are major determinants of a cell's excitability and its capability of vectorial ion transport. These parameters can obviously be regulated on the transcriptional and translational level. In addition, growing experimental evidence indicates that ion
channel proteins can contain intrinsic sequence motifs that control
surface expression by regulating distinct intracellular trafficking
steps (1-3).
Ion channels are sorted via the secretory pathway. After synthesis in
the endoplasmic reticulum
(ER),1 they are forwarded to
the Golgi complex from which they are finally transported to the plasma
membrane. Having reached the cell surface, they might be endocytosed
and either recycled back to the plasma membrane or diverted to
degradation. Protein trafficking between the various intracellular
compartments is accomplished by transport vesicles, which form at sites
to where certain coat proteins have been recruited, a process that
seems rather selective (4-7). Three main types of protein coats have
been identified; clathrin coats vesicles that mediate protein transport
within the endocytic membrane system (8, 9), whereas the coat protein
complexes COPI and COPII are involved in vesicle formation for
anterograde and retrograde protein transport of the early secretory
pathway between the ER and the Golgi complex (10-12). Besides these
classic coat protein complexes, other types of selective vesicular
carriers can be formed, for instance by the clathrin-independent
adaptins AP-3 and AP-4 or the retromer complex (13, 14). Selective recruitment of coat proteins is based on sequence information within
the cytoplasmic domains of the respective cargo proteins. Thus, the
C-terminal amino acid sequence YXX Here, we show that surface expression of the mammalian inward rectifier
potassium channel Kir2.1 is regulated by its cytoplasmic domains at
distinct steps of intracellular protein transport. Whereas its C
terminus bears a previously identified ER export motif (16, 17),
sequence information contained in its N terminus is necessary for
post-Golgi trafficking to the plasma membrane. This N-terminal sequence
information and its function in Golgi export are conserved in Kir4.1,
suggesting that it is of general relevance for Kir channel trafficking.
Gene Construction--
N-terminal fusion constructs of Kir
channel subunits with enhanced green fluorescent protein (EGFP) were
designed by inserting the respective cDNA in-frame into the EGFP-C1
eukaryotic expression plasmid (Clontech) using the
EcoRI and BamHI restriction sites. Channel
mutants were constructed by PCR with oligonucleotides carrying the
desired mutations. A chimera of the Kir4.1 N terminus and Kir2.1 was
constructed by fusing amino acids 1-59 of Kir4.1 to Kir2.1 at amino
acid position 77 using its unique SalI restriction site. An
interior deletion of amino acids 57-69 in Kir2.1 was made following a
site-directed mutagenesis protocol as described (22). The hemagglutinin
(HA) epitope was introduced into the extracellular domain of Kir2.1 at
amino acid position 116. All PCR-derived constructs were verified by sequencing.
Cell Culture and Transfection--
Opossum kidney (OK) cells
(American Type Culture Collection) and tsA201 cells (kindly provided by
P. Dallos, Northwestern University, Evanston, IL) were grown
in Dulbecco's modified Eagle's medium-F12 and Dulbecco's modified
Eagle's medium (Invitrogen), respectively, supplemented with
10% fetal calf serum and 1% penicillin/streptomycin (Invitrogen) at
37 °C and 5% CO2. At ~ 80% confluence, the
cells were transfected with the respective cDNAs using Effectene
reagent (Qiagen) following the supplier's directions. The Golgi
complex was identified by expressing a fusion construct consisting of the 81 N-terminal amino acids of the Golgi resident enzyme
1,4-galactosyltransferase (the cDNA was kindly provided by M. Fukuda, The Burnham Institute, La Jolla, CA) and red fluorescent
protein DsRed-2 (Clontech). In addition, cells were
incubated with 10 µg/ml brefeldin A (BFA; Sigma), which reversibly
fuses the Golgi compartment with the endoplasmic reticulum.
Immunocytochemistry--
For immunocytochemical detection of the
HA epitope, transfected cells were fixed in 4% paraformaldehyde in
phosphate-buffered saline (PBS) for 15 min at 4 °C and pretreated
with 10% normal goat serum (NGS) in PBS with 0.05% Triton X-100
(PBS-T) for 1 h at room temperature to block unspecific antibody
binding. Then they were incubated with a monoclonal anti-HA antibody
(Santa Cruz Biotechnology) diluted 1:200 in 2% NGS/PBS-T for 1 h
at room temperature. Immunoreactivity was visualized by a goat
anti-mouse IgG secondary antibody conjugated to Cy-3 (1:1000 in 10%
NGS/PBS-T). To detect selectively the population of HA-tagged channels
expressed on the cell surface, antibody staining was performed in
vivo without the use of detergents. Antibody incubation was
carried out before fixation in serum-free medium for 15 min at 37 °C
for the primary and secondary antiserum, respectively.
Immunoblot Analysis--
Two days after transfection, cells were
lysed in TEEN-T (50 mM Tris pH 7.6, 1 mM EDTA,
1 mM EGTA, 150 mM NaCl, 1% Triton X-100, and
Complete® protease inhibitor mixture (Roche Applied
Science)) for 30 min on ice. Nuclei and cell debris were
pelleted by low-speed centrifugation at 500 × g for 5 min at 4 °C. The supernatants were separated by reducing SDS-PAGE on
a 10% polyacrylamide gel, transferred onto polyvinylidene difluoride
membranes, and incubated with a mouse monoclonal anti-GFP antibody
(1:5000; Clontech). Following incubation with a
secondary horseradish peroxidase-conjugated anti-mouse IgG antiserum
(1:2500; Santa Cruz Biotechnology), labeled proteins were detected
using ECL-plus reagent (Amersham Biosciences).
Isolation of Detergent-resistant Membrane Fractions--
Lipid
rafts were isolated as described (23). Cells were homogenized in TEEN
(pH 7.6), and cell debris was pelleted by low-speed centrifugation at
500 × g for 5 min at 4 °C. A small fraction of the
supernatant was used to check for equal expression levels of the
respective proteins by immunoblot analysis. Crude membranes were
collected by high-speed centrifugation at 100,000 × g
for 1 h at 4 °C. They were resuspended in 200 µl of TEEN (pH
11) containing 1% Triton X-100 and incubated on ice for 30 min.
Subsequently, the samples were adjusted to 40% sucrose/TEEN overlaid
with 1.5 ml of 36% sucrose/TEEN and 2 ml of 10% sucrose/TEEN.
Following centrifugation at 100,000 × g for 16 h
at 4 °C, six 750-µl fractions were collected from the top, diluted
with 3.5 ml of TEEN (pH 11), and centrifuged again at 100,000 × g for 1.5 h at 4 °C. Triton X-100-insoluble proteins
in the pelleted fractions were resuspended in 2× SDS sample buffer and
separated by reducing SDS-PAGE as above. Successful isolation of lipid
rafts was verified by coexpression of the cell adhesion molecule NCAM
140 (24), which neither interacted with Kir2.1 in coimmunoprecipitation
experiments or affected its subcellular distribution (data not shown).
The cDNA coding for rNCAM 140 and a polyclonal anti-NCAM
antiserum were kindly provided by M. Schachner.
Quantification of Surface Expression--
Two different
experimental approaches were used to quantify plasma membrane
expression of Kir2.1 and the truncation constructs. First, 120 (n) cells of two independent transfections for each experimental group were judged by two investigators without knowledge of the experimental history using the following criteria: ++, strong
plasma membrane fluorescence; +, weak plasma membrane fluorescence; and
Imaging--
Cells were imaged with a confocal laser-scanning
microscope (LSM510, Zeiss) using the following excitation wavelengths
and filter settings: EGFP, excitation 488-nm argon laser/emission BP5
05-530 nm; Cy-3, excitation 543-nm helium laser/emission LP 560 nm.
Kir2.1 Trafficking Is Controlled by Its Cytoplasmic Domains at Two
Distinct Sorting Steps--
As shown in Fig.
1A, the cytoplasmic N- and
C-terminal domains of the inward rectifier potassium channel Kir2.1
regulate surface expression of the channel protein at distinct steps of
the intracellular trafficking pathway. Upon heterologous expression in
OK cells, GFP-fused wild type Kir2.1 (GFPKir2.1) was
predominantly expressed in the Golgi complex and the plasma membrane
(17). Disruption of the previously identified ER export motif in the
C-terminal domain of Kir2.1 (GFPKir2.1-E377/379A) resulted
in accumulation of the channel protein in the ER and significantly
reduced both Golgi and plasma membrane expression. A similar loss of
surface expression was found in a Kir2.1 mutant lacking the N-terminal domain (GFPKir2.1 N-terminal Control of Golgi Export Is Conserved among Other Kir
Channel Subfamilies--
We then investigated whether the need of a
N-terminal domain for post-Golgi trafficking was a broader principle
among other Kir family members. GFP-fused Kir4.1 channel subunits
(GFPKir4.1), which lack the ER export motif found in Kir2.0
channels, showed a homogenous distribution within the ER upon
heterologous expression (Fig. 2).
Insertion of the Kir2.0 ER export sequence (-FCYENEV-) into the Kir4.1
C terminus (GFPKir4.1-export) redistributed the
channel protein both into the Golgi complex and the plasma membrane.
Intriguingly, deletion of the N terminus of
GFPKir4.1-export abolished plasma membrane
fluorescence but left the protein accumulation in the Golgi unaffected,
similar to what had been observed for GFPKir2.1-
To address the question whether specific sequence information within
the N termini of the two Kir channel subunits directed their post-Golgi
trafficking, we designed various deletion mutants and chimeric
constructs and quantified their plasma membrane expression. As can be
derived from Fig. 3, two stretches of
amino acids were identified in the N termini of Kir2.1 and Kir4.1,
respectively, that were necessary for reliable surface expression of
the channel proteins. The deletion of up to 43 N-terminal amino acids
did not affect the predominant localization of GFPKir2.1 to
the plasma membrane. However, the deletion of 46 or more N-terminal
amino acids completely changed its subcellular distribution, resulting in a significant loss of membrane expression and increased accumulation of protein in the Golgi compartment. As an interior deletion of only 13 amino acids also showed reduced surface expression
(GFPKir2.1- Basic Residues Are Critical for Post-Golgi
Trafficking--
Alignment of the identified N-terminal domains
reveals a strikingly high number of basic, positively charged residues
such as arginine and lysine (Fig.
4A). Some of these positive
charges are highly conserved throughout the Kir channel subfamilies.
When we neutralized these residues by mutation to alanine
(GFPKir2.1-R44A,R46A,R67A-K49A,K50A-H53A), the
mutant channel accumulated within the Golgi complex and could not be
detected immunocytochemically on the cell surface anymore, which
demonstrates a critical role of these residues in post-Golgi trafficking (Fig. 4B). This was somewhat surprising, because
basic, positively charged residues have hitherto been implied in
retrograde rather than anterograde sorting signals. Motifs such as
NH2-XXRR-, -RXRR-,
-KKXX-COOH, and -KDEL-COOH function typically as ER
retention/retrieval signals, whereas the known anterograde sorting
signals that favor ER export share the common feature of one or more
diacidic, negatively charged motifs (-DXE-) often neighbored
by hydrophobic residues (1, 3, 19, 30-32). To further narrow down the
trafficking motif, we neutralized single positive charges
in the Kir2.1 N terminus. Whereas single mutations of Arg-44 and
Arg-46 to alanine did not significantly affect surface
trafficking, the double mutant R44A,R46A accumulated in the Golgi
complex, and its surface expression was significantly reduced (Fig.
4C). Though these observations underline a pivotal role for
Arg-44 and Arg-46 in post-Golgi trafficking of Kir2.1, a contribution
of other residues downstream of Arg-46 cannot be excluded, particularly
as the interior deletion construct GFPKir2.1-
Vesicle formation for surface membrane transport does not only involve
the recruitment of coat proteins by the respective cargo proteins but
also the direct interaction of the coat complexes with membrane
phospholipids (33). Thus, the clathrin coat accessory proteins AP180
and epsin have been shown to specifically interact with the negatively
charged head groups of phosphatidylinositol-4,5-bisphosphate (34, 35).
Phosphoinositides are not evenly distributed throughout cellular
membranes but are enriched in lipid raft-like domains, which could
hence represent preferential sites for transport vesicle formation (33,
36, 37). We hypothesized that the cluster of positive charges
identified in the N-terminal domain of Kir2.1 could facilitate Golgi
export by conferring lipid raft localization of the channel via
electrostatic interaction with the negatively charged head groups of
phospholipids. Density centrifugation revealed that wild type
GFPKir2.1 is partially associated with lipid rafts (Fig.
4D), similar to what had been shown previously for a tandem Kir3.1/3.2 channel (23). However, neutralizing the respective charges
in the Kir2.1 N terminus, which had led to the loss of surface
expression, did not disrupt lipid raft association (Fig. 4D). These data exclude a crucial role of these residues in
targeting the channel protein into lipid domains. We assume that the
identified sequence will instead serve as a recognition motif for a
protein coat complex mediating vesicle transport from the Golgi complex to the cell surface.
In summary, we demonstrate that Golgi export of the mammalian Kir
channel subunit Kir2.1 depends on sequence information within its
cytoplasmic N terminus without playing a role in channel
multimerization. We propose that the anterograde transport of ion
channel proteins can be selective not only at the level of ER export
but also at a later stage of the secretory pathway, i.e.
from the Golgi complex to the plasma membrane. Selectivity will most
likely be achieved by coat or mediating adaptor proteins that recognize
sequence information of the cargo protein and then initiate vesicle
formation. The diversity among the signals identified to date might
enable the cell to differentially control surface expression of
different channel proteins at different stages of intracellular transport.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(with X
being any amino acid and
being a bulky, hydrophobic residue)
or di-leucine motifs favor clathrin-mediated endocytosis of membrane
proteins (9), C-terminal di-lysine motifs have been shown to bind COPI (15), and a growing number of N- and C-terminal diacidic motifs render
selective ER export, most likely by binding to COPII coat complexes
(16-19). However, it is still a matter of debate whether sequence
information also facilitates protein transport between the Golgi
compartment and the cell surface or whether this step of the secretory
pathway is constitutive (20, 21).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
, no plasma membrane fluorescence. In a second approach, all deletion
constructs were expressed with extracellular HA epitopes, and surface
expression was quantified by fluorescence intensity measurements of
anti-HA immunocytochemistry without the use of detergents. For each
construct (two independent transfections), pixel intensity values
corrected for background were integrated over 10 areas of 0.2116 mm2 each (Scion Image 4.02). Translation efficiencies of
the constructs were similar as shown by immunoblot analysis (Fig.
3).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1-76) as confirmed by extracellular
epitope tagging (Fig. 1B). In this case, however, the
channel protein accumulated in a juxtanuclear compartment that we
identified as the Golgi complex by the following. (i) Incubation of
transfected cells with the fungal Golgi toxin BFA led to a reversible
redistribution of GFPKir2.1
1-76 to the ER; and (ii) we
found colocalization of GFPKir2.1
1-76 with the red
fluorescent protein DsRed fused to the targeting sequence of the
Golgi-resident enzyme galactosyltransferase I (25) (Fig.
1C). The same observations were made upon
heterologous expression of the respective constructs in tsA201 cells,
another epithelial cell line (data not shown). An effect of the
N-terminal GFP fusion on the sorting behavior of the mutant channel was
excluded because Kir2.1
1-76 tagged with an extracellular HA epitope
and detected by immunocytochemistry showed an identical subcellular localization as GFPKir2.1
1-76 (data not shown). As the
N terminus of Kv potassium channels is thought to be involved in
subunit assembly (26-28), we tested whether deletion of the 76 N-terminal amino acids in Kir2.1 would affect its intracellular
trafficking simply by disturbing channel assembly. Upon coexpression of
HA-tagged Kir2.1 (Kir2.1-HA) and GFPKir2.1
1-76, both
channel subunits did not only colocalize, but Kir2.1-HA was even able
to rescue GFPKir2.1
1-76 into the plasma membrane,
thereby excluding a necessary role of the Kir2.1 N terminus in channel
multimerization (Fig. 1D). This observation is supported by
the earlier findings of Tinker et al., who
assigned the second transmembrane segment and a proximal C-terminal
region to be the assembly domains of Kir channels, with N-terminal
deletions of up to 80 amino acids not affecting coimmunoprecipitation
with homomeric wild type subunits (29). Taken together, whereas the C
terminus of Kir2.1 contains sequence information regulating early
protein trafficking between the ER and the Golgi complex (16, 17), its
N terminus seems to be necessary for later trafficking steps,
i.e. the export from the Golgi complex (Fig.
1E).
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Fig. 1.
The cytoplasmic N- and C termini regulate
surface expression of Kir2.1 at distinct steps of the secretory
pathway. A, representative confocal images of OK
cells expressing GFPKir2.1 and indicated mutations.
B, deletion of the Kir2.1 N terminus
(GFPKir2.1 1-76) leads to a loss of cell surface
expression as determined by extracellular epitope-tagging.
Permeabilization of the cell membrane with Triton X-100 (TX)
is necessary for immunocytochemical detection of the HA-epitope
inserted into the extracellular domain of
GFPKir2.1
1-76. C,
GFPKir2.1
1-76 accumulates in the Golgi complex, which
was identified by the following: (i) BFA-induced redistribution to the
ER; and (ii) colocalization with DsRed-2 fused to the targeting
sequence of galactosyltransferase I (Golgi-DsRed).
D, deletion of the Kir2.1 N terminus does not disturb
channel subunit assembly. Representative confocal images of OK cells
coexpressing GFPKir2.1
1-76 (green) and
Kir2.1-HA detected by immunocytochemistry (red) are shown.
The overlay shows not only colocalization of the two channel subunits
but also rescue of GFPKir2.1
1-76 into the plasma
membrane by Kir2.1-HA. E, schematic drawing of Kir2.1 and
the localization of the proposed anterograde sorting signals for export
from the ER (yellow) and the Golgi complex
(red).
1-76.
Moreover, the Kir4.1 N terminus was able to substitute for the Kir2.1 N
terminus and rescued surface expression of
GFPKir2.1
1-76 (Fig. 2).
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Fig. 2.
The N terminus of Kir4.1 can substitute for
the post-Golgi trafficking effect of the Kir2.1 N terminus.
Representative confocal images of OK cells expressing
GFPKir4.1 and indicated mutations. Note that a deletion of
the Kir4.1 N terminus reduces plasma membrane fluorescence of
GFPKir4.1-export
(Kir4.1-export- 1-59), and fusion of the Kir4.1 N
terminus to Kir2.1 is sufficient to restore surface expression of
GFPKir2.1
1-76 (4.1(1-59)/2.1).
57-69), we conclude that it is not the
length of the N terminus but the specific sequence information that is
necessary for proper post-Golgi targeting of Kir2.1. A similar effect
was observed for the Kir4.1 N terminus, which rescued
GFPKir2.1-
1
76 into the plasma membrane.
Here, membrane fluorescence remained unaffected up to a deletion of 20 N-terminal amino acids but was lost if 40 residues were deleted.
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Fig. 3.
The N termini of Kir2.1 and Kir4.1 contain
specific sequence information that regulates Golgi export of the
proteins to the plasma membrane. A, schematic drawing
of the transfected N-terminal deletion and chimeric mutants.
B, representative confocal images of OK cells expressing the
indicated wild type and mutant channel proteins as GFP fusions.
C, quantification of plasma membrane fluorescence of the
indicated channel mutants (see "Experimental Procedures"). ++,
strong plasma membrane fluorescence; +, weak plasma membrane
fluorescence; , no plasma membrane fluorescence. D,
quantification of cell surface expression of the indicated mutants by
extracellular epitope-tagging (see "Experimental Procedures").
Asterisks mark significant differences from wild type Kir2.1
(p < 0.01, unpaired Student's t test).
Total protein expression of the channel mutants was verified to be
similar by immunoblot analysis. C, untransfected control;
wt (wild type), Kir2.1; lane 1, Kir2.1
1-20;
lane 2, Kir2.1
1-43; lane 3, Kir2.1
1-46;
lane 4, Kir2.1
1-70; lane 5, Kir2.1
57-69;
lane 6, 4.1(1-59)/2.1; lane 7, 4.1(21-59)/2.1;
lane 8, 4.1(41-59)/2.1.
57-69,
which lacks only one positively charged residue (Arg-67), also showed a
loss of surface expression (Fig. 3). Alternatively, impaired surface
trafficking of GFPKir2.1-
57-69 could result from a
conformational change in the N-terminal domain that disturbs the
accessibility of upstream-located positive charges.
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Fig. 4.
Positive charges in the Kir2.1 N terminus are
critical for its cell surface expression. A, alignment
of indicated N-terminal regions of Kir2.1 with other Kir channel
subfamily members in the mouse. Residues conserved between Kir2.1 and
Kir4.1 are indicated by vertical bars. Positively
charged residues conserved among all Kir subfamilies are in
boldface print. B, neutralizing the conserved
positive charges in the Kir2.1 N terminus
(GFPKir2.1-R44A,R46A,R67A-K49A,K50A-H53A) leads to
accumulation of the protein in the Golgi complex and a loss of cell
surface expression as determined by extracellular epitope-tagging. Note
that permeabilization of the cells with Triton X-100 (TX) is
necessary to detect the HA-epitope inserted into the extracellular
domain of the channel mutant. C, single mutations of Arg-44
(R44) and Arg-46 (R46) to alanine do not affect
surface trafficking, whereas the combined mutation R44A,R46A
(R44,46A) shows a significant reduction in surface
expression. Representative confocal images of OK cells expressing
indicated Kir2.1 mutations and quantification of their surface
expression by extracellular epitope-tagging are shown.
Asterisks mark significant differences from wild type Kir2.1
(p < 0.05, unpaired Student's t test).
wt (wild type), Kir2.1; lane 1, Kir2.1-R44A;
lane 2, Kir2.1-R46A; lane 3, Kir2.1-R44A,R46A;
lane 4, Kir2.1-R44A,R46A,R67A-K49A,K50A-H53A.
D, lipid raft association of Kir2.1, however, is not
disrupted by mutation of the identified cluster of positive charges in
its N terminus. An immunoblot analysis of Triton X-100-insoluble
sucrose gradient fractions of tsA201 cells, which were transfected as
indicated, is shown. The cell adhesion molecule rNCAM 140 was used to
identify lipid raft domains (23). Both wild type GFPKir2.1
and mutant GFPKir2.1-R44A,R46A,R67A-K49A,K50A-H53A are
present in lipid raft domains (lanes 3 and 4) and
in the high-density fraction possibly containing
cytoskeleton-associated proteins (lane 6).
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ACKNOWLEDGEMENTS |
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We thank B. Fakler and C. Derst for helpful discussion of the manuscript and G. Heck and G. Kummer for technical assistance.
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FOOTNOTES |
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* The study was supported by Deutsche Forschungsgemeinschaft Grant SFB 430-A2 (to N. K.).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.
Present address: Universitäts-Kinderklinik, Abt. für
Hämatologie, Onkologie, und Immunologie, Im Neuenheimer Feld 156, 69120 Heidelberg, Germany. E-mail:
Clemens.Stockklausner@med.uni-heidelberg.de.
§ To whom correspondence should be addressed. Tel.: 0761-203-5141; Fax: 0761-203-5191; E-mail: nikolaj.kloecker@physiologie.uni-freiburg.de.
Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M212243200
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; GFP, green fluorescent protein; EGFP, enhanced GFP; HA, hemagglutinin; OK, opossum kidney; BFA, brefeldin A; PBS, phosphate-buffered saline; PBS-T, PBS with Triton X-100; NGS, normal goat serum; NCAM, neural cell adhesion molecule; rNCAM, rat NCAM.
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