Surface Expression of Inward Rectifier Potassium Channels Is Controlled by Selective Golgi Export*

Clemens StockklausnerDagger and Nikolaj Klöcker§

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 YXXPhi (with X being any amino acid and Phi  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).

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 -, 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).

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.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 (GFPKir2.1Delta 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 GFPKir2.1Delta 1-76 to the ER; and (ii) we found colocalization of GFPKir2.1Delta 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 Kir2.1Delta 1-76 tagged with an extracellular HA epitope and detected by immunocytochemistry showed an identical subcellular localization as GFPKir2.1Delta 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 GFPKir2.1Delta 1-76, both channel subunits did not only colocalize, but Kir2.1-HA was even able to rescue GFPKir2.1Delta 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 (GFPKir2.1Delta 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 GFPKir2.1Delta 1-76. C, GFPKir2.1Delta 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 GFPKir2.1Delta 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 GFPKir2.1Delta 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).

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-Delta 1-76. Moreover, the Kir4.1 N terminus was able to substitute for the Kir2.1 N terminus and rescued surface expression of GFPKir2.1Delta 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-Delta 1-59), and fusion of the Kir4.1 N terminus to Kir2.1 is sufficient to restore surface expression of GFPKir2.1Delta 1-76 (4.1(1-59)/2.1).

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-Delta 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-Delta 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, Kir2.1Delta 1-20; lane 2, Kir2.1Delta 1-43; lane 3, Kir2.1Delta 1-46; lane 4, Kir2.1Delta 1-70; lane 5, Kir2.1Delta 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.

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-Delta 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-Delta 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).

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.

    ACKNOWLEDGEMENTS

We thank B. Fakler and C. Derst for helpful discussion of the manuscript and G. Heck and G. Kummer for technical assistance.

    FOOTNOTES

* 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.

Dagger 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

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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