From the Glycobiology Program, La Jolla Cancer Research Center, The Burnham Institute, La Jolla, California 92037 and § Division of Ultrastructural Pathology and Cell Biology, Institute of Clinical Pathology, University of Vienna, A-1090, Vienna, Austria
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ABSTRACT |
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Previously, it has been shown that glycoproteins
with ~130-kDa molecular mass react with antisera from patients with
renal vasculitis (Kain, R., Matsui, K., Exner, M., Binder, S.,
Schaffner, G., Sommer, E. M., and Kerjaschki, D. (1995) J. Exp. Med. 181, 585-597). To search for a molecule that reacts
with the antibodies, we screened a gt11 human placental cDNA
library. Two of the isolated clones were found to encode a putative
counterpart of the rodent trans-Golgi network (TGN)
glycoprotein 38, hTGN46, which has the tyrosine containing motif YQRL
shared by mouse and rat TGN38. Moreover, reverse
transcription-polymerase chain reaction analysis of hTGN46
transcripts and genomic analysis of a cDNA deposited as an
expressed sequence tag in dbEST Data Base revealed that additional
cDNAs exist that are produced by alternate usage of 3
-splice sites
of intron III. Alternative splicing results in frame shifts and leads
to novel larger translation products with one (for hTGN48) or two (for
hTGN51) additional tyrosine-containing motifs. hTGN51 expressed in
Chinese hamster ovary cells were localized to the
trans-Golgi network, overlapping with
-1,4-galactosyltransferase even after mutating the
tyrosine-containing motif common to hTGN46. In contrast, mutated hTGN48
and hTGN46 are no longer retrieved to the TGN. These results strongly
suggest that hTGN51 may have a unique function compared with hTGN46 or
hTGN48 in shuttling between the cell surface and the TGN.
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INTRODUCTION |
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The trans-Golgi network (TGN)1 is a tubulo-vesicular structure adjacent to the trans-most cisternae of the Golgi stack. The TGN is the major site where secretory and membrane proteins are sorted to the plasma membrane, lysosomes, endosomes, and secretory granules (1-3). The final stages of processing N-glycans and most likely O-glycans take place in the TGN, and in some instances such unique glycosylation plays a role in sorting to the correct destination (1-3).
TGN38, originally characterized in rat, is a type I integral membrane protein predominantly localized to the TGN (4). The rat cDNA encoding rTGN38 predicts a protein with 357 amino acid residues. The cytoplasmic domain consisting of 33 amino acids contains a YQRL sequence, which has been shown in other proteins to be a signal for the endocytic pathway and for lysosomal targeting (5-9) and a retrieval signal for rTGN38 from the plasma membrane (10, 11). Mutation of this motif resulted in constitutive expression of TGN38 at the plasma membrane (10, 11). It has also been demonstrated that the transmembrane domain of TGN38 plays a role as a retention signal in the TGN, making rTGN38 unique in having both retrieval and retention signals (12).
In rat, a cDNA encoding a translational product different from the original rTGN38 was isolated (13). This cDNA has an insertion of 5 bp, of which 4 bp replace the counterpart of TGN38 cDNA in the region of the cytoplasmic tail. This insertion changes the reading frame such that the three COOH-terminal amino acids of TGN38 are replaced with a different sequence, and the cytoplasmic tail is 23 amino acid residues longer than that of TGN38. This variant, termed rTGN41, also contains the same tyrosine motif as rTGN38. On the other hand, the mouse homologue of rTGN38 has two forms due to allelic differences (14).
Previously, it has been shown that sera from patients with autoimmune
vasculitis were found to react with glycoprotein(s) with a molecular
mass of ~130 kDa (gp130s) (15). Monoclonal and polyclonal antibodies
were raised against gp130s, and monoclonal antibody AG11 was found to
show a staining profile identical to that of sera from patients.
Attempts to isolate cDNA clones using AG11 to screen expression
libraries have been unsuccessful. In contrast, one 130-kDa glycoprotein
has been identified as LAMP-2 in a gt11 library screen using the
antibodies specific to gp130s (15). The subcellular distribution of
LAMP-2 differs from that detected by the AG11 antibody, suggesting that
LAMP-2 is only one of the members of gp130s (15).
By using the antibodies specific to gp130s in the present study, we
screened a gt11 library constructed from poly(A)+ RNA
isolated from human placenta. In addition to cDNAs encoding LAMP-2,
we first isolated cDNA encoding the human homologue of rat TGN38,
designated human TGN46 (hTGN46). We then detected additional transcripts that encode larger proteins than hTGN46. These novel forms
of hTGN46 are produced by different usage of 3
-splicing sites in
intron III. The largest protein, hTGN51, is unique in having two
additional tyrosine containing motifs and a dileucine motif (16, 17),
both of which together we showed to function as a retrieval signal to
the TGN.
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EXPERIMENTAL PROCEDURES |
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Preparation of Rabbit Polyclonal Antibodies Specific to gp130s-- Rabbit polyclonal antibodies specific to gp130s were prepared as described previously (15). Polyclonal antisera specific to gp130s were passed through Escherichia coli lysates immobilized on CNBr-activated Sepharose 4B (Pierce) and purified on gp130s immobilized on CNBr-activated Sepharose 4B (300 µg/ml), as described previously (15).
Isolation of cDNAs Encoding Human TGN46--
A gt11
cDNA term placenta library (18) was screened with affinity purified
rabbit anti-gp130 antibodies. After screening 106 plaques,
9 positive clones were isolated. Among the clones analyzed, 7 clones
were found to harbor cDNAs encoding lamp-2. Using a Blast Search
(19), 2 clones were found to harbor cDNAs with no homology to human
sequences reported at that time, but both were homologous to sequences
encoding the cytoplasmic and transmembrane domains of rat TGN38
(4).
Detection of Multiple Transcripts Encoding hTGN46, hTGN48, and
hTGN51--
PCR was carried out using various cDNA libraries as
templates. For the first PCR, the 5-primer (primer 11)
corresponds to nucleotides 986-1003, and the 3
-primer (primer 10) is
complementary to nucleotides 1347-1367 of hTGN46. For the
second PCR, the nucleotide sequence encoding the lumenal domain
(nucleotides 137-1185) was amplified using primer 1 (nucleotides
137-153) and primer 2 (nucleotides 1168-1185).
Fluorescence in Situ Hybridization Analysis of Human TGN46
Gene--
A human genomic P1 plasmid library was screened by PCR as
described (21). The 5- and 3
-primers for PCR correspond to the sequence of nucleotides 1245 to 1278 (primer 27) and nucleotides 1386 to 1369 (primer 10), respectively, of hTGN46 sequence.
Purified DNA from one of the isolated P1 clones, clone 10508, was
labeled with digoxigenin-dUTP by nick translation. Labeled probe was
hybridized to normal metaphase chromosomes derived from
phytohemagglutinin-stimulated peripheral blood leukocytes under the
conditions as described (22).
Isolation of Genomic Clones Harboring the hTGN46 Gene--
To
isolate genomic clones of TGN46, the above P1 plasmid,
10508, was digested by BamHI and XhoI and cloned
into pBluescript. The resultant colonies were then screened initially
by hybridization with the cDNA clone 8c1 that encompasses
nucleotides 28-2103 as a probe. Five positive clones were then
screened by cDNA encompassing either nucleotides 45 to 153 or
nucleotides 1369 to 1386. Three clones harboring the 5
-half of the
genomic sequence and two clones harboring the 3
-half were
isolated.
Primer Extension Analysis and Promoter Activity
Assays--
5-Primer extension analysis was carried out essentially
as described (23). The primer was complementary to nucleotides +23 to
+46 (46 is at the end of exon 1). Poly(A)+ RNA from HL-60
and human embryonic kidney 293 cells was annealed to the 5
-end-labeled
oligonucleotide and incubated with Superscript II RNase H reverse
transcriptase (Life Technologies, Inc.) as detailed previously
(23).
Isolation of cDNAs Encoding TGN48 and TGN51--
cDNA
was synthesized using reverse transcriptase, poly(A)+ RNA
derived from fetal liver and oligonucleotide r3, shown in Fig. 4,
corresponding to 3-untranslated sequences. Using the cDNAs synthesized as templates, PCR was performed to amplify sequence from
exon 3 to exon 4 under the conditions described (16). In the first PCR,
the 5
-primer (primer 27) corresponds to nucleotides 1245-1278 and the
3
-primer (primer B) is complementary to nucleotides 1348-1372 of the hTGN51 sequence. This PCR reaction yielded
128- and 87-bp products derived from the transcripts for
TGN51 and TGN48 (Fig. 2C). For
isolation of cDNA encoding TGN51, another PCR was carried out to
isolate the 3
-region of the sequence using a 5
-primer (primer
A) corresponding to nucleotides 1301-1321 and a 3
-primer
(primer 54) complementary to nucleotides 1518-1547. This PCR product
and the 128-bp product described above were then used as a template for
PCR using primers 27 and 54 as described (16), resulting in cDNA
encoding nucleotides 1245-1547 of hTGN51.
Northern Blot Analysis of Various Human Tissues--
A human
multiple fetal tissue Northern blot (CLONTECH
Laboratories) was hybridized with a gel-purified cDNA fragment
(nucleotide 45 to nucleotide 153) of pcDNA3-hTGN46
after labeling with [
-32P]dCTP by one PCR cycle (25).
The same blot was also hybridized with 32P-labeled
oligonucleotide 59 or primer 54, as described previously (26).
Oligonucleotide 59 (nucleotides 1342-1318) hybridizes only with the
transcript of hTGN51, whereas primer 54, described above,
hybridizes with all three of the TGN glycoprotein
transcripts.
Mutation of Tyrosine Residue 430 in the Cytoplasmic
Domain--
Site-directed mutagenesis was carried out by PCR using
pcDNAI-hTGN46 as a template as described (16). The
5-primer for this PCR (primer 30) was used in the isolation of
pcDNA3-hTGN46. The 3
-primer was
5
-TCTGTTAGGACTTCTGGTCCAAACGTTGGTTGTCACT-3
, complementary to nucleotides 1280-1318 in hTGN46, in
which the tyrosine codon was changed to asparagine, as shown by double
underlines. The PCR product was blunt-ended by vent DNA polymerase,
digested by BamHI, and cloned into the BamHI and
EcoRV sites of pcDNA3, resulting in
pcDNA3-TGN46YN.
Preparation of Anti-TGN46/48/51 Antibodies--
The fragment of
cDNA encoding part of the lumenal domain of hTGN46 was excised from
clone 4a1 with BamHI and HindIII (position 862)
(see Fig. 1) and ligated in-frame into pMALc2 (New England Biolabs).
Transformed E. coli DH5 were induced to express a soluble maltose binding fusion protein (15). Purified recombinant fusion protein was digested with factor Xa, resulting in precipitates that
almost exclusively contained hTGN46 peptide. One hundred µg of this
protein were used for the initial immunization and for further boosts.
Sera were passed through a column of bacterial proteins produced in
E. coli DH5
and then purified by adsorbing on protein
A-Sepharose (Pierce) as described (15, 27).
Expression of hTGN46, hTGN48, hTGN51, and Corresponding Mutant
Forms in CHO Cells--
To express hTGN46 and related glycoproteins,
pcDNA3-hTGN46 was transfected into Chinese hamster ovary
(CHO) cells grown on coverslips using LipofectAMINE (Life Technologies,
Inc.) as described (27). In some experiments, TGN glycoproteins were
co-expressed with human -1,4-galactosyltransferase. For this,
pcDNAI-GalT (28) and pcDNAI-hTGN46,
hTGN48, hTGN51, or corresponding mutant proteins
were transiently transfected into CHO cells. Rabbit anti-hTGN46 antibodies and a mouse monoclonal antibody specific to human
-galactosyltransferase (29) were used in the assay. Samples were
visualized as described (27, 30). When cell surface proteins were to be
detected, saponin was omitted throughout the procedure.
Immunocytochemical Staining for Electron Microscopy-- Indirect immunoperoxidase staining was performed as described previously (15, 27, 31). For gold labeling, ultrathin frozen sections of paraformaldehyde/lysine/periodate-fixed normal human kidneys were incubated with purified rabbit anti-hTGN46 antibodies followed by goat anti-rabbit IgG-10-nm gold conjugate (1:20, Auroprobe, Amersham Corp), as described (15). Controls were performed either by omitting the primary antibody or by replacing it with rabbit preimmune serum.
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RESULTS |
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Cloning of cDNA Encoding Human TGN46 Glycoprotein--
A
gt11 cDNA expression library constructed from
poly(A)+ RNA of human term placenta was screened with
affinity purified rabbit anti-gp130s antibodies. After screening
106 plaques, 9 positive clones were isolated. Among them,
seven clones were found to harbor cDNAs encoding LAMP-2, whereas
two other clones were found to have no homology to any human sequences
in the data base at that time.
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Characterization of cDNAs Related to hTGN46--
To determine
whether variants of hTGN46 are expressed in placenta and kidney,
cDNA sequences of hTGN46 were amplified using various
cDNA libraries as templates. When the sequence encoding the lumenal
domain was amplified by PCR, only one product (~1.0 kb), that
expected from hTGN46 sequence, was obtained (Fig.
2B). In contrast, two bands
were obtained when PCR was carried out with primers to the lumenal
domain and 3-untranslated sequence (Fig. 2A). Of these
products, the shorter one (402 bp) corresponds to the hTGN46
sequence (nucleotides 998-1399). The longer product is larger than
this shorter product by approximately 60 bp.
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The hTGN46 Gene Is Mapped to Chromosome 2 Band p11.2-- Different forms of hTGN46 described above could be produced by different genes or by alternative splicing of mRNA encoding hTGN46. To determine whether more than one gene encodes hTGN46 and related sequences, the hTGN46 gene was localized on chromosomes using fluorescence in situ hybridization procedures. These experiments demonstrated that hTGN46 gene is localized in chromosome 2. Measurement of 10 specific hybridizations of chromosome 2 demonstrated that 10508 is located at a position 10% the distance from the centromere to the telomere of chromosome 2 arm p, an area corresponding to chromosome 2, band p11.2 (Fig. 3). The results strongly suggest that only one gene encodes different forms of hTGN46, although tandemly arranged multiple genes would provide the same results.
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Isolation of Genomic Clones Harboring the hTGN46 Gene-- The above results suggest that different transcripts of hTGN46 are produced by alternate splicing. To determine whether variant transcripts are produced from the same gene, we isolated genomic clones from the P1 plasmid, as detailed in "Experimental Procedures." Two representative genomic clones were obtained, and clone 6 contained exon 1 to intron III sequence, whereas clone 24 contained intron III to exon 4 sequence. The genomic organization of hTGN46 as shown in Fig. 1 is derived from this analysis.
Primer extension analysis showed that the transcription start site lies 62 bp upstream from the initiation methionine codon. In addition, the 5Differential Usage of 3-Splice Sites in Intron III--
The
results shown in Fig. 2, A and B, also indicate
that a portion of the sequences present in introns II or III may be
transcribed in longer forms of hTGN46. We were particularly
interested in determining whether the 5
-splice or 3
-splice site of
intron III differs in those larger transcripts.
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Isolation of cDNA Encoding Novel Forms of hTGN46--
The
above results indicate that cDNA encoding novel forms may be
isolated by PCR amplification of sequences between exon 3 and exon 4. However, the majority of PCR products amplified by primers 10 and 11 shown in Fig. 1 was derived from hTGN46 mRNA, probably
because the transcripts of the variants are present in low abundance.
The sequence from nucleotides 1300 to 1547 was thus obtained by RT-PCR
using primers A and 54 shown in Fig. 4. This PCR
product, representing the 3-half of the sequence, and the 128-bp PCR
product shown in Fig. 2C (right lanes) were used as templates to obtain a cDNA sequence encompassing nucleotides 1245-1547 using primers 27 and 54. This cDNA fragment was digested with PflMI and XbaI and cloned into the
corresponding portion of pcDNA3-hTGN46, resulting in
pcDNA3-hTGN51 (see Fig. 4).
Expression of hTGN46, hTGN48, and hTGN51-- Rabbit antiserum was raised against the lumenal domain of hTGN46. TGN38 endogenously expressed in CHO cells did not react with the above prepared antiserum, consistent with the fact that hTGN46/48/51 and rodent TGN38/41 have no homology in the NH2 terminus region. CHO cells were thus used as recipient cells for introducing cDNA encoding TGN46 and its variants.
In the first set of experiments, pcDNAI-human GalT (28) and cDNA encoding hTGN46, hTGN48, or hTGN51 were expressed together in CHO cells. The results, as shown in Fig. 6 (top panel), demonstrated that most of the
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Mutation of the Tyrosine Motif at Residues 430-433-- As shown in Fig. 4, TGN46, TGN48, and TGN51 share the same amino acid sequences up to residue 436, indicating that all forms of these related proteins have a common tyrosine-containing motif from residue 430 to residue 433. TGN48 and TGN51 contain one and two additional tyrosine motifs, respectively (Fig. 4).
To determine whether these additional tyrosine-containing sequences in hTGN48 and hTGN48 function as retrieval signals, tyrosine residue 430 was mutated to asparagine in TGN46, TGN48, and TGN51, and the mutated proteins were then expressed in CHO cells. As shown in Fig. 6 (lower panel), mutated TGN46 and TGN48 were localized at the plasma membrane, and a very small portion was retrieved to the TGN. In contrast, TGN51 was still retrieved to the TGN even after the tyrosine-containing motif at residues 430-433 was mutated (Fig. 6, lower panel). To confirm the results shown in Fig. 6, wild type and mutant TGN46, TGN48, and TGN51 were expressed in CHO cells, and those cells expressing the proteins were examined without permeabilization. As shown in Fig. 8 A, C and E, wild type TGN46, TGN48, and TGN51 were faintly expressed on the cell surface. In contrast, mutant TGN46 and TGN48 were substantially expressed on the cell surface, whereas only a small amount of mutant TGN51 was expressed on the cell surface (Fig. 8, B, D, and F). These results indicate that the tyrosine motif at residues 430-433 is critical for retrieving TGN46 and TGN48, and that Tyr-Ser-Ser-Gly (residues 444-447) in TGN48 does not function well as a retrieval signal. In contrast, Tyr-Val-Leu-Ile (residues 437-440) and Tyr-Ile-Pro-Leu (residues 461-464) sequences in TGN51 function as a retrieval signal to the TGN.
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DISCUSSION |
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In the present study, we have cloned cDNAs encoding human
TGN46 (hTGN46) using antibodies reacting with glycoproteins of ~130 kDa. The deduced amino acid sequence of the isolated cDNA shows that the COOH terminus is highly homologous to rat and mouse TGN38 (4,
14). The subcellular distribution of hTGN46 still differs from AG11
(15), and it is not clear if this is a good candidate for autoimmune
antigens in vasculitis. Human TGN46 was not cloned when we started our
studies; however, a sequence almost identical to that described here
but shorter on both the 5- and the 3
-ends has been reported recently
(32).
In the present study, we have also isolated novel forms of TGN46. The
presence of novel forms was apparent when the sequence between the
lumenal domain and 3-untranslated region was amplified by PCR using
various cDNA libraries as templates (Fig. 2A). Genomic analysis and sequence comparison of the intron III sequence and EST
data bases revealed that these novel transcripts are produced by
alternative usage of the 3
-splice of intron III (Fig. 4). We show that
the two novel forms are 58 and 17 bp larger than that of hTGN46. More
importantly, these alternative usages of 3
-splice sites result in
frame shifts resulting in one (TGN48) or two (TGN51) additional
tyrosine-containing motifs.
Expression of hTGN46, hTGN48 and hTGN51 demonstrated that these proteins reside mostly in the TGN (Figs. 6 and 7). Although TGN46 is ubiquitously present in various tissues, TGN51 is more abundant in fetal lung and kidney, whereas TGN48 is barely present in embryonic kidney 293 and promyelocytic HL-60 cells (Figs. 2 and 5). We also demonstrated that the additional tyrosine-motif found in hTGN51 functioned as a retrieval signal to the TGN, since mutation of the tyrosine-motif shared by all three of these forms did not alter its traffic to the TGN. In contrast, the second tyrosine motif in hTGN48 functioned poorly as a retrieval signal (Figs. 6 and 8). The additional tyrosine motif in hTGN51 contains the amino acid sequences of Tyr-Val-Leu-Leu and Tyr-Ile-Pro-Leu, whereas that in hTGN48 is Tyr-Ser-Ser-Gly. The results obtained in mutant hTGN48 and hTGN51 are consistent with previous results demonstrating that a tyrosine motif for the endocytic pathway should contain a bulky hydrophobic amino acid at the fourth position from the tyrosine (5-11). Apparently, Tyr-Ser-Ser-Gly in hTGN48 does not function as a retrieval signal through an endocytic pathway.
In addition to tyrosine-containing motif, hTGN51 contains a Leu-Leu motif at residues 475-476 (Fig. 4). It has been shown that this motif also serves as an endocytic pathway and lysosomal targeting signal (16, 17, 33-35). It is thus possible that hTGN51 contains a fourth retrieval signal in addition to three tyrosine-containing motifs. In this regard, it is noteworthy that rat TGN41 also contains a Leu-Val motif (13). This motif worked as a lysosomal targeting signal when the last 9 amino acids of rat LimpII sequence was replaced with human mannose-6-phosphate receptor sequences containing a Leu-Val sequence (16). These results strongly suggest that Leu-Leu and Leu-Val in hTGN51 and rTGN41, respectively, may work as retrieval signals for these proteins. On the other hand, it has been suggested that other amino acids such as acidic amino acids or phosphorylation sites are required in order for Leu-Leu (Val) motif to function (36, 37). Further studies will be necessary to determine whether the Leu-Leu and Leu-Val motif in hTGN51 and rTGN41 work as retrieval signals to the Golgi.
It is noteworthy that the transmembrane domains are also well conserved among human TGN46/48/51 and rodent TGN38/41. It has been shown that the transmembrane portion of TGN38 is critical as a Golgi retention signal (12). Similarly, the transmembrane domain was shown to be critical as a retention signal for Golgi resident proteins such as glycosyltransferases (28, 38-41). These results strongly suggest that the transmembrane domain of hTGN46, hTGN48, and hTGN51 play a role as a Golgi retention signal. In addition to the above two domains, the amino acid sequence upstream from the transmembrane domain (residues 314-384, Fig. 1) is also conserved well among human, rat, and mouse TGN glycoproteins. This sequence is enriched in acidic amino acid residues, but the role(s) of this acidic region is not known.
In human TGN46 variants, transcripts diverge three codons after the
tyrosine-containing motif shared by all three forms of TGN
glycoproteins. By comparing rat TGN38 and TGN41 sequences (4, 13), it
is possible to infer that these two different transcripts start after
one codon from the tyrosine motif, shared by rTGN38 and rTGN41.
Although the genomic organization of rTGN38/41 has not been elucidated,
it is possible that these two forms arise from different usage of the
3-splice sites as demonstrated for human TGN46,
-48, and -51 in the present study.
It has been reported that chicken Lamp-2 variants differ in the transmembrane domain and the cytoplasmic tails due to alternative splicing (42). These variations in the transmembrane and cytoplasmic domain were found to influence the rate of internalization of Lamp-2, resulting in different levels of cell surface expression exhibited by variants of Lamp-2 (43). These results strongly suggest that hTGN46 variants may also differ in the rates of retrieval to the TGN. Such a difference may suggest different functions for different forms of hTGN46 on the cell surface and the TGN.
It has been proposed that both rTGN38 and rTGN41 together associate with p62 and Rab6 and that this complex is involved in budding of exocytic vesicles (44). Other studies have shown that the tyrosine motif in rTGN38 associates with AP-1 present in clathrin-coated pits (45). Moreover, the tyrosine-containing and dileucine motifs are apparently recognized by different cytoplasmic carriers (46). These results point toward the possibility that hTGN48 and hTGN51 in particular may have unique function in association with hTGN46 in the process of both endocytic and exocytic pathways. The discovery of two novel forms of human TGN glycoproteins hTGN48 and hTGN51 will allow us to determine the roles that TGN glycoproteins play in these intricate intracellular movements.
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ACKNOWLEDGEMENTS |
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We thank Drs. Jose Luis Millan, Hisashi
Narimatsu, and Michiko N. Fukuda for providing the placenta gt 11 library, mouse monoclonal anti-human galactosyltransferase antibody,
and pcDNAI-GalT, respectively; Genome Systems, Inc., for
chromosomal localization; Helga Poczewski for technical assistance in
immunoelectron microscopy; Dr. Ellen Elise Lamar for critical reading
of the manuscript; and Susan Greaney for organizing the manuscript. We
also thank Drs. Ken Watanabe and Anders Aspberg for useful discussions;
and Dr. Edward Monosov, Tristan Williams, and Muizz Hasham for various technical support.
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FOOTNOTES |
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* This work was supported by Grant CA48737 awarded by NCI, the National Institutes of Health.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U62390, AF027515, AF027516, AF029313-029316.
Recipient of the Max Kade Foundation fellowship and Erwin
Schrödinger fellowship.
¶ To whom correspondence should be addressed: The Burnham Institute, 10901 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-646-3144; Fax: 619-646-3193.
1 The abbreviations used are: TGN, trans-Golgi network; TGN38, trans-Golgi network glycoprotein 38; PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; CHO, Chinese hamster ovary; EST, expressed sequence tag; kb, kilobase pair(s); bp, base pair(s); RACE, rapid amplification of cDNA ends.
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REFERENCES |
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