From the
TGN38 and TGN41 are isoforms of an integral membrane protein
that is predominantly localized to the trans-Golgi network
(TGN) in rat cells. They have been proposed to form a heterodimer and
to be involved in the budding of exocytic transport vesicles from the
TGN. By cDNA cloning and analysis using polymerase chain reaction, we
found that there were two TGN38 isoforms in a strain of mouse (ICR),
whereas other strains examined (BALB/c, DBA/2, and C57BL/6) had only
one TGN38. The major difference between the two isoforms was in the
number of characteristic octapeptide repeats. Apart from this, there
were several nucleotide substitutions between them. The two isoforms
appeared to be derived from two distinct genes but not from one gene
via alternative splicing. Furthermore, we failed to show the presence
of TGN41 in all the strains examined. This result suggests that TGN38
may function as a monomer or a homodimer in mouse cells.
The trans-Golgi network (TGN)
TGN38 is a type I integral membrane protein
predominantly localized to the TGN (for review, see Luzio and Banting,
1993; Stanley and Howell, 1993). Its cDNA first isolated from rat liver
(Luzio et al., 1990) is deduced to encode a protein with a
17-amino acid signal peptide, a 286-residue luminal domain having
characteristic octapeptide repeats and a 57-residue acidic region, a
21-residue transmembrane domain, and a 33-residue cytoplasmic tail.
Recently, an isoform of TGN38, designated TGN41, has been identified in
rat (Reaves et al., 1992) and suggested to form a heterodimer
with TGN38 (Jones et al., 1993; Stanley and Howell, 1993). The
only difference between the cDNAs encoding TGN38 and TGN41 is an 8-bp
change, including a 5-bp insertion in TGN41, in the region encoding the
cytoplasmic tail. The insertion changes the reading frame such that the
three COOH-terminal amino acids of TGN38 are not present, and instead
an extra 23 amino acids are added in TGN41. Although TGN38/41 is
concentrated in the TGN, recent studies have shown that it also cycles
between the TGN and the cell surface (Ladinsky and Howell, 1992; Reaves et al., 1993; Bos et al., 1993; Ponnambalam et
al., 1994; Chapman and Munro, 1994). Extensive studies have
revealed that a tyrosine-containing motif, YQRL, within the cytoplasmic
tail serves as a signal for both the TGN localization and the retrieval
from the cell surface to the TGN (Bos et al., 1993; Humphrey et al., 1993; Wong and Hong, 1993). Recently, Jones et
al.(1993) have shown that the cytoplasmic tail of TGN38/41 binds a
cytosolic complex of a 62-kDa protein (p62) and some small GTP-binding
proteins including Rab6 and have suggested that the dynamic interaction
between the cytosolic complex and TGN38/41 is essential for the budding
of exocytic vesicles from the TGN (Stanley and Howell, 1993).
To
learn more about the function of TGN38 and TGN41, we first attempted to
clone their cDNAs. However, we unexpectedly obtained evidence for the
presence of two isoforms of TGN38 in mouse. We therefore began to
investigate these TGN38 isoforms in detail.
To obtain a rat TGN38 cDNA fragment, we carried out reverse
transcriptase-PCR of total RNA isolated from rat pheochromocytoma PC12
cells using a set of primers flanking the characteristic octapeptide
repeats (see ``Experimental Procedures''). As shown in Fig. 1, an amplified cDNA fragment with an expected size
(
Fig. 3shows the
composite nucleotide sequences of the TGN38A and B cDNAs and their
deduced amino acid sequences. TGN38A and B were deduced to consist of
353 and 363 amino acids, respectively (also see Fig. 4). The
major differences in the coding sequence between them were
deletions/insertions of 6 and 24 bp. The latter was within the
characteristic octapeptide repeat region (also see Fig. 2and Fig. 4). Consequently, TGN38A and B have six and seven repeats,
respectively. There were also 11 nucleotide substitutions throughout
the coding sequence, six of which caused amino acid substitutions.
Comparison of the mouse TGN38 sequences with the rat one (Luzio et
al., 1990) revealed that the NH
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Drs. George Banting and Yukio Ikehara for
critical reading of the manuscript and Dr. Kaichiro Yanagisawa for
encouragement.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)is a tubuloreticular structure immediately apposed to
the trans-most face of the Golgi stack and functions as a
sorting machinery for proteins within the secretory pathway. Newly
synthesized proteins delivered through the Golgi stack to the TGN are
packaged into different types of transport vesicles which are directed
to the final destinations, including different domains of the plasma
membrane, lysosomes, endosomes, and regulated secretory granules (for
review, see Griffiths and Simons, 1986; Burgess and Kelly, 1987;
Mellman and Simons, 1992; Rothman and Orci, 1992; Bauerfeind and
Huttner, 1993). The molecular mechanism that underlies the sorting on
the TGN is not well understood. One well characterized exception is the
sorting of lysosomal hydrolases, which bind to mannose 6-phosphate
receptor via their mannose 6-phosphate residues, are packaged into
clathrin-coated vesicles and are delivered to lysosomes (for review,
see Kornfeld and Mellman, 1989; Pearse and Robinson, 1990). Interaction
of sorting receptors with cytosolic proteins must be required for the
protein sorting on the TGN. For example, mannose 6-phosphate receptor
has been shown to interact with HA-I/AP-1 clathrin adaptor via its
cytoplasmic tail (Glickman et al., 1989; Le Borgne et
al., 1993).
Animals
All mice used in the present
study were purchased from CLEA Japan, Inc. (Tokyo, Japan).
Cloning of Mouse TGN38 cDNAs
Mouse TGN38
cDNA fragments were obtained by reverse transcriptase-polymerase chain
reaction (PCR) as follows. Single-stranded cDNA generated from ICR
mouse brain total RNA using a first-strand cDNA synthesis kit
(Pharmacia-LKB Biotechnology, Uppsala, Sweden) was subjected to PCR
amplification using primers synthesized based on the rat TGN38 cDNA
sequence (Luzio et al., 1990): 5`-GATAAAGAAGGCCAGGACAAGAC-3`
(primer 1; corresponding to nucleotides 201-223) and
5`-GTTTCCACATCTTCAGTAGGCTC-3` (primer 2; complementary to nucleotides
717-739). DNA amplification using a GeneAmp AmpliTaq PCR kit
(Perkin-Elmer) was performed in a Perkin-Elmer thermal cycler with 40
cycles of denaturation (94 °C, 2 min), annealing (55 °C, 2
min), and extension (72 °C, 50 s). When electrophoresed on a 5%
polyacrylamide gel, two DNA fragments, designated A and B, around the
expected size (540 bp) were observed. Both fragments were
extracted from the gel, ligated into the SmaI site of
pBluescript II (Stratagene, La Jolla, CA), and subjected to sequence
analysis. Screening of
3
10
and
2
10
phages from ICR mouse brain and liver cDNA libraries,
respectively, in the
gt10 vector (Hosaka et al., 1994)
using the B fragment as a probe gave rise to 10 and 3 positive clones,
respectively. The cDNA inserts subcloned into pBluescript II were
subjected to sequence analysis. None of the cDNA inserts thus obtained
contained the 5`-end of the coding sequence. To obtain the missing
5`-end of the TGN38 transcripts, we performed extension of ICR mouse
brain RNA by the 5`-RACE (rapid amplification of cDNA ends) method
(Frohman et al., 1988). Primer 2 was used for the first round
of PCR amplification and primer 3, 5`-CCAAACTGTGATCGGTCGCG-3`
(complementary to nucleotides 299-318 in Fig. 3), for the
second round. Five PCR-derived clones containing the 5`-end of the
TGN38 cDNAs (three for TGN38A and two for TGN38B) were sequenced to
rule out potential PCR-generated mutations.
Figure 3:
Composite nucleotide and deduced amino
acid sequences of mouse TGN38A and B. The overall nucleotide and amino
acid sequences of mouse TGN38B are shown. The nucleotides and amino
acids of TGN38A which differ from those of TGN38B are also shown. Gaps
introduced into the alignment are indicated by hyphens.
Northern Blot Analysis
Ten micrograms of
total RNAs isolated from tissues of ICR mouse were electrophoresed on
an agarose gel and blotted onto a Nylon membrane as described
previously (Hatsuzawa et al., 1990). The blot was hybridized
with a 308-bp HincII-DraI fragment of the mouse
TGN38A cDNA and then washed under conditions as described previously
(Hatsuzawa et al., 1990).
540 bp) was observed. At the same time, reverse transcriptase-PCR
of total RNA from ICR mouse brain was also performed as a control. In
this case, however, we unexpectedly found two amplified DNA fragments
around 540 bp. We labeled the smaller and larger fragments as A and B,
respectively. Two intense bands at
1.2 and
1.5 kilobases were
also detected but were confirmed to be nonspecific by sequence analysis
(data not shown). As shown in Fig. 2, the nucleotide and deduced
amino acid sequences of the A and B fragments were very similar but not
identical to each other and were significantly homologous to those of
rat TGN38/41 (Luzio et al., 1990). The differences were a
24-bp deletion/insertion and five nucleotide substitutions. Therefore,
we tentatively designated the proteins encoded by the A and B fragments
as TGN38A and TGN38B, respectively.
Figure 1:
Presence of two TGN38 isoforms in
mouse. Total RNAs isolated from rat pheochromocytoma PC12 cells and ICR
mouse brain were subjected to reverse transcriptase-PCR using a set of
primers for rat TGN38. Experimental details are described under
``Experimental Procedures.'' DNA bands corresponding to mouse
TGN38A and B are indicated.
Figure 2:
Nucleotide and deduced amino acid
sequences of PCR-amplified cDNA fragments for mouse TGN38. The
nucleotide and amino acid sequences of mouse (m) TGN38A and B
are aligned with the pertinent region of rat (r) TGN38
sequences. Nucleotides in the two mouse sequences which differ from
each other are shown in italics. Amino acid residues conserved
between at least two members are shown in dark boxes. Gaps
introduced into the alignment are indicated by hyphens. The
sequences of the octapeptide repeats are double
underlined.
In an attempt to obtain
full-length cDNAs for TGN38A and B, cDNA libraries of ICR mouse brain
and liver were screened using the B fragment as the probe. Ten and
three positive clones were obtained from the brain and liver libraries,
respectively. By restriction endonuclease mapping and partial
nucleotide sequencing, four of the brain clones were deduced to code
for TGN38A and six for TGN38B, whereas all of the liver clones were for
TGN38A. It should be emphasized that no cDNA clone thus obtained
encoded a mouse counterpart of the TGN41 isoform (Reaves et
al., 1992); this is discussed below. Since none of the clones thus
obtained covered the putative translation initiation codon (the longest
clones for TGN38A and B started at nucleotides 59 and 30, respectively,
in the sequences shown in Fig. 3), we applied the 5`-RACE method
(Frohman et al., 1988) using a primer corresponding to the
nucleotide sequence common to both types of the cDNAs (see
``Experimental Procedures'').
-terminal half (amino
acids 18-194 in mouse TGN38B) including the octapeptide repeats
is not well conserved; the amino acid identity is only 60% between
mouse TGN38B and rat TGN38 (Fig. 4). In contrast, the
COOH-terminal half (amino acids 195-363 in mouse TGN38B)
including the acidic region, the transmembrane domain, and the
cytoplasmic domain with the tyrosine-containing TGN localization motif
is highly conserved with an amino acid identity of 94%; notably, the
sequences containing the transmembrane and the cytoplasmic domains are
identical to each other. These data lead to the speculation that the
COOH-terminal region plays more important roles in the function and
localization of TGN38 than the NH
-terminal one does,
although the roles of the octapeptide repeat and acidic regions are
currently unknown. A part of this speculation is supported by the
recent data of Ponnambalam et al.(1994) showing that not only
the cytoplasmic domain but also the transmembrane domain contains a TGN
localization signal.
Figure 4:
Sequence comparison of mouse (m)
TGN38 isoforms with rat (r) TGN38. Amino acid residues of
mTGN38A and rTGN38 identical with those of mTGN38B are indicated by dots. Gaps introduced into the alignment are indicated by hyphens. Predicted signal peptide and transmembrane domain
sequences are shown in dark boxes. The octapeptide repeat
sequences are shadowed, and the acidic regions are underlined. The tyrosine-containing motifs are double
underlined.
We then examined tissue and cellular
distribution of TGN38 transcripts because there has been no report on
the TGN38 distribution. Northern blotting of RNAs from mouse tissues
and from cultured cell lines of mouse origin revealed that TGN38 mRNAs
were expressed ubiquitously (Fig. 5), suggesting that TGN38 plays
a fundamental role in most, if not all, types of cells. Three
transcripts with different sizes (4.5,
3.3, and
1.8
kilobases) were observed. This size difference can be ascribed to the
difference in the length of the noncoding region rather than to one for
TGN38A and others for TGN38B or vice versa because cDNA clones with
sizes of >1.8 kilobases were obtained for both TGN38A and B (data
not shown).
Figure 5:
Northern blot analysis. Total RNAs from
various ICR mouse tissues and mouse cell lines were probed with a
TGN38A cDNA fragment as described under ``Experimental
Procedures.''
To determine whether both TGN38 isoforms are expressed
ubiquitously, we then performed reverse transcriptase-PCR of RNAs from
ICR mouse tissues and mouse cell lines using a set of primers flanking
the octapeptide repeats; in this and the following experiments, primers
synthesized on the basis of the mouse TGN38 sequence were used in place
of those for the rat TGN38 sequence used in the above experiments
because there were some mismatched nucleotides between sequences of the
rat primers and the mouse TGN38 cDNA. As shown in Fig. 6A, DNA fragments for both TGN38A and B were
observed in all examined tissues, indicating widespread expression of
both isoforms; two intense bands at 420 and
500 bp were also
detected but were confirmed to be nonspecific (data not shown).
However, we unexpectedly found only one fragment corresponding to the
position of TGN38A from RNAs from cultured cell lines of mouse origin,
NIH3T3, AtT-20, and L929. One possibility to which this observation is
ascribed is that the presence of the two TGN38 isoforms is a
strain-specific event because the cell lines used originate from
different mouse strains. To address this possibility, we performed
reverse transcriptase-PCR of liver RNAs from different mouse strains.
As shown in Fig. 6B, only TGN38A was present in the
BALB/c, C57BL/6, and DBA/2 strains, whereas both isoforms were in the
ICR strain. We also performed PCR of genomic DNAs from BALB/c and ICR
strains to determine whether the absence of the TGN38B transcript is
for lack of the TGN38B gene itself or for lack of its transcription. As
shown in Fig. 6C, although two bands for TGN38A and B
were detected in the ICR strain, one band for TGN38A was in the BALB/c
strain. Sequence analysis of the amplified DNA fragments revealed not
only that the two genomic fragments of ICR coded for TGN38A and B, and
the one of BALB/c for TGN38A, but also that there were no intervening
sequence within the amplified region including the octapeptide repeats
(data not shown). These data indicate that the presence of TGN38B is a
strain-specific event. Furthermore, taken together with the fact that
substitutions between TGN38A and B disperse throughout the nucleotide
sequence (see Fig. 3), these data indicate that the two isoforms
in ICR mouse derive from two distinct genes but not from the same gene
via alternative splicing. To understand the origin of the two TGN38
genes, a phylogenetically based survey of laboratory and wild mice will
be required.
Figure 6:
Strain-specific presence of TGN38B. Panel A, single-stranded cDNAs synthesized from RNAs of ICR
mouse tissues and mouse cell lines were subjected to PCR using a set of
primers: 5`-GTCAACCCCAAACTGGAAAAGTC-3` (primer 4; corresponding to
nucleotides 387-409 in Fig. 3) and
5`-TTTTTCAGTTGGCTGGACTTTGTCA-3` (primer 5; complementary to nucleotides
626-650 in Fig. 3). DNA amplification using a GeneAmp AmpliTaq
PCR kit was performed in a thermal cycler with 35 cycles of
denaturation (94 °C, 2 min), annealing (65 °C, 2 min), and
extension (72 °C, 45 s). The PCR products were electrophoresed on a
5% polyacrylamide gel. Panel B, single-stranded cDNAs
synthesized from liver RNAs of BALB/c, C57BL/6, DBA, and ICR strains
were subjected to PCR under conditions described above. Panel
C, genomic DNAs of BALB/c and ICR strains were subjected to PCR
under conditions described above.
As described above, we could not obtain a cDNA for
TGN41; amino acid sequences deduced from all 13 cDNA clones of mouse
brain and liver possessed a TGN38-type cytoplasmic tail sequence. This
led to the possibility that TGN41 does not exist in mice. To address
this possibility, cDNAs synthesized from RNAs of various mouse tissues
and cell lines, and mouse genomic DNAs were subjected to PCR analysis
using primers flanking the sequence where TGN38 and TGN41 should differ
from each other (Fig. 7A). In rat, there is an 8-bp
change including a 5-bp insertion in TGN41. Therefore, using these
primers, the TGN38 sequence should give a 76-bp fragment, whereas the
TGN41 sequence, if present, should give an 81-bp fragment. As shown in Fig. 7B, only one fragment around 76 bp was amplified
from RNAs of ICR and BALB/c mouse tissues and mouse cell lines, and
from genomic DNAs of ICR and BALB/c mice. Identical results were
obtained using liver RNAs of C57BL/6 and DBA/2 mice (data not shown). A
rare possibility that two DNA fragments with a difference of 5 bp in
length could migrate with the same mobility in the gel can be excluded
by the following three criteria. (i) By HindIII digestion, the
TGN38 PCR product should be cut into two fragments of 34 and 42 bp,
whereas the TGN41 product, if present, should be cut into 39- and 42-bp
fragments (see Fig. 7A). As shown in Fig. 7C, the endonuclease digestion gave rise to 34- and
42-bp fragments but not a 39-bp one. (ii) The sequence alteration and
5-bp insertion in rat TGN41 cDNA introduce a HinfI restriction
site that is not found in rat and mouse TGN38 cDNAs (see Fig. 7A). However, all of the PCR products examined
remained intact after the HinfI digestion (data not shown).
(iii) Finally, we subcloned the PCR products from liver RNAs and from
genomic DNAs of both ICR and BALB/c mice, determined the sequences of
some independent clones in each case, and found that all of the clones
had a TGN38-type sequence. It is also of note that the PCR products
from the genomic DNAs did not contain a putative intervening sequence;
this excludes the possibility that a TGN41-type transcript could be
generated via alternative splicing. Our data thus suggest the absence
of a TGN41 transcript and its gene in mouse, although further studies
will be required for us to draw a firm conclusion.
Figure 7:
Absence of TGN41. Panel A,
nucleotide and amino acid sequence of the pertinent region of mouse (m) mTGN38A and mTGN38B and rat (r) rTGN38 and
rTGN41. Primers used for PCR analysis are also shown. Gaps introduced
into the alignment are indicated by hyphens, and nucleotides
that differ between TGN38 and TGN41 are shadowed. Recognition
sequences for HindIII and HinfI are underlined and double underlined, respectively. Panel B,
single-stranded cDNAs synthesized from RNAs of ICR and BALB/c mouse
tissues and mouse cell lines, and genomic DNAs of ICR, and BALB/c mice
were subjected to PCR using primers 6 and 7. DNA amplification using a
GeneAmp AmpliTaq PCR kit was performed in a thermal cycler with 35
cycles of denaturation (94 °C, 1 min), annealing (68 °C, 2
min), and extension (72 °C, 45 s). The PCR products were
electrophoresed on a 15% polyacrylamide gel. Panel C, the PCR
products as above were digested with (+) or without (-) HindIII and were electrophoresed on a 20% polyacrylamide
gel.
Reaves et
al.(1992) have cloned a cDNA encoding TGN41 from a rat liver
library and shown the presence of its transcript in rat epididymis by
reverse transcriptase-PCR analysis. On the basis of experimental
evidence obtained using a rat liver Golgi fraction, Howell and
colleagues have proposed that TGN38 and TGN41 form a heterodimer and
are involved in the budding of exocytic transport vesicles from the TGN
by forming a complex with cytosolic proteins (Jones et al. 1993; Stanley and Howell, 1993). In the present study, however, we
failed to show the presence of TGN41 in mouse. A possible explanation
for the present observations is that TGN38 functions as a homodimer or
a monomer in mouse cells. Since the sequence common to both TGN38 and
TGN41 is thought to have the signal that is essential for their TGN
localization as discussed by Reaves et al. (1992), TGN41 may
be dispensable. To address whether the presence of TGN41 is a
rat-specific event, studies are under way to examine whether other
species have TGN41.
/EMBL Data Bank with accession number(s) D50031 and
D50032.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.