(Received for publication, December 13, 1994; and in revised form, May 5, 1995)
From the
Four variant AE1 anion exchangers with predicted molecular
masses of 99,
102,
104, and
108 kDa are expressed
in chicken erythroid cells. These variant polypeptides differ in
sequence only at the N terminus of their cytoplasmic domains. Molecular
analyses have shown that transcripts derived from both of the
erythroid-specific promoters, P1 and P2, encode all four of these AE1
anion exchanger variants. However, quantitative RNase protection
analyses have shown that the transcripts derived from the P1 promoter
are much more prevalent than those derived from the P2 promoter.
Reverse transcriptase polymerase chain reaction studies have indicated
that the extensive diversity in the transcripts derived from the AE1
gene occurs both in primitive and definitive lineage erythroid cells.
Transient transfection analyses using human erythroleukemia cells have
investigated the functional significance of the alternative sequences
at the N terminus of these variant exchangers. These studies have shown
that the erythroid AE1 variants are sorted to different membrane
compartments in these cells. The
99- and
102-kDa variants are
primarily sorted to the plasma membrane, whereas the
108-kDa
variant is retained in a perinuclear compartment. These results suggest
that the alternative N-terminal cytoplasmic sequences of these
polypeptides may serve as signals to direct these variant transporters
to different membrane compartments within cells.
The erythroid AE1 anion exchanger (band 3) possesses dual
functions that are segregated into distinct domains. The C-terminal
membrane-spanning region of the polypeptide primarily mediates the
electroneutral exchange of bicarbonate for chloride(1) . This
electroneutral exchange reaction allows the CO generated in
respiring tissues to be carried to the lungs in the form of bicarbonate
where it is expelled. The N-terminal cytoplasmic domain of the AE1
anion exchanger provides an attachment site for the erythroid membrane
cytoskeleton through its interaction with ankyrin(2) , protein
4.1(3) , and protein 4.2(4) .
Recent analyses have indicated that the gene that encodes the erythroid AE1 anion exchanger also encodes the electroneutral anion exchanger that has been characterized in the basolateral membrane of the A-intercalated cell of the kidney collecting duct(5) . These studies have shown that the AE1 anion exchanger transcripts that accumulate in human(6) , mouse(7) , rat(8) , and chicken (9) kidney differ from the erythroid AE1 transcripts in the sequences present at their 5` ends, resulting in polypeptides that only differ from their erythroid counterparts in the sequences present at the N terminus of their cytoplasmic domains. This type of N-terminal cytoplasmic diversity has also been shown to exist among the AE1 anion exchangers expressed in chicken erythroid cells. Two variant erythroid AE1 anion exchanger transcripts, AE1-1 and AE1-2, which are derived by alternative transcriptional initiation and differential RNA splicing(10, 11) , encode polypeptides with alternative N-terminal cytoplasmic domains.
The studies described
here, in conjunction with previous analyses, have indicated that a
total of 14 AE1 anion exchanger transcripts accumulate in chicken
erythroid cells. Twelve of these transcripts are derived from the two
erythroid-specific promoters of the AE1 gene, P1 and P2 (11) .
The other two transcripts, AE1-3 and AE1-5, whose expression is
regulated by the P3 promoter of the AE1 gene, ()accumulate
both in erythroid and in kidney cells(9) . Although 12 AE1
transcripts are derived from the two erythroid-specific promoters, they
encode only four different structural variants with predicted molecular
masses of
99,
102,
104, and
108 kDa. This observed
diversity is consistent with immunoblotting studies that have shown
that four anion exchanger species of
95,
99,
102, and
108 kDa accumulate in chicken erythroid membranes(9) .
Like the variant AE1 anion exchangers that have been previously
characterized, the polypeptides encoded by these variant AE1
transcripts differ only in the sequences present in their N-terminal
cytoplasmic domains. Transfection analyses have indicated that the
108-kDa AE1 variant is primarily retained in a perinuclear
compartment when transiently expressed in human erythroleukemia (HEL) (
)cells. In contrast, the
99- and
102-kDa AE1
variants primarily accumulate in the plasma membrane of transfected HEL
cells. These results suggest that the alternative N-terminal
cytoplasmic sequences of these variant transporters may be involved in
targeting these polypeptides to different membrane compartments within
cells.
Figure 5:
Quantitation of the abundance of the four
structural AE1 anion exchanger variants by RNase protection analyses.
The relative abundance of the four AE1 anion exchanger structural
variants was determined by RNase protection analyses. The structure of
each construct used to generate antisense RNA probes is illustrated in A. The black boxes in each construct represent vector
sequences. P-labeled antisense transcripts corresponding
either to exon 4 (B, lanes 4-6), exons 3 and 4 (B, lanes 1-3), or exons 3, 4, 5, and 6 (B, lanes 7-9) were synthesized in vitro and hybridized to 1 µg of poly(A)
RNA
isolated from 10-day embryonic erythroid cells (lanes 2, 5, and 8). Following hybridization, samples were
digested with RNase, and the products were resolved on a 6%
polyacrylamide, 7 M urea gel. In B, lanes 1, 4, and 7 correspond to the probe alone, and lanes
3, 6, and 9 correspond to control hybridizations
and digestions carried out in the presence of the probe and tRNA alone.
The protected fragments corresponding to exon 4; exons 3 and 4; exons
4, 5, and 6; and exons 3, 4, 5, and 6 are indicated on the right
hand side of the figure. Markers in B correspond
to in vitro transcripts of known
size.
Figure 1: Structure of the variant chicken AE1 anion exchangers. The exon organization of the variant chicken AE1 anion exchangers is illustrated diagrammatically in A. The predicted site of translation initiation of each variant, as well as the location of the antisense primer (arrow in AE1-1) used for the RT-PCR RACE analyses are indicated. Boxes containing different patterns at the 5` ends of the AE1-3, AE1-4, and AE1-5 transcripts correspond to the variant specific exons of these transcripts. The boxes that are shaded are held in common among all of the variant transcripts. The location of the peptide, pAE1A, that was used to generate AE1 anion exchanger-specific antibodies is indicated. The nucleotide and predicted amino acid sequence of exons 5 and 6 is illustrated in B. The arrows in B mark the location of splice junctions.
To determine whether additional diversity exists at the 5` ends of the chicken erythroid AE1 transcripts, RT-PCR RACE using an antisense primer corresponding to nucleotides 298-318 of the AE1-1 transcript has been used to amplify the 5` ends of the erythroid AE1 transcripts. The location of the antisense primer used for these analyses is indicated by the arrow in Fig. 1A. The amplification products resulting from this protocol were subcloned into a pGEM-3 vector, and 20 clones were isolated and sequenced. Eight clones were identical to the sequence of the AE1-1 and AE1-2 transcripts, and their 5` ends corresponded to various sites within exon 4 of the AE1 gene (Fig. 1A). These clones presumably resulted from premature termination during the reverse transcriptase reaction. Six clones were identical to the AE1-1 transcript, and their 5` ends corresponded to the nucleotide immediately 3` of the previously defined transcription start site of the erythroid AE1-1 transcript (11) . Four clones extended further 5` in the AE1 gene than the putative transcription initiation site of the AE1-1 transcript(11) , with the largest clone extending 21 nucleotides upstream of this site. Whether the erythroid AE1-1 transcripts initiate at a site upstream of that previously determined (11) will be addressed in the RNase protection analyses described below.
Two additional clones were isolated that contained
a novel 153-nucleotide insert between exon 4 and exon 10. Exon 10 is
the first exon that is held in common among all of the variant
erythroid and kidney AE1 transcripts (Fig. 1). Data base
searches have shown that the amino acid sequence encoded by this
153-nucleotide insert (Fig. 1B) is not homologous to
the sequence of any of the previously characterized anion exchanger
gene members. Sequence analysis of the region of the AE1 gene that
contains this 153-nucleotide insert has shown that this sequence is
derived from two exons, 5 and 6, that are separated by an intron of 102
nucleotides (Fig. 2A). Exons 5 and 6 lie immediately
upstream of the exons at the 5` ends of the variant kidney AE1
transcripts, AE1-3, AE1-4, and AE1-5.
Figure 2:
Analysis of the structure of AE1
transcripts containing exons 5 and 6 by RT-PCR. Poly(A) RNA isolated from 10-day embryonic erythroid cells was reverse
transcribed using a primer complementary to nucleotides 3017-3037
of the erythroid AE1-1 transcript(10, 11) . This first
strand cDNA was PCR amplified using an antisense primer complementary
to nucleotides 74-95 of exon 6 (B, lanes
1-6) or an antisense primer complementary to nucleotides
54-74 of exon 10 (B, lanes 7-12). Each of
these antisense primers was used in combination with three different
sense primers; 1S (B, lanes 1, 2, 7, and 8), 1`S (B, lanes 3, 4, 9,
and 10), or 2S (B, lanes 5, 6, 11, and 12). The location of the sequences in the AE1
gene corresponding to each primer is illustrated in A. The even-numbered lanes in B correspond to PCR
amplifications carried out in the absence of first strand cDNA
template. The amplification products were electrophoresed on a 1.5%
agarose gel and stained with ethidium bromide (B).
Alternatively, the amplification products were transferred to
nitrocellulose and probed with a
P-end-labeled
oligonucleotide corresponding either to nucleotides 11-31 in exon
3 (C), nucleotides 14-34 in exon 4 (D), or
nucleotides 1-21 in exon 6 (E). The organization and
size of the exons at the 5` end of the AE1 gene are depicted in A. The filled box at the 5` end of exon 2 corresponds
to the 21 nucleotides 5` of the previously defined transcription
initiation site of the erythroid AE1-1 transcript(11) . Open boxes correspond to introns in the variant erythroid AE1
transcripts and are not drawn to scale. Exons 7, 8, and 9, which are
not illustrated in this diagram, are not part of the transcripts
derived from the P1 and P2 promoters. The markers in B correspond to pGEM-3 DNA digested with HinfI restriction
endonuclease.
The 5` termini of
both of the cDNAs that contained exons 5 and 6 resided within exon 4 of
the AE1 gene (Fig. 1A). Because the AE1-1 and AE1-2
transcripts, which are derived from the P1 and P2 promoters,
respectively, each contain exon 4 of the AE1 gene, it was possible that
transcripts containing exons 5 and 6 initiated transcription from one
or both of these erythroid-specific AE1 promoters. To further
investigate the structure of transcripts containing exons 5 and 6,
RT-PCR analyses have amplified the sequence at the 5` end of AE1
transcripts present in RNA isolated from 10-day embryonic erythroid
cells. Poly(A) erythroid RNA was reverse transcribed
using a primer complementary to nucleotides 3017-3037 of the
erythroid AE1-1 transcript. This first strand cDNA was PCR amplified
using an antisense primer complementary to a sequence in exon 6 (6AS in Fig. 2A) in combination with one of three different
sense primers. The first sense primer, 1S, corresponded to nucleotides
11-31 of the erythroid AE1-1 transcript (10, 11) . The second sense primer, 1`S, corresponded
to the 21 nucleotides 5` of the putative transcription initiation site
of the erythroid AE1-1 transcript in the AE1 gene(11) . The 1`S
sequence was contained in one of the cDNA clones originally isolated by
RT-PCR RACE. The third sense primer, 2S, corresponded to a sequence in
the 5` untranslated first exon of the erythroid AE1-2
transcript(11) . The location of these sense primers is
illustrated in Fig. 2A. Interestingly, amplifications
using either the 1S (Fig. 2B, lane 1), 1`S (Fig. 2B, lane 3), or 2S (Fig. 2B, lane 5) sense primer resulted in two
amplification products.
To determine the origin of these species,
the amplification products were transferred to nitrocellulose, and the
filters were probed with P-end-labeled oligonucleotides
corresponding either to nucleotides 11-31 in exon 3 (Fig. 2C) or to nucleotides 14-34 in exon 4 (Fig. 2D) of the AE1 gene. Both of the amplification
products in each lane hybridized with the exon 4-specific
oligonucleotide (Fig. 2D, lanes 1, 3,
and 5), whereas only the larger product hybridized with the
exon 3-specific oligonucleotide (Fig. 2C, lanes
1, 3, and 5). The observation that the exon
3-specific oligonucleotide weakly hybridized with the amplification
products generated with the 1S and 1`S sense primers (Fig. 2C, lanes 1 and 3) was not
observed when an oligonucleotide corresponding to nucleotides
50-70 in exon 3 was used as a probe. The hybridization pattern
obtained with the oligonucleotide corresponding to nucleotides
50-70 in exon 3 mirrored the ethidium bromide staining pattern
observed for the larger amplification product in each lane (data not
shown). Sequence analysis of the PCR products in Fig. 2B (lanes 1-6) that hybridized with the exon 3- and
exon 4-specific oligonucleotides verified that these products were
derived from AE1 transcripts that contain exons 4, 5, and 6, and either
lack or contain exon 3.
These results indicate that some of the transcripts initiating with the 5`-untranslated first exon of both the AE1-1 and the AE1-2 transcripts contain exons 5 and 6. In addition, they indicate that AE1 transcripts derived from both the P1 and P2 promoters in 10-day embryonic erythroid cells either lack (Fig. 2, lower band in each amplification) or contain (Fig. 2, upper band in each amplification) exon 3 of the AE1 gene. Finally, the data indicate that at least some of the transcripts derived from the P1 promoter initiate transcription upstream of the previously defined transcription initiation site(11) , because an identical profile of PCR amplification products was obtained when using the 1S or 1`S sense primers (compare lanes 1 and 3 of Fig. 2B).
To
further investigate the structure of AE1 transcripts containing exon 6,
200 ng of poly(A) erythroid RNA was electrophoresed on
a formaldehyde-agarose gel, transferred to nitrocellulose, and probed
either with a
P-labeled antisense transcript complementary
to nucleotides 2790-3068 of the erythroid AE1-1 transcript (Fig. 3, lane 1; this sequence is present in all the
previously characterized chicken AE1 transcripts) or with a
P-labeled antisense transcript complementary to
nucleotides 1-95 of exon 6 (Fig. 3, lane 2). This
analysis revealed that the exon 6-specific probe hybridizes with a
major transcript of
4.7 kilobases, as well as minor species that
range in size from
4.8 to
5.1 kilobases (Fig. 3, lane 2). In contrast, a diffuse RNA species that was centered
at
4.6 kilobases was detected with the probe that recognizes all
of the AE1 variants (Fig. 3, lane 1). The major exon
6-containing transcript comigrates with the slower migrating
transcripts recognized by the probe that detects all of the variants,
consistent with the additional 153 nucleotides contained within exons 5
and 6. This result suggested that like the previously characterized AE1
transcripts, the transcripts containing exons 5 and 6 may only differ
from the other variants in the sequences present at their 5` ends. To
insure this was the case, cDNAs have been cloned by RT-PCR using an
exon 6-specific sense primer and an antisense primer complementary to a
sequence in the 3` untranslated region of all the chicken erythroid and
kidney AE1 cDNAs that have been characterized. Sequence analysis of
these clones has indicated that the sequence 3` of exon 6 is identical
to the sequence of the other AE1 variants.
Figure 3:
RNA blotting analysis of the exon
6-containing transcripts in chicken erythroid cells. 200 ng of
poly(A) RNA isolated from 10-day embryonic erythroid
cells was electrophoresed on a formaldehyde-agarose gel and transferred
to nitrocellulose. These nitrocellulose filters were probed either with
a
P-labeled antisense transcript complementary to
nucleotides 2790-3068 of the erythroid AE1-1 transcript (lane
1) or with a
P-labeled antisense transcript
complementary to nucleotides 1-95 of exon 6 (lane 2).
Following washing, the hybridizing species were detected by
autoradiography using an intensifying screen. Markers correspond to transcripts of known
size.
The analyses described
above have indicated that transcripts containing exons 5 and 6 are
derived from both the P1 and P2 promoters of the AE1 gene, and these
transcripts either lack or contain exon 3. Additional RT-PCR studies
have examined whether similar complexity exists among the AE1
transcripts that lack exons 5 and 6. In this experiment, the first
strand cDNA was amplified with the same three sense primers described
above, 1S, 1`S, and 2S (Fig. 2A), and with an antisense
primer complementary to nucleotides 54-74 in exon 10 (10AS in Fig. 2A), which is the first exon held in common among
all of the variant AE1 transcripts(9, 10) . Analysis
of the resulting amplification products revealed two major ethidium
bromide-stained species (Fig. 2B, lanes 7, 9, and 11). To determine the origin of these species,
the amplification products were transferred to nitrocellulose and
probed with P-labeled oligonucleotides corresponding to a
sequence in exon 3 (Fig. 2C), a sequence in exon 4 (Fig. 2D), or a sequence in exon 6 (Fig. 2E) of the AE1 gene. The two major species (Fig. 2B, lanes 7, 9, and 11) in each amplification correspond to transcripts that lack
exons 5 and 6 and either lack (the faster migrating species) or contain
(the slower migrating species) exon 3. Additional species are also
visible in the autoradiograms that correspond to transcripts containing
exons 5 and 6. Longer exposure of the autoradiogram in Fig. 2C has indicated that the slower migrating species
that hybridizes with the exon 6-specific probe (Fig. 2E, lanes 7, 9, and 11) also hybridizes with the exon 3-specific probe (data not
shown). The faster migrating species that hybridizes with the exon
6-specific probe (Fig. 2E, lanes 7, 9, and 11) presumably corresponds to transcripts
containing exons 5 and 6 that lack exon 3. As mentioned above, the
difference in the extent of hybridization of the PCR products derived
from transcripts from the P1 (Fig. 2C, lanes 7 and 9) and P2 (Fig. 2C, lane 11)
promoters with the exon 3-specific probe is not observed when another
exon 3-specific oligonucleotide is used as a probe (data not shown).
These results indicate that PCR reactions using each of these sense
primers result in four amplification products, indicating that at least
four erythroid AE1 transcripts initiate transcription from both the P1
and P2 promoters.
The exon organization at the 5` end of each of the erythroid AE1 transcripts is illustrated in Fig. 4. Because the four variants derived from each of the promoters have identical coding sequences, we have named the variants AE1-1a, AE1-1b, AE1-1c, AE1-1d, AE1-2a, AE1-2b, AE1-2c, and AE1-2d. The 1 and the 2 in the nomenclature refer to the promoter from which the transcript was derived, and a, b, c, and d refer to the four different structural variants. Finally, four additional AE1 transcripts, AE1-1`a, AE1-1`b, AE1-1`c, and AE1-1`d, are expressed in erythroid cells. These transcripts initiate transcription at least 21 nucleotides upstream (filled box at the 5` end of exon 2 in Fig. 2A) of the previously defined transcription initiation site of the AE1-1 transcript. Whether all of the AE1-1 variants initiate transcription from this site will be examined in the RNase protection analyses described below.
Figure 4: Exon organization at the 5` end of the variant transcripts derived from the erythroid-specific promoters of the chicken AE1 gene. The exon organization at the 5` end of the variant transcripts derived from the P1 and P2 promoters of the AE1 gene is illustrated. The 1 and 2 in the nomenclature refer to the promoter from which the transcript was derived, and a, b, c, and d refer to the four different structural variants. The filled box at the 5` end of the variant AE1-1 transcripts represents the sequence present in transcripts that initiate from the minor transcription initiation site of the P1 promoter. The regions that are shaded are held in common among all of the variant transcripts.
Densitometric scanning of these protection analyses has revealed
that transcripts containing exons 5 and 6 comprise 12% of the
erythroid AE1 transcripts. The value obtained for the percentage of AE1
transcripts containing exons 5 and 6 from this quantitative protection
analysis is much higher than the PCR analyses had suggested. In
addition, these experiments have shown that the transcripts lacking
exon 3 comprise
37% of the total AE1 transcripts.
Additional
RNase protection analyses have determined the percentage of exon 3- and
exon 4-containing transcripts that are derived from the P1 and P2
promoters of the AE1 gene. For these studies, constructs containing
exons 3 and 4 were generated that initiated either with the
5`-untranslated first exon of transcripts derived from the P2 promoter
or with the 5`-untranslated first exon of transcripts derived from the
P1 promoter (Fig. 6C). The RNA probe corresponding to
transcripts derived from the P1 promoter possesses 21 additional
nucleotides at its 5` end that are not present in the previously
characterized AE1-1 transcript(11) . These additional
nucleotides correspond to the 21 nucleotides in the AE1 gene that are
immediately upstream of the putative transcription initiation site of
the AE1-1 transcript(11) . P-labeled in vitro transcripts derived from these constructs were hybridized to 1
µg of poly(A)
RNA isolated from 10-day embryonic
erythroid cells. Following RNase digestion, protected fragments of 92
and 168 nucleotides were observed with each probe (Fig. 6, A and B, lanes 2). These fragments correspond to
exon 4 alone and exons 3 and 4 together. Again these protection
fragments are slightly larger than predicted due to the conserved
sequences at the 3` end of each of the exons. In addition, a
full-length protected product was observed in each of the protections.
However, the full-length protected fragments corresponding to
transcripts derived from the P1 promoter (Fig. 6B,
2-3-4 and 2`-3-4) are much more prominent than the
full-length protected fragment corresponding to the transcripts derived
from the P2 promoter (Fig. 6A, 1-3-4). The
additional bands in the protection analyses in Fig. 5and Fig. 6that were absent in control lanes were reproducibly
observed, and at this time their origin is unknown. Densitometric
scanning of the protections in Fig. 6has indicated that
99% of the exon 3- and exon 4-containing transcripts initiate from
the P1 promoter, whereas only
1% initiate from the P2 promoter.
Furthermore, this analysis has revealed that 5% of the transcripts
derived from the P1 promoter initiate transcription at a site upstream
of the previously defined start site for transcription (protected
fragment marked 2`-3-4 in Fig. 6B), whereas
95% of the transcripts derived from this promoter initiate
transcription from the previously defined start site (11) (protected fragment marked 2-3-4 in Fig. 6B).
Figure 6:
Quantitation of the relative abundance of
the transcripts derived from the two erythroid-specific AE1 promoters
by RNase protection analyses. Two constructs were generated in the
pGEM-3 vector that contained exons 3 and 4 (C). The first
construct initiated with the 5`-untranslated first exon of the variant
AE1-2 transcripts (A, lanes 1-3), and the
second construct initiated with the 5`-untranslated first exon of the
variant AE1-1 transcripts (B, lanes 1-3). The cross-hatched box corresponds to the 21 nucleotides 5` of the
previously defined transcription initiation site of the erythroid AE1-1
transcript in the AE1 gene(11) , and the black boxes in each construct represent vector sequences (C). P-labeled antisense transcripts were generated from each
construct and hybridized to 1 µg of poly(A)
RNA
isolated from 10-day embryonic erythroid cells (A and B, lanes 2). Following hybridization, samples were
digested with RNase, and the products were resolved on a 6%
polyacrylamide/7 M urea gel. Lanes 1 in A and B correspond to the probe alone. Lanes 3 in A and B correspond to control hybridizations and
digestions carried out in the presence of the probe and tRNA alone. The
protected fragments corresponding to exon 4; exons 3 and 4; exons 1, 3,
and 4; exons 2, 3, and 4; and exons 2`, 3, and 4 are indicated on the right hand side of the figures. Markers in A and B correspond to in vitro transcripts of
known size.
Figure 7:
Expression of the variant AE1 transcripts
in chicken erythroid cells of the primitive lineage. Poly(A) RNA isolated from 3-day-old chicken embryos was reverse
transcribed using a primer complementary to nucleotides 3017-3037
of the erythroid AE1-1 transcript(10, 11) . This first
strand cDNA was PCR amplified using an antisense primer complementary
to nucleotides 74-95 of exon 6 (lanes 1-6) or an
antisense primer complementary to nucleotides 54-74 of exon 10 (lanes 7-12). Each of these antisense primers was used
in combination with three different sense primers: 1S (lanes
1, 2, 7, and 8), 1`S (lanes 3, 4, 9, and 10), or 2S (lanes 5, 6, 11, and 12). The location of the sequence
in the AE1 gene corresponding to each primer is illustrated in Fig. 2A. The even-numbered lanes correspond to
PCR amplifications carried out in the absence of first strand cDNA
template. The amplification products were electrophoresed on a 1.5%
agarose gel and stained with ethidium bromide (A).
Alternatively, the amplification products were transferred to
nitrocellulose and probed with a
P-end-labeled
oligonucleotide corresponding either to nucleotides 11-31 in exon
3 (B), nucleotides 14-34 in exon 4 (C), or
nucleotides 1-21 in exon 6 (D). The markers correspond to pGEM-3 DNA digested with HinfI restriction
endonuclease.
Figure 8:
Immunolocalization of the variant
erythroid AE1 anion exchangers in transfected HEL cells. cDNAs encoding
the 99- (A),
102- (B), and
108-kDa (C) erythroid AE1 variants were subcloned into the pcDNA3
vector and transfected into HEL cells. 48 h following transfection, the
cells were fixed, permeabilized in 0.5% Triton X-100 in PBS, and
incubated with the pAE1A antibody. Following washing in PBS, the cells
were incubated with donkey anti-rabbit IgG conjugated to lissamine. The
images in A-C correspond to 0.5-µm optical sections
collected on a Bio-Rad confocal laser scanning microscope. Each image
is approximately 1.5 µm from the top of the cell. The arrows in C mark the location of the plasma membrane in this
transfected cell. The fluorescence surrounding the plasma membrane of
the cell in B is due to the background staining of immediately
adjacent, nontransfected cells. The bar is 3
µm.
These analyses revealed that the variant AE1 anion exchangers were
targeted to different membrane compartments in transfected HEL cells (Fig. 8). The 99-kDa anion exchanger, which contains exon
4, accumulated exclusively in the plasma membrane of transfected HEL
cells (Fig. 8A). In >80% of the cells expressing the
102-kDa anion exchanger, which contains exons 3 and 4, this
variant transporter also accumulated exclusively in the plasma membrane (Fig. 8B). Although the vast majority of staining for
the remainder of the cells expressing the
102-kDa variant was also
restricted to the plasma membrane, some staining was observed in a
perinuclear compartment of these cells (data not shown). In contrast to
the patterns of localization observed for the
99- and
102-kDa
variants in transfected HEL cells, the
108-kDa anion exchanger,
which contains exons 3, 4, 5, and 6, accumulated in a perinuclear
compartment of transfected cells (Fig. 8C). The
vesicular perinuclear staining observed for the
108-kDa variant
was often in the form of a cap that was restricted to one side of the
nucleus. However, the staining was sometimes seen to entirely surround
the nucleus (Fig. 8C). Identical results were obtained
in four separate experiments in which at least 100 cells were scored
for each of the variant constructs. In addition, immunolocalization
analyses using secondary antibody alone or carried out in the presence
of the peptide against which the pAE1A antiserum was generated
exhibited background levels of staining, whereas a nonspecific peptide
had no effect on the staining patterns described above (data not
shown). At this time, the identity of the intracellular compartment
where the
108-kDa AE1 variant accumulates is not known.
Nonetheless, these data suggest that the alternative N-terminal
sequences of the erythroid AE1 anion exchangers serve as signals to
direct these variant transporters to different membrane compartments
within cells.
The results described here have indicated that transcripts
encoding polypeptides of 99,
102,
104, and
108 kDa
are derived from both of the erythroid-specific promoters, P1 and P2,
of the chicken AE1 gene. The predicted sizes of these polypeptides
agree reasonably well with previous immunoblotting analyses that have
demonstrated the presence of four AE1 anion exchanger species of
95,
99,
102, and
108 kDa in membranes isolated from
chicken erythroid cells(9) . This cDNA cloning analysis has
indicated that the diversity observed among the four variant chicken
erythroid AE1 anion exchanger polypeptides is restricted to the N
terminus of their cytoplasmic domains. Similar N-terminal cytoplasmic
diversity has also been observed among the three variant anion
exchangers, AE1-3, AE1-4, and AE1-5, that are encoded by the AE1 gene
in chicken kidney(9) . Immunolocalization analyses have
suggested that the alternative N-terminal cytoplasmic sequences of the
kidney AE1 anion exchangers may be involved in targeting these variant
transporters to different membrane compartments in kidney epithelial
cells. (
)Similarly, the different patterns of intracellular
localization exhibited by the variant erythroid AE1 anion exchangers
that have been transiently expressed in HEL cells suggest that the
alternative N-terminal sequences of these polypeptides may serve as
signals to target these transporters to different membrane compartments
within these cultured cells.
Previous studies have shown that murine
erythroid AE1 anion exchangers exclusively accumulate in a pre-Golgi
compartment rather than in the plasma membrane of transfected human
embryonic kidney cells(18) . Anion transport assays (18) and the ability of the transfected murine AE1 anion
exchanger to be coimmunoprecipitated with the anion exchanger binding
domain of ankyrin (19) have suggested that the retention of the
murine AE1 anion exchanger in this perinuclear compartment is not due
to misfolding. The data presented here have shown that the 99- and
102-kDa chicken AE1 variants are transported to the plasma
membrane of transfected HEL cells, which upon stimulation can
differentiate into erythroid cells, whereas the
108-kDa AE1
variant is retained in a perinuclear compartment. The observation that
some of the AE1 variants undergo transport to the plasma membrane in
transfected HEL cells indicates that HEL cells, unlike the human
embryonic kidney cells, contain all of the components necessary for the
plasma membrane targeting of these anion exchangers. Furthermore, the
demonstration that the cellular sorting machinery of HEL cells can
target these electroneutral transporters to the plasma membrane
suggests that the
108-kDa AE1 variant, which accumulates in a
perinuclear compartment of transfected HEL cells, may be specifically
retained in the endoplasmic reticulum or Golgi.
Immunolocalization
analyses of other investigators have shown that antibodies generated
against a peptide corresponding to the C terminus of the murine
erythroid AE1 anion exchanger recognize a polypeptide of 115 kDa
that is localized to the Golgi complex of a variety of cell
types(20) , suggesting that AE1 anion exchanger-like
polypeptides may participate in the maintenance of pH equilibrium in
this organelle. The observation that the
108-kDa erythroid AE1
variant is preferentially retained in a perinuclear compartment of
transfected HEL cells, whereas variants lacking exons 5 and 6 are
primarily transported to the plasma membrane, suggests that the amino
acid sequence of exons 5 and 6 may serve as a signal for retention in
the endoplasmic reticulum or Golgi. Short cytoplasmic sequences have
previously been shown to serve as retrieval signals for some type I
membrane proteins to the endoplasmic reticulum (21, 22) and the trans Golgi
network(23, 24) . However, these sequences share no
obvious similarity with the sequence of exons 5 and 6. The recent
demonstration that isoforms of ankyrin (25) and spectrin (26) are localized to the Golgi complex suggests the
possibility that variants containing exons 5 and 6 associate with the
Golgi-specific isoforms of these membrane cytoskeletal polypeptides,
thereby retaining these variants in this intracellular compartment.
Alternatively, the sequence of exons 5 and 6 could act by inhibiting
the ability of variants containing this sequence to interact with
elements of the HEL transport machinery involved in transport to the
plasma membrane, or these cells may simply lack the elements required
for the plasma membrane transport of this variant. Although we cannot
rule out that the retention of variants containing exons 5 and 6 in
this as yet undefined perinuclear compartment of HEL cells is due to
misfolding of the polypeptides, the fact that the variants lacking
exons 5 and 6 are sorted to the plasma membrane suggests that this
possibility is unlikely.
Previous studies have shown that the
extreme N terminus of the human erythroid AE1 anion exchanger is one of
several regions of this polypeptide that are involved in mediating its
association with erythroid ankyrin(27, 28) .
Furthermore, recent studies have shown that the alternative N-terminal
sequences of the variant chicken kidney AE1 anion exchangers affect
their capacity to associate with the detergent insoluble membrane
cytoskeleton. At this time there is no evidence that the
diversity at the N terminus of the variant chicken erythroid AE1 anion
exchangers affects their ability to associate with the membrane
cytoskeleton. However, it is possible that the alternative N-terminal
sequences of these variant transporters may regulate their interaction
with the membrane cytoskeleton. Regulating this interaction may in turn
provide a mechanism for targeting these variant polypeptides to
different membrane compartments within cells.
The studies described above have shown that transcripts encoding each of the variant AE1 anion exchanger polypeptides are derived from both the P1 and P2 promoters of the AE1 gene. RNase protection analyses have shown that the transcripts derived from the P1 promoter are much more prevalent than the transcripts derived from the P2 promoter. In addition, these studies have shown that transcripts derived from the P1 promoter initiate transcription from two sites. The primary site of transcription initiation from the P1 promoter corresponds to that previously defined from primer extension analyses(11) . Although the secondary transcription initiation site from the P1 promoter has not been precisely mapped, cDNA cloning and RNase protection analyses have shown that it lies at least 21 nucleotides upstream of the primary transcription start site. Initiation of transcription from multiple sites has also been shown to occur at the erythroid-specific promoter of the AE1 anion exchanger gene in both mouse (29) and rat(30) . Interestingly, the transcripts that initiate from the secondary initiation site of the P1 promoter also encode each of the four AE1 structural variants. The diversity observed among the transcripts derived from the P1 and P2 promoters is made even more remarkable by the fact that the 12 variant AE1 transcripts encode only four different structural polypeptides.
Although transcription from alternative promoters that results in the production of polypeptides that differ at their N terminus has been reported for several genes(13, 17, 31) , the chicken erythroid AE1 anion exchanger transcripts are unique in that variant transcripts encoding identical structural polypeptides are simultaneously derived from two separate promoters. Previous studies have also shown that the use of alternative promoters can be developmentally regulated(17) . To investigate whether the two erythroid-specific AE1 promoters, P1 and P2, are differentially regulated during development, RT-PCR analyses have determined the array of AE1 transcripts expressed in erythroid cells in 3-day-old and 10-day-old chicken embryos. The erythroid cells isolated from 10-day-old chicken embryos are almost entirely of definitive lineage in origin, whereas the erythroid cells in 3-day-old embryos are exclusively derived from cells of the primitive lineage (16) . These studies have shown that each of the 12 variant AE1 transcripts that accumulate in erythroid cells isolated from 10-day-old embryos also accumulate in erythroid cells isolated from 3-day-old embryos. Because the erythroid AE1 anion exchanger is involved in the maintenance of pH equilibrium in the plasma, the redundancy observed in the gene products derived from the two erythroid-specific AE1 promoters may provide a mechanism to insure the high level expression of these electroneutral transporters during all stages of chicken embryonic development. Alternatively, these two promoters may differentially respond to different environmental stimuli, thereby increasing the capacity of erythroid cells to regulate the expression of the anion exchanger under different environmental conditions.