From the
We have constructed cDNA clones encoding various portions of the
human red cell anion transporter (band 3), a well characterized
integral membrane protein with up to 14 transmembrane segments. The
biosynthesis, stability, cell surface expression, and functionality of
these band 3 fragments were investigated by expression from the cRNAs
into microsomal membranes using the reticulocyte cell-free translation
system and in Xenopus oocytes. Co-expression of the pairs of
recombinants encoding the first 8 and last 6 transmembrane spans
(8+6) or the first 12 and last 2 spans (12+2) of band 3
generated stilbene disulfonate-sensitive anion transport in oocytes.
When the pairs of fragments 8+6 or 12+2 were co-expressed
with glycophorin A (GPA), translocation to the plasma membrane of the
fragment corresponding to the first 12 or the first 8 transmembrane
spans was greater than in the absence of GPA. Only the fragment
encoding the first 12 transmembrane spans showed GPA-dependent
translocation when expressed in the absence of its complementary
fragment. A truncated form of band 3 encoding all 14 transmembrane
spans but lacking the carboxyl-terminal 30 amino acids of the
cytoplasmic tail did not induce anion transport activity in oocytes and
was not translocated to the plasma membrane but appeared to be degraded
in oocytes. Our results suggest that there is no single signal for the
insertion of the different transmembrane spans of band 3 into membranes
and that the integrity of the loops between transmembrane spans
8-9 or 12-13 is not essential for anion transport function.
Our data also suggest that a region of transmembrane spans 9-12
of band 3 is involved in the process by which GPA facilitates the
translocation of band 3 to the surface.
The human erythrocyte anion transporter (band 3, AE1) is the
most abundant integral membrane protein in red cells (reviewed in Refs.
1-6). This 911-amino acid protein comprises two domains that are
structurally and functionally distinct: the amino-terminal 43-kDa
cytoplasmic domain binds cytoskeletal and other peripheral membrane
proteins, whereas the carboxyl-terminal 52-kDa integral membrane domain
is responsible for red cell anion exchange. A combination of hydropathy
analysis on the amino acid sequence, proteolysis, and various chemical
and antibody binding studies suggest that the polypeptide chain
traverses the lipid bilayer up 14 times (reviewed in Refs. 2-4, 6
and 7).
Our current knowledge of the three-dimensional structure of
integral membrane proteins is very limited in comparison with the
wealth of data that is available for soluble proteins. Most of the
structures that have been obtained at high resolution (with the
exception of the bacterial porins) have indicated that these proteins
are based upon bundles of hydrophobic transmembrane
In this
paper, we describe the expression of two pairs of cRNAs representing
fragments of band 3 and show that, while the individual fragments are
not competent for transport, the pairs of fragments are able to
assemble to form a structure that carries out anion transport with the
stilbene-disulfonate sensitivity that is characteristic of band 3.
The recombinants encoding fragments containing
the amino-terminal portion of the protein ( i.e. truncated at
the carboxyl terminus) were prepared by incorporating a translation
termination codon into the coding sequence of band 3 and deleting the
region of the cDNA on the 3`-side of this termination codon. Each
antisense PCR primer contained the sequence 5`-GGCGGTAACCTCA-3` (which
includes a BstEII restriction enzyme site and a termination
codon), followed by the particular 17-20-nucleotide sequence,
which perfectly matches the band 3 cDNA encoding the 5-7 amino
acids of band 3 immediately prior to the required carboxyl-terminal
truncation. For example, the antisense primer used to prepare construct
b3(1:12), which is carboxyl-terminally truncated after residue
Tyr
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy For each of these PCR reactions, the sense primer was
5`-TCCCTGGCCCTGCCCTT-3`, which anneals to the portion of the band 3
cDNA encoding amino acid residues 856-861 and which is upstream
of the BstEII site in the 3`-untranslated region of
There combinantsen coding fragments containing the
carboxyl-terminal portion of the protein ( i.e. truncated at
the amino terminus) were prepared by inserting a methionine codon and
deleting there gion of the band 3 cDNA upstream of this protein
initiator codon. Eachsense PCR primer contained the sequence
5`-CCGCCATGG-3` (which includes an NcoI restriction enzyme
site and a methionine codon), followed by the particular
15-20-nucleotide sequence, which perfectly matched the portion of
band 3 cDNA encoding the first 5-6 amino acids of band 3 that
would follow in-frame with the initiator methionine of the
amino-terminally truncated protein. For example, the sense primer used
to prepare construct (13:14), which initiates at residue 825 in the
loop between transmembrane spans 12 and 13, was as follows.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy For each of these PCR reactions, the antisense primer
5`-TAGTCTGTGGCTGTTGCCT-3` was used. This anneals to the portion of the
band 3 cDNA encoding amino acid residues 46-41 of the protein and
downstream of the NcoI site of pBSXG1.b3, which includes the
codon corresponding to the initiator methionine of band 3. In each
case, the amplified cDNA was digested with NcoI,
recircularized, and cloned.
The COOH-terminally truncated mutant
b3(1:14*) was constructed from pBSXG1.b3 by site-directed mutagenesis
(Amersham Sculptor kit) using the oligonucleotide
5`-AGGAACGTGTAGCTTCAGT-3`, which mutates the GAG codon that corresponds
to amino acid Glu
The
sequences of cDNA constructs were verified using either a DuPont
Genesis 2000 automated sequencer or by manual double-stranded dideoxy
sequencing using Sequenase (U. S. Biochemical Corp.) according to the
manufacturer's protocols.
The band 3 membrane domain
fragments mem(1:8) and mem(1:12) were similar to the intact band 3
mutants b3(1:8) and b3(1:12), except that in these constructs the
cytoplasmic domain of band 3 (amino acid residues 1-359) was
replaced by a single initiator methionine residue.
Cell-free translation
of the amino-terminal fragments b3(1:12) and b3(1:8) in the presence of
canine pancreatic microsomes resulted in two closely spaced bands on
SDS-PAGE, with the higher molecular weight band predominating
(Fig. 2 a). Inclusion of the N-linked
glycosylation acceptor NLT peptide at 30 µ
M in the
translation mixture caused the intensity of the lower band to increase
and the upper band to decrease in each case, corresponding to a
50-75% inhibition of core N-glycosylation, similar to
the effect of this peptide on intact band 3
(12) . Cell-free
translation of each fragment in the absence of microsomes resulted in a
single band on SDS-PAGE, running in the same position as the lower
(unglycosylated) band obtained in the presence of microsomes. These
results demonstrate that the amino-terminal fragments are all
N-glycosylated at a single site, similarly to intact band 3.
A lower yield of each of
the four fragment proteins was obtained from cell-free translation in
the absence of microsomes. This suggests that the integration of
nascent proteins into microsomes is important for efficient protein
synthesis. A similar effect is observed in the absence of microsomes on
translation of intact band 3 (data not shown).
A parallel set of microsomal
membranes containing the protein were treated with the detergent Triton
X-100 prior to trypsin digestion to examine the susceptibility of
solubilized band 3 to proteolysis. In the detergent-treated samples
that had been digested either with 3 or 10 µg/ml trypsin, the
47-kDa band 3 membrane domain and the products running at approximately
32 and 26 kDa were not visible (Fig. 3 a, lanes 4 and 6), but the 21-kDa fragment was not affected by prior
solubilization with Triton X-100. Immunoprecipitation with BRIC 155
confirmed that the band 3 membrane domain was degraded by the trypsin
under these conditions in the presence of detergent
(Fig. 3 a, lanes 10 and 12).
The 43-kDa cytoplasmic domain was more resistant to proteolysis in the
presence of Triton X-100 than the membrane domain and was only digested
partially by 10 µg/ml trypsin in the presence of detergent
(Fig. 3 a, lane 6). These results
indicate that detergent solubilization causes the membrane domain of
band 3 to become susceptible to digestion by trypsin at various sites
that are normally cryptic to the enzyme when the protein is present in
microsomal membranes.
In a subsequent experiment, each of intact
band 3, b3(1:12), b3(1:8), (9:14), and (13:14) was translated and
incorporated into microsomes from their respective cRNAs using the
cell-free system. GPA cRNA was co-expressed with the band 3 fragment in
each of the translations. Although we have shown previously that
co-expression of GPA with band 3 does not affect sensitivity to trypsin
and hence the overall structure of the intact protein
(11) , we
could not exclude the possibility that GPA might influence the initial
folding of protein fragments. Furthermore, it is clear that the
presence or absence of GPA does affect the eventual conformation and
functional activity of the mature protein in the red cell
(16) .
A trypsin concentration of 5 µg/ml was selected for this analysis
of the topography and folding of the newly synthesized amino- and
carboxyl-terminal fragments on the basis of the results of the
experiment shown in Fig. 3 a. The products of trypsin
digestion were analyzed by SDS-PAGE and autoradiography (Fig.
3 b). GPA was not digested by trypsin, and the GPA monomer may
be seen in each lane running at approximately 25 kDa.
The
carboxyl-terminally truncated band 3 fragments b3(1:12) and b3(1:8)
both yielded proteolysis products that were in common with the
trypsin-treated intact protein, migrating at positions corresponding to
approximately 43 kDa (demonstrated by immunoprecipitation with BRIC
170, which reacts with the amino terminus of band 3, to be the
cytoplasmic domain (data not shown) and at 26 kDa
(Fig. 3 b, lanes 1-3).
Superimposed upon this pattern was a ``ladder'' of
distinctive proteolysis products running on the gel at apparent
molecular masses as follows: intact band 3, 51 kDa (demonstrated to
include the carboxyl terminus of band 3 by immunoprecipitation with
BRIC 155; data not shown); b3(1:12), 41 kDa; and b3(1:8), 32 kDa. These
proteolysis products correspond in size to the intact membrane domain
portion of each carboxyl-terminally truncated fragment, resulting from
cleavage after amino acid residue 359. The band 3 and b3(1:12) lanes
also contained another proteolysis product that was not present in
b3(1:8), running at approximately 35 kDa. This probably represents the
proteolysis product from the trypsin cleavage site at amino acid 360 to
that after residue 742. This tentative identification is supported by
the presence of faint bands at lower apparent molecular weights in both
band 3 and b3(1:12), which may represent the polypeptide from amino
acid 743 to the carboxyl terminus. Very small amounts of residual
undigested proteins may be seen running on the gel at apparent
molecular masses as follows: band 3, 97 kDa; b3(1:12), 86 kDa; and
b3(1:8), 70 kDa.
Trypsin treatment of the amino-terminally truncated
fragment (9:14) yielded a major band migrating at approximately 24 kDa
(Fig. 3 b, lane 6) that was slightly more
intense than the equivalent band derived from intact band 3 (Fig.
3 b, lane 7) and may contain amino acids
743-911. Trypsin digestion of intact band 3 yielded a product
migrating at approximately 28 kDa, which was of similar mobility to the
undigested fragment (9:14). Other bands were obtained migrating with
apparent molecular masses of 17 kDa in the case of (13:14) and at about
21 kDa in (9:14) and intact band 3.
The trypsin susceptibility of
both the amino- and carboxyl-terminally truncated fragments is
comparable with that of intact band 3, and these fragments are
therefore likely to be folded in a manner similar to that in the intact
protein. Furthermore, in the chymotrypsin cleavage experiment described
in Fig. 8(below), fragments b3(1:12) and b3(1:8) were cleaved at
the same site as intact band 3, with no indication of cleavage at
additional sites. These observations, together with the analysis of the
N-glycosylation of the fragments (Fig. 2), provide
further evidence that both the amino- and carboxyl-terminally truncated
band 3 fragments insert into the membrane with the correct topography
and form tightly folded structures.
None of the truncated
fragments b3(1:12), b3(1:8), (9:14), or (13:14) individually exhibited
a measurable level of DNDS-sensitive chloride uptake into the oocytes
(Fig. 4 a). However, co-injection of the pair of cRNAs
encoding fragments b3(1:8) and (9:14) and the pair of fragments
b3(1:12) and (13:14) did give rise to DNDS-sensitive chloride transport
(significant at the 0.1% level in t tests). In the case of
b3(1:12) and (13:14), the level of DNDS-sensitive transport induced in
the oocytes was almost as high as that obtained by injection of an
equal weight of the cRNA encoding intact band 3. These results show
that these two pairs of band 3 fragments are able to complement each
other to generate a protein structure that is competent for anion
transport, which shows the stilbene disulfonate sensitivity
characteristic of intact band 3.
Fig. 5
also shows that although a
modest level of DNDS-sensitive chloride transport was obtained with the
intact band 3 and intact band 3 membrane domain controls in the absence
of GPA, a very low level of DNDS-sensitive chloride influx was given by
all of the four pairs of fragments when GPA was not co-expressed. To
investigate whether GPA is essential for the expression of anion
transport activity of the fragments, higher concentrations (>8-fold)
of the cRNAs for b3(1:12) and (13:14) and for b3(1:8) and (9:14) were
expressed in oocytes in the absence of GPA cRNA (Fig. 6). A measurable
level of DNDS-sensitive anion transport was obtained under these
conditions by co-expression of b3(1:12) and (13:14) (significant at the
0.1% level); however, even using these high concentrations of cRNA, no
significant band 3-mediated chloride transport was detected in the
oocytes when the pair of fragments b3(1:8) and (9:14) was expressed
without GPA.
Groups of oocytes were injected with an equimolar amount of
the cRNA encoding either b3 (1:12) or b3(1:8) in either the presence or
absence of their respective complementary partners (13:14) and (9:14)
and the presence or absence of GPA. After pulse-chase labeling with
The results of the
immunoprecipitations are shown in Fig. 8. As expected
(11, 13) , the expression of intact band 3 at the cell
surface was markedly GPA dependent and attained a high level in the
presence of GPA (Fig. 8, lanes 9 and
10). Similarly high proportions of b3(1:12) and b3(1:8) were
expressed at the cell surface when expressed as complementary pairs
with (13:14) and (9:14), respectively, in the presence of GPA
(Fig. 8, lanes 4 and 8). Co-expression
of b3(1:12) with GPA in the absence of (13:14) also resulted in the
expression of b3(1:12) at the cell surface (Fig. 8, lane 2), although the level was reduced to about 60% of that
in the presence of both (13:14) and GPA (Fig. 8, lane 4). In contrast, co-expression of b3(1:8) and GPA in the
absence of (9:14) did not result in a significant amount of b3(1:8)
being expressed at the cell surface (Fig. 8, lane 6). This result was in marked contrast with the high
level of cell surface expression of b3(1:8) in the presence of both
(9:14) and GPA (Fig. 8, lane 8).
The
co-expression of GPA substantially increased the proportion of b3(1:12)
(Fig. 8, lanes 3 and 4) or b3(1:8)
(Fig. 8, lanes 7 and 8) expressed at
the cell surface when their respective complementary partners (13:14)
and (9:14) were also present. The extent of the increase in cell
surface expression induced by GPA was similar to that observed with
intact band 3 (Fig. 8, lanes 9 and
10). In the absence of (13:14), the proportion of b3(1:12)
expressed at the cell surface was markedly enhanced by the
co-expression of GPA (Fig. 8, lanes 1 and
2). This result shows that GPA is able to facilitate the
translocation of b3(1:12) to the plasma membrane in a manner similar to
that with the intact protein and that (13:14) is not essential for the
surface expression of b3(1:12). In contrast, very little b3(1:8) was
translocated to the plasma membrane when expressed without (9:14) in
either the presence of absence of GPA (Fig. 8, lanes 5 and 6).
Analysis by the chymotrypsin
digestion assay showed that the carboxyl-terminally truncated fragment
b3(1:14*) is not translocated to the plasma membrane of oocytes. The
amount of b3(1:14*) recovered in these experiments suggests this
construct is degraded in the oocytes, even though the more extensively
truncated fragments b3(1:8) and b3(1:12) are expressed stably in
oocytes. This low yield of b3(1:14*) was observed when the protein was
expressed in either the presence (Fig. 8, lane 12) or absence (Fig. 8, lane 11)
of GPA. Absence of the carboxyl terminus may cause the inappropriate
exposure of a site in the protein (presumably in the region of
transmembrane spans 13 and 14, between amino acid residues 825 and
881), which results in the intracellular retention and probable
degradation of the protein by the oocyte. The cytoplasmic
carboxyl-terminal tail of band 3 may be required for the expression at
the cell surface of a band 3 construct containing all 14 transmembrane
spans.
In this paper, we demonstrate that fragments of band 3
containing the first 8 and last 6 transmembrane spans co-expressed from
separate cRNAs in Xenopus oocytes generate chloride transport
activity in the oocyte plasma membrane with the stilbene disulfonate
sensitivity characteristic of intact band 3. Similarly, reconstitution
of anion transport activity can be achieved by co-expression of
fragments of band 3 containing the first 12 and last 2 transmembrane
spans from separate cRNAs. None of the individual fragments induced
anion transport in oocytes without their complementary partner, and
co-expression of GPA was required for high levels of cell surface
expression to be observed, as is found with the intact protein
(11, 13) . These results show that anion transport
activity is not sensitive either to the introduction of a breakage in
the polypeptide chain within the loops between transmembrane spans 8
and 9 or between spans 12 and 13 or to the consequent generation of
-COO
The properties of the fragment (13:14) are of some interest
since it contains two rather short hydrophobic segments. In the intact
protein, the two ends of this fragment are located in the cytoplasm
(reviewed in Refs. 4 and 7), while the region around Lys
The translocation of b3(1:12) to the cell
surface is faster when (13:14) is co-expressed than when b3(1:12) is
expressed without (13:14). Similarly, b3(1:8) moves faster to the cell
surface in the presence of (9:14) than in its absence. These results
suggest that the complementary pairs of fragments first associate in
internal membranes rather than at the plasma membrane and that the
complex is better able to translocate to the plasma membrane than
either b3(1:12) or b3(1:8) individually.
In the absence of (9:14),
fragment b3(1:8) was poorly expressed at the cell surface whether or
not GPA was co-expressed. However, the movement of b3(1:12) to the
plasma membrane in the absence of (13:14) was markedly accelerated by
the presence of GPA. These results, which were also obtained in a
duplicate set of experiments, suggest that the first 12 transmembrane
segments of band 3 are sufficient for GPA-dependent translocation to
the cell surface but that the first 8 spans alone will not suffice.
Since it was possible to restore GPA-dependent translocation of b3(1:8)
to the cell surface by complementation with (9:14), we conclude that a
portion of the region containing transmembrane segments 9-12 is
involved in the interaction between band 3 and GPA, which results in
the enhanced movement of band 3 to the oocyte surface.
GPA has been
shown to facilitate the translocation to the oocyte cell surface of the
mutant form of band 3 (band 3 SAO) present in Southeast Asian
ovalocytosis
(23) . Band 3 SAO (reviewed in Ref. 5) contains a
deletion of amino acid residues 400-408 (located at the
cytoplasmic boundary of the first transmembrane segment of normal band
3), which probably results in the mis-assembly of the membrane domain.
The deletion causes the loss of anion transport function and the
inability to bind stilbene disulfonates
(24) . However,
extracellular cleavage of band 3 SAO by chymotrypsin appears to occur
at the same site as normal band 3
(25, 26) , suggesting
that the loop between transmembrane spans 5 and 6 has the normal
extracellular location. It is likely that some or all of the
amino-terminal four transmembrane segments are mis-folded in this
variant form of band 3. The observation that the translocation of band
3 SAO to the plasma membrane is facilitated by GPA like the native
protein is consistent with a portion of the region around membrane
spans 9-12 being required for the GPA-dependent translocation of
band 3. However, it is possible that some part of the band 3 membrane
domain within transmembrane spans 5-8 is also involved in the
interaction with GPA.
A ``two-stage model'' has been
proposed to explain the process by which polytopic integral membrane
proteins attain their folded structure
(27) . This postulates
that transmembrane
The
complementary fragments we have prepared are able to generate stilbene
disulfonate-sensitive chloride transport and show several of the
properties of the intact protein (GPA-dependent translocation to the
surface, time-dependent accumulation at the cell surface, and
inhibitor-sensitivity of anion transport). The individual fragments
appear to take up tightly folded native-like structures, which can
combine with their complementary partners to give a structure similar
to that of intact band 3. While the association of the fragments may
involve some minor structural rearrangements (for example, movements of
the transmembrane helices), it seems unlikely that major unfolding and
refolding events occur because of the high energy barriers involved.
Our results are consistent with the ``two-stage model'' for
membrane protein biosynthesis
(27) , in which individual
transmembrane helices fold independently on insertion into the membrane
and retain this fold in the fully assembled protein.
We thank Dr. David Anstee for antibodies, Peter Martin
and Julian Ng for assistance with preparation of the mutants, and
Rhiannan Jowers for assistance with automated DNA sequencing.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-helices, and
the transmembrane spans of band 3 have been shown to be almost entirely
-helical in conformation
(8) . A low resolution (20
Å) two-dimensional model for structure of the membrane domain of
band 3 obtained from the study of two-dimensional crystals indicated
the presence of a mobile subdomain in this region of the protein
(9) . However, a recent three-dimensional map of the membrane
domain suggests that this mobile region is made up from the portions of
the membrane domain located in the cytoplasm
(10) .
Construction of Band 3 Fragments
The
cDNA clones encoding human band 3 (pBSXG1.b3), the band 3 membrane
domain (pBSXG1.b3 mem) and glycophorin A (pBSXG.GPA) have been
previously described
(11) . In these constructs, the
protein-coding cDNA is flanked by the 5`- and 3`-untranslated regions
of Xenopus -globin. cDNAs encoding fragments of band 3
truncated at the amino or carboxyl terminus of the protein were
prepared from pBSXG1.b3 and pBSXG1.b3 mem by PCR
(
)
as detailed below. In each case, the pairs of
oligonucleotide primers were designed to anneal to the band 3 cDNA with
a calculated T
of approximately
56-60 °C.
in the loop between transmembrane spans 12 and 13,
was as follows.
-globin. After amplification, the cDNAs were digested with
BstEII, recircularized, and cloned into Escherichia coli strain TG2.
to a TAG terminator codon.
Preparation of cRNAs and Expression of Band 3
Fragments
Protocols for synthesis of cRNA, cell-free
translation using rabbit reticulocyte lysate with canine pancreatic
microsomes, inhibition of N-glycosylation using the acceptor
tripeptide N-benzoyl-Asn-Leu-Thr- N-methylamide (NLT
peptide), purification of microsomes, trypsin treatment of microsomally
expressed proteins, oocyte isolation and cRNA injection, S
amino acid labeling and immunoprecipitation of proteins, chymotrypsin
treatment of oocytes, and chloride transport assay methods were all as
previously detailed
(11, 12, 13) . SDS-PAGE was
by the method of Laemmli
(14) or using the tricine buffer
system of Schägger and von Jagow
(15) .
Preparation of cDNAs
The PCR was used
to delete various portions of the coding sequence from pBSXG1.b3 and
pBSXG1.b3 mem cDNAs
(11) , which encode, respectively, for the
entire human band 3 (b3) and the membrane domain alone of band 3 (b3
mem, amino acids 360-911 of band 3). The amino- and
carboxyl-terminal truncations were each positioned in a loop that was
thought to be located in the cytoplasm in the model of 14 transmembrane
spans for the band 3 monomer
(4, 7, 10) . Band 3
fragments were prepared as ``complementary'' pairs, which
together contained all 14 putative transmembrane spans of the entire
band 3 membrane domain. The protein fragments derived from the
expression of these recombinants are illustrated in Fig. 1, which also
gives the exact locations of the truncations and nomenclature used for
each fragment. For example, fragment b3(1:12) is truncated after amino
acid residue 824 and includes the cytoplasmic domain and transmembrane
spans 1-12; likewise, the complementary fragment (13:14)
initiates with a methionine residue followed by amino acid residues
825-911 and includes transmembrane spans 13 and 14 and the
carboxyl terminus of the protein.
Characterization of Band 3 Fragments by Cell-free
Translation
For initial characterization of the fragments,
cRNA was prepared from each of the fragment cDNAs and translated in the
rabbit reticulocyte lysate cell-free system. Analysis by SDS-PAGE and
autoradiography demonstrated that the fragments gave the expected
Min each case (Fig. 2).
Figure 2:
Expression of band 3 fragments in the
rabbit reticulocyte lysate cell-free system. Various band 3 fragment
cRNAs (50 ng) were translated individually (in 6.25-µl reaction
mixtures), as described in Ref. 11. For each cRNA, parallel
translations were carried out in the presence or absence of canine
pancreatic microsomes and in the presence or absence of the
N-glycosylation acceptor NLT peptide at 30 µ
M final concentration, as described in Ref. 12. Samples (0.5 µl)
of each translation were analyzed by SDS-PAGE either on 9% Laemmli gels
(for larger fragments) ( a) or 10% Tricine gels (for smaller
fragments) ( b), followed by
autoradiography.
Cell-free translation of the carboxyl-terminal fragments (13:14) and
(9:14) resulted in a single band on SDS-PAGE, the position of which was
not affected by the addition of NLT peptide or by the absence of
microsomes from the translation (Fig. 2 b). These results
confirm that, as expected, the carboxyl-terminal fragments do not
contain an N-glycosylation site.
Trypsin Treatment of Intact Band 3 and the Band 3
Fragments
Intact band 3 was synthesized from the cRNA (in
the absence of GPA cRNA) and incorporated into microsomes using the
reticulocyte cell-free translation system. Microsomes were then
isolated and treated with trypsin at 0, 3, or 10 µg/ml (Fig.
3 a). When the band 3 in microsomes was treated with trypsin
either at 3 or at 10 µg/ml (Fig. 3 a, lanes 3 and 5) the amino-terminal cytoplasmic domain
(running at 43 kDa) was cleaved from the carboxyl-terminal membrane
domain (running at 47-51 kDa). Very small quantities of intact
band 3 and of a 75-kDa fragment were visible after treatment with 3
µg/ml trypsin, but these were degraded fully with 10 µg/ml
trypsin. Fragments with apparent molecular masses of approximately 32,
26, and 21 kDa were obtained at both trypsin concentrations, in
addition to smaller fragments that ran at the SDS-PAGE gel front.
Figure 3:
Trypsin digestion of band 3 and the band 3
fragments expressed in microsomes by the rabbit reticulocyte lysate
cell-free system. a, band 3 cRNA (0.5 µg) was translated
(in a 50-µl reaction mixture) and microsomes were purified by
Airfuge centrifugation. The pelleted membranes were resuspended and
divided into six portions (8 µl each), and 1 µl of 10% (w/v)
Triton X-100 was added to each of three tubes. Trypsin was then added
(to a final concentration of 0, 3, or 10 µg/ml) to both
detergent-treated and untreated samples (10 µl, final volume), and
all six tubes were incubated at 4 °C for 1 h, as described in Ref.
11. b, equimolar amounts of the various band 3 fragment cRNAs
were cotranslated with GPA cRNA. The amounts of cRNA (in 30-µl
reaction mixtures) were 45 ng, band 3 and b3(1:12); 37.5 ng, b3(1:8);
15 ng, (13:14), (9:14), and GPA. Microsomes were purified and then
treated with trypsin at 5 µg/ml, as described above. Samples were
separated by SDS-PAGE on 10% Laemmli gels ( a) or 4-20%
Laemmli ``Ready-Gels'' (Bio-Rad) ( b), followed by
autoradiography. The apparent molecular weights of the various tryptic
digestion products and residual undigested proteins are detailed in the
text. Key: mem, 44-51-kDa band 3 membrane domain;
cyt, 43-kDa band 3 cytoplasmic domain; A, 25-kDa
glycophorin A monomer. Other numbers indicate the apparent molecular
mass (kDa) of the various proteolytic
fragments.
Immunoprecipitation by the monoclonal antibody BRIC 155 (which
reacts with the carboxyl terminus of band 3) was used to probe for this
region of the polypeptide in the various tryptic fragments. When
samples were treated either with 3 or 10 µg/ml trypsin
(Fig. 3 a, lanes 9 and 11),
the polypeptide corresponding to the entire band 3 membrane domain was
the major proteolytic product that was immunoprecipitated. A small
amount of immunoreactive material migrated at the gel front, probably
representing very small peptide fragments containing the carboxyl
terminus of the protein. This indicates that the other fragments
(observed in Fig. 3 a, lanes 3 and
5) do not contain the carboxyl terminus of band 3. In the
immunoprecipitations obtained after treatment with 3 µg/ml trypsin
(Fig. 3 a, lane 9), traces of both
intact band 3 and the 75-kDa fragment were visible, indicating that
both of these products contained the carboxyl terminus. The 75-kDa
polypeptide therefore corresponds to band 3 cleaved at a site within
the amino-terminal cytoplasmic domain.
Figure 8:
Immunoprecipitation of band 3 fragments
from chymotrypsin-treated oocytes. Oocytes were co-injected with an
equimolar quantity of either band 3 cRNA or of one or more band 3
fragment cRNAs, in the presence ( even-numbered lanes) or
absence ( odd-numbered lanes) of 1.5 ng of GPA cRNA. The band
3-derived cRNAs injected were (per oocyte) lanes 1-4,
4.5 ng of b3(1:12); lanes 3 and 4, 1.5 ng of (13:14);
lanes 5-8, 3.75 ng of b3(1:8); lane 7 and
8, 1.5 ng (9:14); lanes 9 and 10, 4.5 ng of
band 3; lanes 11 and 12, 4.5 ng of b3(1:14*). Oocytes
were pulse labeled with S-labeled amino acids for 24 h at
20 °C and then chased with unlabeled amino acids for a further 24
h. Groups of 10 oocytes were treated with chymotrypsin for 1 h at 4
°C and then immunoprecipitated with BRIC 170 (against the
amino-terminal domain of band 3) as detailed in Ref. 11. Each lane represents immunoprecipitated protein from 10 oocytes, separated
by SDS-PAGE on 9% Laemmli gels. Fluorographs were exposed for
18-48 h. The proportion of each band 3 fragment that was cleaved
by chymotrypsin (to yield the 60-kDa amino-terminal portion of band 3)
was determined by scanning densitometry of the SDS-PAGE fluorographs.
Expressed protein was estimated from the areas under the peaks after
allowing for the different amounts of radioactivity incorporated into
the various cleaved and uncleaved fragments, based on their methionine
and cysteine contents. In a parallel immunoprecipitation using BRIC 155
(directed against the COOH terminus of band 3), oocytes injected with
intact band 3 and GPA cRNAs showed 42% cleavage of band 3 by
chymotrypsin, which is comparable with the result obtained using BRIC
170 ( lane 10). nd indicates cleavage was too low to
be determined ( lanes 11 and
12).
Expression of Band 3-mediated Anion Transport from
Pairs of Band 3 Fragments
Xenopus oocytes were
injected with cRNAs encoding various amino- and carboxyl-terminally
truncated recombinant fragments of band 3, either individually or by
co-injection of the complementary pairs of cRNAs, which together encode
the entire protein sequence of the membrane domain. In each case, the
cRNA coding for GPA was also co-expressed, as this has been shown to
increase the amount of band 3 translocated to the plasma membrane of
oocytes by up to 5-fold
(11, 13) . After 24 h of
incubation of the oocytes to allow the proteins to be expressed, the
influx of Cl
into the oocytes was
measured over a 1-h period in either the presence or absence of the
band 3-specific inhibitor DNDS (Fig. 4). The difference between the
chloride influx values in the absence and presence of inhibitor (the
DNDS-sensitive chloride influx) was used to provide an estimate of the
band 3-specific anion transport in each case.
Figure 4:
Chloride influx into oocytes expressing
band 3 fragments. Oocytes were injected with 1.5 ng ( a) or 5
ng ( b) of the band 3-derived cRNAs indicated, together with
1.5 ng of GPA (9 µl each) cRNA. Chloride influx (over 60 min) was
measured 24 h after injection using groups of 12-15 oocytes,
either in Barths saline ( hatched bars) or in Barths saline
containing 400 µ
M DNDS ( shaded bars), as
described in Ref. 11. The bar shows the standard error of the
chloride influx in each case. The DNDS-sensitive chloride influx may be
derived from the difference between the mean values in the presence and
absence of DNDS.
Co-expression of Pairs of Overlapping Recombinant
Band 3 Fragments
We also co-injected oocytes with cRNAs
encoding b3(1:12) and (9:14) at a higher concentration (5 ng each
cRNA/oocyte) together with GPA cRNA. When anion transport activity was
measured in the oocytes 24 h after injection, the level of chloride
influx in the absence of DNDS was about 1.7 times greater than in the
presence of DNDS (significant at the 5% level in a t test)
(Fig. 4 b). Although this amount of chloride transport activity
obtained with b3(1:12) and (9:14) was not as high as that observed with
the complementary fragments b3(1:8) and (9:14) under the same
conditions, these overlapping fragments (which duplicate transmembrane
helices 9-12, amino acid residues 696-824) may be able to
complement each other (poorly) to restore a low level of DNDS-sensitive
chloride transport activity.
Effect of the Amino-terminal Cytoplasmic Domain and
GPA on Band 3-mediated Anion Transport Activity by Co-expressed
Fragments
The amino-terminal cytoplasmic domain of band 3
is structurally and functionally distinct from the membrane domain and
is not essential for the expression of band 3-mediated anion transport
in oocytes
(11, 17) . We co-injected oocytes with the
pairs of cRNAs encoding either mem(1:8) and (9:14) or b3(1:8) and
(9:14) and with the pairs of cRNAs encoding either mem(1:12) and
(13:14) or b3(1:12) and (13:14). When GPA cRNA was also co-injected,
all four sets of fragments expressed a moderate level of DNDS-sensitive
anion transport activity in comparison with the equivalent intact band
3 or band 3 membrane domain controls (Fig. 5). This confirms that the
cytoplasmic domain is not involved in the process of folding and
association of the band 3 fragments to generate the functional protein
at the plasma membrane.
Figure 5:
The effect of the band 3 cytoplasmic
domain on the DNDS-sensitive chloride influx into oocytes expressing
band 3 fragments. Oocytes were injected with equimolar quantities of
cRNAs encoding either band 3 or the band 3 membrane domain (b3 mem) or
various fragments of band 3 or b3 mem in either the presence or absence
of GPA cRNA. The amounts of each cRNA injected were (per oocyte) 1.5
ng, band 3, b3(1:12), or GPA; 1.25 ng, b3(1:8); 1.0 ng, b3 mem or
mem(1:12); 0.75 ng, mem(1:8); 0.5 ng, (13:14) or (9:14). Chloride
influx (over 60 min) was measured 24 h after injection as described in
Ref. 11. Each value shows the mean DNDS-sensitive chloride influx
estimated from the difference between the means of groups of
13-14 oocytes, which were assayed in the presence and absence of
400 µ
M DNDS. The bar shows the standard error of
the mean DNDS-sensitive chloride influx in each case. Cross-hatched
bars, with GPA absent; shaded bars, with GPA
present.
Time-dependent Accumulation of Band 3 Fragments at
the Cell Surface
We have shown previously that functional
intact band 3 accumulates steadily in the plasma membrane of oocytes
with time after injection of the cRNA
(13) . Groups of oocytes
were co-injected with equimolar quantities of the cRNAs encoding either
b3(1:12) and (13:14) or b3(1:8) and (9:14) or intact band 3. GPA cRNA
was also co-expressed in each case. The injected oocytes were incubated
at 20 °C, and the the DNDS-sensitive chloride uptake into the
oocytes was measured at various times over a 3-day period (Fig. 7). The
results show that the expressed pairs of complementary fragments
mediate increasing levels of DNDS-sensitive anion transport with the
time of expression in a similar manner to the intact protein. In
contrast, oocytes injected with a cRNA encoding b3(1:14*), which lacks
30 amino acids at the carboxyl-terminal cytoplasmic tail of band 3
(containing amino acid residues 1-881) (Fig. 1), induced no
significant transport activity even after 3 days of expression in the
oocytes.
Figure 1:
Structure of fragments of band 3.
Various fragments of band 3 were prepared as described under
``Materials and Methods.'' The amino acid ( aa)
residues and the nomenclature used throughout this paper are indicated.
Putative transmembrane segments are represented by analogy with those
of the 14 span model for intact band 3. The arrows indicate
the extracellular chymotrypsin cleavage site in each
case.
Cell Surface Expression of Band 3
Fragments
Chymotrypsin cleavage has been used as a probe
for the band 3 expressed at the surface of oocytes
(11) . Band 3
in red cells is cleaved by chymotrypsin at a single extracellular site
in the loop between putative transmembrane helices 5 and 6 to yield an
amino-terminal 60-kDa fragment and a carboxyl-terminal 35-kDa fragment
(18) . We used this assay to investigate the factors affecting
the cell surface expression of each of the recombinant fragments that
contains this chymotrypsin cleavage site. Since this method provides an
assay for plasma membrane-expressed protein that is independent of the
anion transport function, it also enabled us to investigate whether the
individual fragments of band 3 (which do not mediate chloride
transport) were translocated to the plasma membrane, rather than being
retained in internal membranes or targeted for degradation in the
cells.
S-labeled amino acids, the intact oocytes were treated
with chymotrypsin. Subsequently, the radiolabeled protein was
immunoprecipitated using the monoclonal antibody BRIC 170. The
immunoprecipitated proteins were separated by SDS-PAGE to distinguish
the intact fragments from the 60-kDa chymotryptic cleavage product,
which originates from the fraction of the protein that is present at
the oocyte cell surface. The percentage of the band 3 construct that
had been cleaved by chymotrypsin was used to estimate the proportion of
the protein located at the cell surface.
or -NH
charged groups at the new termini of the fragment proteins within these
loops. When designing the positions of our truncated band 3 constructs,
we were careful to avoid introducing significant alterations to the
charge distribution between cytoplasmic and extracellular sides of the
fragment, since these could lead to an altered topology in the membrane
(19, 20) . Furthermore, all of the fragments we have
studied show a distribution of protease cleavage sites that is similar
to that of intact band 3, suggesting that the topology of the truncated
mutants (when expressed without their complementary partner) is similar
to that of the same regions of the intact protein. The results suggest
that there is no single signal sequence for inserting the different
transmembrane spans of band 3 into the endoplasmic reticulum. It is
clear that the band 3 fragments examined in this study can insert
independently into this membrane so that each fragment contains
sufficient information in its protein sequence to carry out this
process.
is accessible from the extracellular surface. Lys
is a site of intramolecular cross-linking of band 3 by
extracellular 4,4`-diisothiocyanato-2,2`-dihydrostilbene disulfonate at
high pH, and small soluble peptides can be released from this region by
proteolysis
(21) . In addition, the mutation Pro
Leu results in the formation of a new extracellular
antigen on band 3
(22) . In the intact protein, the ends of the
two short hydrophobic segments are accessible at both surfaces of the
membrane. This could be achieved by these segments adopting either an
extended structure or rather short helices at a location within the
structure of the protein that is shielded from contact with the lipid
by the remainder of the polypeptide
(22) . Our results with
fragment (13:14) indicate that this fragment inserts into microsomal
membranes by itself and shows a resistance to proteolysis that is
similar to that of the other fragments and to intact band 3. This
suggests that the fragment is tightly folded in the absence of the
remainder of the protein. If the membrane-penetrating portion of
(13:14) takes up a predominantly helical conformation when expressed by
itself, it seems unlikely that it would be able to span the bilayer
completely. However, (13:14) efficiently forms a transport-active
structure when co-expressed with fragment b3(1:12), and it is likely
that (13:14) adopts the same membrane-spanning structure in the
associated complex as in the intact protein. This suggests that the
integral membrane portion of (13:14) may undergo substantial
rearrangement of its structure when it associates with the remainder of
the polypeptide chain.
-helices fold independently on insertion into
the membrane, stabilized by main chain hydrogen bonds and interaction
with the lipid bilayer, and that subsequently these autonomous
-helical domains associate to yield the final structure without
significant rearrangement of their secondary structure (reviewed in
Refs. 28-30). In support of this hypothesis, several workers have
generated functional bacterial membrane proteins by co-expression or
reconstitution of two or more complementary protein fragments (reviewed
in Ref. 31). Bacteriorhodopsin was refolded from three peptides
comprising the first transmembrane span, the second span, and the
remaining five spans, and after addition of retinal, a functional pump
was reconstituted
(32) . Similarly, lactose transport was
restored by co-expression of fragments of the lac permease of
E. coli containing 2 and 10 spans
(33) or two 6-span
fragments
(34) . Iron (III) hydroxamate transport was restored
by co-expression of two complementary fragments of the the FhuB protein
(35) . There are a few examples where this complementation
approach has been extended to the functional expression of eukaryotic
membrane proteins. The
adrenergic receptors have been
co-expressed in oocytes from fragments containing 5 and 2 spans
(36) , and human and rat muscarinic acetylcholine receptors have
been expressed in COS cells, also from a 5- and a 2-span fragment
(37) . Recently, the glucose transporter GLUT1 was expressed in
the baculovirus system from fragments corresponding to the two
homologous 6-span fragments that are a feature of this family of
transporters
(38) , and human P-glycoprotein activity has been
reconstituted by co-expression of two polypeptides each containing six
transmembrane spans
(39) . In many of these cases, the
individual fragments were expressed as stable proteins but were not
functional unless both fragments were co-expressed.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.