(Received for publication, January 22, 1997)
From the Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218
Subunit interactions of the 1- and
1-subunits of the chicken Na,K-ATPase were explored with the yeast
two-hybrid system. Gal4-fusion proteins containing domains of the
1-
and
1-subunits were designed for examining both intersubunit and
intrasubunit protein-protein interactions. Regions of the
- and
-subunits known to be involved in
-
-subunit assembly were
positive in two-hybrid assay, supporting the validity of the assays. A
library of
-subunit ectodomains with C-terminal truncations was
screened to find the maximal truncation retaining an interaction with
the
-subunit extracellular H7H8 loop (where H7 refers to the seventh membrane span, and so on). The maximal truncation removed all the
cysteines involved in disulfide bridges, leaving only 63 amino acids of
the
-subunit ectodomain. Scanning alanine mutagenesis led to
identification of an evolutionarily conserved sequence of four amino
acids (SYGQ) in the extracellular H7H8 loop of the
-subunit that is
crucial to
-
-intersubunit interactions. Oligomerization studies
with single domains failed to detect self-association of either of the
two large cytosolic loops (H2H3 and H4H5) within the
-subunit.
However, evidence was found for an interaction between these two
cytoplasmic loops.
The Na,K-ATPase is an integral membrane protein that plays a
central role in ionic homeostasis in animals by mediating the translocation of Na+ and K+ ions across the
plasma membrane against their electrochemical gradients. The active
Na,K-ATPase is a heterodimer comprised of a 100-kDa -subunit that
spans the plasma membrane 10 times, and a 40-60-kDa glycoprotein
-subunit that has a short cytoplasmic N-terminal domain, a single
transmembrane domain, and a large extracellular domain. Both subunits
are required for Na+ and K+ ion transport
(1-3). The
-subunit contains the cation binding sites and the sites
of ATP binding and phosphorylation, and it is therefore sometimes
referred to as the catalytic subunit. The
-subunit is involved in
the structural and functional maturation of the holoenzyme (4, 5) and
transport to the plasma membrane (2, 6), and it appears to influence
K+ sensitivity (7, 8). The
- and
-subunits assemble
in a noncovalent, glycosylation-independent manner during or soon after biosynthesis (9, 10), and assembly is required for exit from the
endoplasmic reticulum (11).
Identification of domains involved in assembly of Na,K-ATPase subunits
has been approached in previous studies by immune precipitation experiments that have involved co-expression of truncated -subunits (12, 13) and chimeras between the Na,K-ATPase
-subunit and either
sarcoplasmic/endoplasmic Ca-ATPase (14-16) or the gastric H,K-ATPase
catalytic subunit (17-19). Expression of Na,K/Ca-ATPase chimeric
catalytic subunits together with the avian
-subunit in mammalian
cells, usually in the T7 RNA polymerase-based expression system (20),
has allowed us to define a 26-amino acid segment within an
extracellular loop of the Na,K-ATPase
-subunit that is necessary and
sufficient for assembly of the chimeras with the Na,K-ATPase
-subunit. To define further the specific amino acids involved in
Na,K-ATPase subunit interactions, we have employed the yeast two-hybrid
assay system (21, 22).
There is evidence that the Na,K-ATPase exists as an
(-
)2 heterotetramer in cell membranes, at least
during some portion of the transport cycle (23-25). Thus, one would
expect that, in addition to sites of
-
-subunit assembly, there
must be sites at which
-
-subunit heterodimers interact to form
the native tetramers. Blanco et al. (26) demonstrated that
-
oligomers containing two different isoforms of the
-subunit
could be purified by immune precipitation from detergent-solubilized
rat brain membranes and that
-
dimers formed when two
-subunit
isoforms were co-expressed in Sf-9 insect cells. In studies reported
here, we used the yeast two-hybrid system to screen for intrasubunit
interactions in the major cytosolic domains of the
-subunit and in
the extracellular domain of the
-subunit.
In the two-hybrid system assay, plasmids are constructed that
encode two hybrid proteins: one consists of the DNA-binding domain of
the transcription factor Gal4 fused to one test protein, X,
and the other consists of the Gal4 activation domain fused to another
test protein, Y. These plasmids are transformed into a
Saccharomyces cerevisiae strain that contains reporter genes whose regulatory region contains Gal4 binding sites. Either hybrid protein alone must be unable to activate transcription of the reporter
genes. The DNA-binding domain hybrid should not activate transcription
because it does not provide the activation function, whereas the
activation domain hybrid also should not activate transcription because
it cannot localize to the Gal4 binding sites. Interaction of the two
test proteins reconstitutes the function of the Gal4 transcription
factor and results in expression of the reporter genes, which are
detected by assays for the reporter gene products. For our studies, a
set of plasmids encoding Gal4-fusion proteins that contained elements
of the Na,K-ATPase - and
-subunits were constructed and used in
the two-hybrid system to explore intersubunit interactions and to look
for intrasubunit interactions. The results of these experiments are
presented in this report.
All of
the hybrid constructs were created using amplification by polymerase
chain reaction (PCR).1 The PCR reactions
contained 10 ng of template pBluescript SK+ plasmid
(Stratagene) containing a cDNA encoding either the chicken Na,K-ATPase 1- or
1-subunit, 100 ng of each primer (see below), 1 unit of Perkin-Elmer Taq DNA polymerase, 50 mM
KCl, 20 mM Tris-HCl, pH 8.3, 1.5 mM
MgCl2, 0.001% gelatin, and 0.2 mM of each of
the four deoxynucleotide triphosphates (Pharmacia Ultrapure), in a reaction volume of 0.1 ml, overlaid with 50 µl of mineral oil (Sigma). Amplification was performed for 30 cycles with a temperature profile of 1 min at 95 °C, 1 min at 42 or 50 °C, and 1 min at 72 °C.
All of the PCR fragments were digested with the appropriate restriction
enzymes (BamHI, BglII, NcoI, and/or
SmaI) overnight at room temperature for SmaI
digests or at 37 °C for the other restriction enzymes. The digested
PCR products were purified by agarose gel electrophoresis,
electroeluted, ligated overnight at 15 °C into both the pAS2 and
pACT2 vectors for use in the yeast two-hybrid system (27), and
transformed into Escherichia coli DH5 competent cells
(Boehringer Mannheim). Ampicillin-resistant colonies were screened for
the presence of the PCR fragment by restriction analysis of their
plasmids. The nucleotide sequences of candidate plasmids were
determined, and the desired plasmids were used in the yeast
transformations described below.
DNA encoding the -subunit cytoplasmic loop 1 (H2H3) was constructed
using the PCR primers TC126 (5
-GATCCCGGGCAAGAAGCGAAGAGTTCGAAG-3
) and
TC121 (5
-GATCGGATCCCTCCATGGCAATGGGAG-3
). This loop contains amino
acids Gln143 to Glu280 of the chicken
Na,K-ATPase
1-subunit, numbering residues beginning with N-terminal
glycine of the mature protein as residue 1. The DNA encoding the
-subunit cytoplasmic loop 2 (H4H5) was constructed using the PCR
primers TC128 (5
-GATCCCGGGGTAACGGTATGTCTGACACTA-3
) and TC119
(5
-GATCGGATCCCAGGTTATCAAAGATCA-3
). This loop contains amino acids
Val335 to Leu765 of the chicken Na,K-ATPase
1-subunit. The DNA encoding the EC49 loop (H7H8) was constructed
using the PCR primers TC130 (5
-GATCCCGGGATGGCAGAGAATGGGTTCTTG-3
) and
TC120 (5
-GATCAGATCTGGCTGTATG GCAAGTGAATTC-3
). This loop contains
amino acids Met866 to Ala914 of the chicken
Na,K-ATPase
1- subunit.
The DNA encoding the -subunit ectodomain was constructed using the
PCR primers TC150 (5
-ACGTCCCGGGGAATTTGAACCCAAGTAC-3
) and TC110
(5
-ATCGGGATCCGCTGCTTTTTATGTCAAATT-3
). This domain contains amino
acids Glu63 to Ser304 of the chicken
Na,K-ATPase
1-subunit, numbering from the N-terminal Ala of the
mature protein. The DNA encoding the extracellular
X149 domain was
constructed using PCR primers TC150 and TC191 (5
-TACGTCCCGGGATCCTGGACAGGGATGAGATAGGGGTTG-3
). This domain is a
93-amino acid C-terminal truncation of the
1 extracellular domain
and contains amino acids Glu63 to Val211 of the
chicken Na,K-ATPase
1-subunit. The DNA encoding the extracellular
X96 domain was constructed using PCR primers TC150 and TC190 (5
-TACGTCCCGGGATCCCAGTTCTCCAGCCACTCACGTTTG-3
). This domain is a
146-amino acid C-terminal truncation of the
1 extracellular domain
containing amino acids Glu63 to Asn158 of the
chicken Na,K-ATPase
1-subunit.
For alanine scanning mutagenesis, both strands of DNA were synthesized
and hybridized together to form double-stranded DNA fragments for
cloning into the pAS2 and pACT2 vectors. The DNA encoding the EC49 Ala1
domain was constructed by creating two unique restriction sites within
the encoding DNA and replacing the intervening region with hybridized
oligonucleotides TC1C1F (5-GTGGGATGACCGATGGATTAATGCAGCGGCCGCTAGCTATGGACAGCATGGACCTTCGAACAGAGGAAAATTGTGG-3
) and TC1C1R
(5
-AATTCCACATTTTCCTCTGTTCGAAGGTCCATTGCTGTCCATAGCTAGCGGCCGCTGCATTAATCCATCGGTCATCCCACTGCA-3
). The resultant DNA encoded amino acids Met866 to
Ala914 of the chicken Na,K-ATPase
1-subunit with amino
acid sequence DVED converted to AAAA. The DNA encoding the EC49 Ala2
domain was constructed in an analogous fashion but using hybridized
oligonucleotides TC1DF
(5
-GTGGGATGACCGATGGATTAATGATGTTGAAGACGCAGCGGCCGCTCAATGGACCTTCGAACAGAGGAAAATTGTGG-3
) and TC1DR
(5
-AATTCCACAATTTTCCTCTGTTCGAAGGTCCATTGAGCGGCCGCTGCGTCTTCAACATCATTAATCCATCGGTCATCCCACTGCA-3
). The resultant DNA encoded amino acids Met866 to
Ala914 of the chicken Na,K-ATPase
1-subunit with SYGQ
converted to AAAA. The DNA encoding the EC49 Ala3 domain was
constructed using hybridized oligonucleotides TC1EF
(5
-GTGGGATGACCGATGGATTAATGATGTTGAAGACAGCTATGGACAGGCAGCGGCCGCTGAACAGAGGAAAATTGTGG-3
) and TC1ER
(5
-AATTCCACAATTTTCCTCTGTTAGCGGCCGCTGCCTGTCCATAGCTGTCTTCAACATCATTAATCCATCGGTCATCCCACTGCA-3
). This encodes amino acids Met866 to Ala914
of the chicken Na,K-ATPase
1-subunit with QWTF converted to AAAA.
The DNA encoding the EC49 Ala4 domain was constructed using hybridized
oligonucleotides TC1FF
(5
-GTGGGATGACCGATGGATTAATGATGTTGAAGACAGCTATGGACAGCAATGGACCTTCGCAGCGGCCGCTATTGTGG-3
) and TC1FR
(5
-AATTCCACAATAGCGGCCGCTGCGAGGGTCCATTGCTGTCCATAGCTGTCTTCAACATCATTAATCCATCGGTCATCCCACTGCA-3
). This encodes amino acids Met866 to Ala914
of the chicken Na,K-ATPase
1-subunit with EQRK converted to AAAA.
The S. cerevisiae strains SFY526 (MATa, ura3-52, his3-200, ade2-101, lys 2-801, trp 1-901, leu 2-3, 112, canr, gal4-542, gal80-538, URA3::Gal1-lacZ) and HF7c (MATa, ura3-52, his3-200, lys 2-801, ade2-101, trp1-901, leu2-3, 112, gal4-542, gal80-538, LYS2::GAL1-HIS3, URA3::(GAL4 17-mers)3-CYC1-lacZ) (CLONTECHMatchmakerTM) were used for all assays. Yeast cultures were grown at 30 °C in either YPD medium (1% yeast extract, 2% peptone, and 2% glucose) or SD minimal medium (0.5% yeast nitrogen base without amino acids, 2% glucose, and 1% desired amino acid dropout solution).
Yeast Transformation andFusion
genes were introduced into a yeast reporter strain by the lithium
acetate transformation procedure of Gietz et al. (28).
Transformants were allowed to grow at 30 °C, usually for 2-4 days,
until colonies were large enough to assay for -galactosidase activity. Transformant cells were then plated directly onto sterile Whatman number 1 filters that had been layered onto selective growth
media. After colonies had grown, the filters were assayed for
-galactosidase activity. The cells were permeabilized by a cycle of
freezing the filters in liquid nitrogen and thawing to room
temperature. Each filter is then soaked with 2 ml of Z-buffer (CLONTECHMatchmakerTM protocol manual) containing
5-bromo-4-chloro-3-indolyl-
-D-galactoside. The filters
were then placed in a covered plastic container at room temperature and
checked periodically for the appearance of blue colonies. Blue colonies
appeared between 30 min and 12 h. The filters were then dried and
photographed to record the data.
Truncations of the DNA encoding the extracellular
domain of the Na,K-ATPase -subunit were performed on the pACT
plasmid. Exonuclease III digests were performed for 12 min during which samples were removed and the reactions quenched every 2 min. Plasmid DNA was digested with two restriction enzymes, cleaving the DNA just 3
of the
-subunit coding DNA and leaving a protective 3
overhang at
the termination sequence and a 5
overhang in the direction of the
-subunit coding sequence. The 3
overhang is resistant to
exonuclease III digestion. The cut DNA, 5 µg, was dissolved in 50 µl of reaction buffer (New England Biolabs) (66 mM
Tris-HCl, pH 8.0, and 0.66 mM MgCl2), 100 units
of exonuclease III enzyme (New England Biolabs) were added, and the
reaction mixture was incubated at 37 °C. The exonuclease III
digestion rate under these conditions is approximately 300 bases per
min. At 2-min intervals, 5 µl of aliquots were removed, mixed with STOP solution (0.2 M NaCl, 5 mM EDTA, pH 8.0),
and incubated at 70 °C for 10 min to inactivate the exonuclease III
enzyme. The digested DNA was then ethanol-precipitated and resuspended
in 20 µl of mung bean nuclease buffer (New England Biolabs) (50 mM sodium acetate, pH 5.0, 30 mM NaCl, 1 mM ZnSO4). Ten units of mung bean nuclease (New
England Biolabs) were added, and the samples were incubated at 30 °C
for 15 min to digest the single-stranded DNA and create blunt ends for
ligation. The DNA was again precipitated in ethanol and resuspended in
20 µl of Tris-EDTA buffer, and blunt-end ligations were performed
overnight at 16 °C to generate small libraries of pACT
truncations. Yeast cells were co-transformed with aliquots of these
pACT
truncations and pAS
EC49.
Previous immunoprecipitation studies using chimeric
Ca-ATPase/Na,K-ATPase -subunits and chimeric DPPIV/Na,K-ATPase
-subunits have demonstrated that a stretch of 26 amino acids (called
EC26) (15), within the H7H8 extracellular loop of the
-subunit, and the ectodomain of the
-subunit (13) are required for
-
-subunit assembly. In the present study, initial two-hybrid experiments with
Gal4-fusion proteins containing the entire H7H8 extracellular loop
(called EC49) of the
-subunit and the extracellular domain of the
-subunit showed that these fusion proteins form protein-protein interactions resulting in activation of transcription of the
-galactosidase reporter gene. Fig. 1 is a diagram of
the Na,K-ATPase, indicating the regions of the
- and
-subunits
that were analyzed in two-hybrid assays. Table I shows
the results indicating that neither pAS
EC49 nor pACT
alone
activates transcription. Table II shows that when both
the pAS
EC49 and pACT
are co-expressed,
-galactosidase transcription is activated. These results demonstrate that these
and
peptides as fusion proteins in yeast retain their ability to
assemble and therefore are compatible for use in the two-hybrid system.
|
|
Hamrick et al. (13) showed that the
avian Na,K-ATPase -subunit, truncated by 92 or 146 residues from the
C terminus, remained competent to form
·
complexes when
expressed in mammalian cells. As an extension of these experiments, we
constructed GAL4-fusion proteins containing deletions in the
extracellular
domain that were the same as those of Hamrick
et al. (13), that is deletions of 92 and 146 amino acids
from the C terminus. These
deletions were named
X149 and
X96
because they retain 149 and 96 aminoacyl residues of the
-subunit
ectodomain, respectively (see Fig. 2). These deletion
constructs co-expressed with the
EC49 (H7H8) fusion protein yielded
positive results in the two-hybrid assay (Table II), suggesting that
the N-terminal 96 amino acids of the extracellular domain of the
-subunit (Glu63-Asn158) are sufficient to
form a protein-protein interface with the EC49 extracellular loop
(H7H8) of the
-subunit.
Further Truncations of the
To determine the minimal C-terminal extent of the
-subunit involved in protein-protein interactions with the
EC49
(H7H8)
-domain, we performed a series of exonuclease III digests on the pACT
construct from the 3
end. The pools of pACT
deletions were transformed into yeast together with the pAS
EC49 (H7H8) construct. The co-transformants were plated onto media lacking histidine. If the co-expressed fusion proteins interacted, they would
cause transcription of a histidine reporter gene, allowing those
co-transformants to grow on histidine-deficient medium. Two separate
experiments were performed, resulting in a total of 25 transformant
colonies after 12 min of exonuclease III digestion. The DNA from each
of these 25 colonies was isolated and used as a template in PCR
reactions primed by the primers for the
X96 construct (TC150 and
TC190). This PCR screen was used to look for any deletion that encoded
less than the 96 amino acids of the extracellular
domain already
identified. The results of this screening indicated that three of the
deletions contained fewer than 96 amino acids of the extracellular
domain. Then, a set of nested oligonucleotide primers was used to
estimate the extent of coding region remaining in the three
deletion clones. From this experiment, it appeared that the shortest
truncation encoded no more than 63 amino acids and possibly as few
as 61. Finally, to test this conclusion, site-directed mutagenesis was performed on the pACT
X96 plasmid, introducing a stop codon after the
codon for Asp125 of the
-subunit ectodomain. This
construct, pACT
X63, co-expressed with pAS
EC49, yielded positive
results in the yeast two-hybrid system, confirming that
Glu63 to Asp125 is a sufficient extent of the
extracellular
-subunit domain for interaction with the
-subunit
EC49 loop (H7H8). These results suggest that the three disulfide loops
of the
-subunit are not required for interaction with
EC49 (H7H8)
and that no more than the 63 amino acids adjacent to the transmembrane
domain are necessary for this interaction. This region is
shaded in the diagram of the
-subunit in Fig. 2.
To
identify individual amino acids involved in the -
intersubunit
interaction, we constructed Gal4-fusions with altered forms of the EC49
-subunit domain (H7H8) that contained amino acid mutations to
alanine. We chose to use the EC49 context since the EC26 fusion protein
activated transcription on its own (Table I). Sequence alignments of
the aminoacyl resides in the EC49 (H7H8) domain of the
-subunit
revealed a cluster of residues well-conserved among all the known
-subunits of the Na,K-ATPase and H, K-ATPase families (the families
in which
-
heterodimers appear to be the functional units) (Fig.
3).
To identify aminoacyl residues within the H7H8 loop that might be
involved in -
-subunit interactions, a set of four alanine scanning variants of the EC49 region were constructed (see Fig. 3), and these constructs were co-expressed with the
Gal4-
-subunit constructs pACT
, pACT
X149, and pACT
X96
in yeast two-hybrid assays. Table III shows the results
of the two-hybrid assays. The fusion proteins EC49 Ala1, EC49 Ala3, and
EC49 Ala4 all demonstrate positive protein-protein interactions with
all three
fusion proteins. These results suggest that the 12 mutated aminoacyl residues may not be crucial to protein-protein
interactions between the
- and
-subunits. However, the fusion
protein EC49 Ala2 did not show positive protein-protein interactions
with any of the three Gal4-
-subunit fusion proteins. These results
suggest that the group of four highly conserved amino acids (SYGQ) is
directly involved in protein-protein interactions between the
- and
-subunits.
|
There is substantial evidence that the Na,K-ATPase
exists largely as (-
)2 dimers in cell membranes. The
yeast two-hybrid system was used to seek elements of the
-subunit
that might be involved in
-
interactions; similarly, we sought
evidence for
-
interactions. The two largest individual cytosolic
domains of the
-subunit (see Fig. 1) were tested for
self-association:
cytoplasmic loop 1 (H2H3) with itself, and
cytoplasmic loop 2 (H4H5) with itself. The results in Table
IV demonstrate that in two-hybrid assays no
-
self-associations occurred. Likewise, tests for oligomerization
involving the ectodomain of the
-subunit were mostly negative (Table
IV). Only the
X96 fusion protein demonstrated any oligomerization
interactions with itself. These results are inconclusive as to whether
biologically significant
-
-subunit interactions occur. The lack
of positive results with the larger ectodomain suggests that
-
interactions are not biologically meaningful. The full-length
-subunit ectodomain could not be tested for
-
interactions
because the
-subunit ectodomain fused to the Gal4 DNA-binding domain
was positive when expressed in yeast by itself.
|
The two large cytoplasmic loops of the -subunit
were also tested for interaction with each other in the yeast
two-hybrid system. The
cytoplasmic loop 1 (H2H3) and
cytoplasmic loop 2 (H4H5) were positive for interaction when
cytoplasmic loop 1 (H2H3) was fused to the activation domain and
cytoplasmic loop 2 (H4H5) was fused to the DNA-binding domain of Gal4
but not when in the reverse orientation (Table IV). These results do
support the occurrence of interactions between the H2H3 and the H4H5
loops of the
-subunit but are less compelling than would be the case
if positive results had been obtained with the loops in both fusion
configurations.
While the major application of the two-hybrid system has been for
screening cDNA libraries to find clones encoding proteins that bind
some target protein, the same methodology is useful for identifying
domains or amino acids involved in interactions between proteins that
are known to interact. Many combinations of proteins have been used
successfully in the two-hybrid assay. These combinations include many
nuclear, cytoplasmic, mitochondrial, and viral proteins but only a few
membrane-associated proteins (22). To our knowledge, this study is the
first to utilize the two-hybrid system to define domains of subunit
interaction in a multi-subunit plasma membrane protein. It is important
to note some caveats about the studies reported here. First, - and
-subunit interactions that are essential to the processes involved
in assembly of the Na,K-ATPase in the endoplasmic reticulum need not be
entirely the same as sites of subunit interaction in the mature, active enzyme. Second, while studies on assembly identify domains and residues
that are important to the
and
contacts, they do not distinguish
those elements that indirectly regulate the
-
interface from
those that are the interface. This same caveat, of course, applies to
virtually all studies that involve analysis of perturbations in protein
structure. Third, while these experiments reveal some domains and
residues that are at interfaces, they do not identify all relevant
interfaces. We know, for example, that subunit assembly of the
Na,K-ATPase is more efficient when the
-subunit includes its
cytoplasmic and membrane-spanning domains, although these are not
absolutely necessary for assembly (13, 30).
To
define features of the -subunit necessary and sufficient for
assembly with the
-subunit, Renaud et al. (12) began by making mutations in the avian
1-subunit and assaying for the ability
of mutant
-subunits to assemble with mouse
-subunits in
transfected mouse L-cells. These experiments showed that the cytosolic
33 amino acids at the N terminus of the
-subunit were not required
for assembly. A set of small deletions in the membrane-spanning region
of the
-subunit also failed to inhibit assembly, although most of
these deletions prevented the assembled
·
complexes from
leaving the endoplasmic reticulum. Hamrick et al. (13) expanded these studies by examining the assembly potential of C-terminally truncated
-subunits. Truncation of the
-subunit by
92 or 146 amino acids failed to abolish assembly with the
-subunit, although the yields of assembled complexes in the immunoprecipitation experiments were clearly decreased. These results suggested a region
between aminoacyl residues 126 and 170 might be especially important
for assembly. However, subunit assembly was not detected between avian
-subunits and a chimera consisting of the cytosolic and membrane
spanning domains of DPPIV and the
-subunit extracellular domain
truncated by 146 amino acids. The combined effect of replacement of the
cytosolic and membrane-spanning domains together with truncation of the
C terminus by 146 residues either decreased the efficiency of subunit
assembly or the stability of the assembled complexes during isolation
(i.e. solubilization and immunoprecipitation) to the point
where no evidence of assembly remained. However, the ectodomain of the
-subunit, as a secretory protein, remained capable of forming stable
complexes with the
-subunit (31).
In the experiments reported here, an attempt was made to define the
minimal -
interaction domains further, by using the yeast
two-hybrid system, which might be sensitive enough to detect weaker
interactions. Indeed, positive results were found when Gal4 fusions
containing deletions in the extracellular
domain matching those
created by of Hamrick et al. (13) were co-expressed with the
EC49 fusion protein. This is the first time that the regions of the
- and
-subunit that had separately been defined as sufficient for
subunit assembly were actually shown to interact with each other.
Previously it had been found (a) that
-subunits truncated
at their C terminus by 92 or 146 residues could assemble with the
entire
-subunit, and (b) that a Na,K-ATPase/Ca-ATPase chimera in which the only residues contributed by the Na,K-ATPase were
26 amino acids in the H7H8 loop could assemble with the entire extracellular domain of the
-subunit (15, 16). The results of the
two-hybrid assay indicate that these minimal assembly domains do
interact with each other. That is the segment from Glu63 to
Asn158 of the
-subunit is sufficient to form a
protein-protein interface with the EC49 extracellular loop (H7H8) of
the
-subunit.
By screening a library of cDNAs encoding C-terminal truncations of
the -subunit ectodomain, we found that an even shorter segment of
the
-subunit contained an
-subunit binding site. This region was
estimated by PCR amplification to correspond approximately to residues
Glu63 to Phe123. These results were
substantiated by showing that Glu63 to Asp125
interacts with the EC49
-domain (H7H8). These results are remarkable in showing that none of the three disulfide loops of the
-subunit is
critical in protein-protein interactions with the EC49 loop (H7H8) of
the
-subunit.
There are several isoforms of each subunit of the
Na,K-ATPase (see reviews 25, 32, 33). Assembly between the various isoforms and
isoforms from the same species has been demonstrated
directly with immunological methods (16, 34-37) and indirectly by
detection of functional pumps (11, 36-39). Formation of interspecies
hybrid
·
complexes has also been demonstrated (2, 7, 12, 13, 40-42). Furthermore, co-expression of Na,K-ATPase
1-subunits and H,K-ATPase
-subunits resulted in functional hybrid
·
complexes (6, 39). Finally, Na,K-ATPase/Ca-ATPase catalytic subunit chimeras containing only 26 amino acids of the Na,K-ATPase H7H8 loop
were shown to assemble with the gastric H,K-ATPase
-subunit as well
as with the
1 and
2 isoforms of the Na,K-ATPase
-subunit (16).
The results of these studies suggest common assembly domains in the
Na,K-ATPase and the H,K-ATPase subunits, with the
-subunit domain
lying within the H7H8 loop. There is a stretch of 26 aminoacyl residues
within this domain that is well conserved in evolution between
Na,K-ATPase and H,K-ATPase
-subunits. Twelve of the 26 residues are
identical in all
-subunits. These residues might be expected to
include a general motif for interaction with the ectodomain of
-subunits. The replacement of critical aminoacyl residues by alanine
should eliminate protein-protein interactions involving these
contacting amino acids.
Protein-protein interfaces are made up of a mixture of hydrophobic and hydrophilic residues. They are usually well paired so that hydrogen bond donors and acceptors are matched along with the hydrophobic groups (43). Alanine is chosen as a generic replacement residue because it is the most common amino acid in proteins, and it is found within buried and exposed positions and in all manner of secondary structures (43). Alanine does not supply new hydrogen bonding, sterically bulky, or unusually hydrophobic side chains (43). Alanine substitutions reduce the functional comparisons among the mutants to a common standard state. To increase the efficiency of analysis one can mutate amino acid groups to alanine in clusters ranging from 2 to 5 residues within segments of 10 to 15 residues (43). This allows one to determine quickly which clustered mutants are most disruptive and subsequently dissect them to identify the important residues. The scanning mutational approach directly tests only the importance of side chains; information about main chain interactions remains unknown. Main chain interactions are common among protein-inhibitor complexes but less so in subunit-subunit and antibody-antigen interactions (43).
With this alanine-scanning strategy, the fusion proteins EC49 Ala1,
EC49 Ala3, and EC49 Ala4 all demonstrate positive protein-protein interactions with the full -subunit ectodomain and the two truncated
fusion proteins. However, the fusion protein EC49 Ala2 did not evidence interaction with any of the three
fusion proteins. This
result suggests that the group of four highly conserved amino acids,
SYGQ, in the
-subunit H7H8 loop includes residues directly involved
in protein-protein interactions between the
- and
-subunits.
Although structural studies of the sodium pump support
a subunit stoichiometry of one -subunit to one
-subunit, the
exact quaternary structure is still in debate. The formation of a
higher order enzyme complex is supported by studies of
-
interactions among the Na,K-ATPase isoforms in rat brain and among rat
-subunits expressed in virally infected Sf-9 insect cells (26).
Expression of a truncated
1 isoform with the full-length
-subunit
demonstrated that the C-terminal half of the
-subunit is required
for
-
-subunit oligomerization in insect Sf-9 cells. Through the
use of chimeras between the catalytic
-subunits of the Na,K-ATPase
and H,K-ATPase, the region involved in
-
interaction was further
defined as lying between residues Gly554 and
Pro785 in the central, cytosolic loop (44). Cross-linking
studies by Sarvazyan et al. (45) indicate
-
associations between the N-terminal H1-H2 and C-terminal H8-H10
segments of the Na,K-ATPase
-subunit, with the most probable
interacting helices being the H1-H10 pair and the H2-H8 pair. It is not
known whether these contacts involve intra- or inter-
-subunit
interactions.
The results of -
oligomerization experiments performed with the
two-hybrid system did not reveal any self-association of either large
cytoplasmic loop of the
-subunit, even though
cytoplasmic loop 2 contained the residues Gly554 to Pro785,
implicated in
-
oligomerization by Koster et al. (44).
One possible rationalization of the differing results is that
-
dimerization involves elements of
-subunit structure that are not
intrinsic to an isolated
-subunit cytosolic loop, such as would be
present in Gal4 fusion proteins. There is evidence to support this
interpretation in recent experiments of Froehlich and colleagues (46),
experiments that suggest that dimerization of
·
complexes to
form (
·
)2 complexes may involve conformational states that occur transiently during the transport cycle.
The cytoplasmic loop 2 of the -subunit (H4H5)
contains the phosphorylation and nucleotide binding sites. However,
some mutations within cytoplasmic loop 1 (H2H3) influence the ATPase
activity and vanadate sensitivity of the Na,K-ATPase, suggesting that
loop 1 may interact with loop 2 (H4H5). The possibility of loop 1-loop 2 interaction was tested in two-hybrid assays. The assays were positive
when cytoplasmic loop 1 (H2H3) was fused to the activation domain of
Gal4 transcription factor, and cytoplasmic loop 2 (H4H5) was fused to
the DNA-binding domain but not vice versa. In two-hybrid assays of defined protein combinations, one orientation of the hybrids
(i.e. protein X fused to the DNA-binding domain
and protein Y to the activation domain) often activates
transcription much more efficiently than the reverse hybrids (29). This
may reflect differences between the levels of expression or stability
of hybrids containing X and those containing Y.
Transcription is optimal when the activation domain hybrid is in excess
over the DNA-binding domain hybrid (29). When the reverse is true,
DNA-binding domain hybrids bound to the reporter gene promoters are
less likely to be engaged in the X-Y
protein-protein interaction and therefore may not give positive
results. Since
-subunit loop 1 and loop 2 interactions were seen
with only one orientation, this constitutes positive but weak evidence
for inter-loop interaction.
There are additional protein-protein
interactions within the Na,K-ATPase that can be approached with
two-hybrid studies. For example, the present studies do not include a
search for interactions involving the cytosolic domain of the
-subunit nor the N-terminal, C-terminal, and H6H7 and H8H9 cytosolic
loops of the
-subunit. In addition, the Na,K-ATPase is known to
interact with ankyrin (47-49), and there is evidence that the
2
isoform
-subunit expressed by glial cells may interact with
"receptors" on some central nervous system neurons (34). The yeast
two-hybrid system appears to be a promising approach not only for
defining the subunit assembly domains more completely but also for
observing other protein-protein interactions that involve the
Na,K-ATPase.
We thank Dee Thomas, Ben Hwang, Dr. Yuanyi Feng, Amy Lawson, Mitch Kostich, Dr. Shawn Robinson, Christine Hatem, and Delores Somerville for helpful discussions and occasional assistance in this research and Dr. Stanley Fields and Dr. Steven Elledge for gifts of two-hybrid vectors.