Báo cáo khoa học: The highly conserved extracellular peptide, DSYG(893–896), is a critical structure for sodium pump function docx

The highly conserved extracellular peptide, DSYG(893–896), is a
critical structure for sodium pump function
Susanne Becker*, Heike Schneider* and Georgios Scheiner-Bobis
Institut fu
¨
r Biochemie und Endokrinologie, Fachbereich Veterina
¨
rmedizin, Justus-Liebig-Universita
¨
t Giessen, Germany
The peptide sequence DSYG(893–896) of the s heep sodium
pump a1 subunit is highly conserved among all K
+
-trans-
porting P-type ATPases. To obtain information about its
function, single mutations were introduced and the mutants
were expressed in yeast and analysed for enzymatic activity,
ion recognition, and a/b subunit interactions. Mutants of
Ser894 or Tyr895 were all active. Conservative phenylalan-
ine and tryptophan mutants of Tyr895 displayed properties
that were similar to the properties of the wild-type enzyme.
Replacement of the same amino acid by cysteine, however,
produced heat-sensitive enzymes, indicating that the
aromatic group contributes to the stability of the enz yme.
Mutants of the neighbouring Ser894 recognized K
+
with altered apparent affinities. Thus, the Ser894fiAsp
mutant displayed a threefold higher apparent affinity
for K
+
(EC

50
¼ 1.4 ± 0.06 m
M
) than the wild-type
enzyme (EC
50
¼ 3.8 ± 0.33 m
M
). In contrast, the mutant
Ser894fiIle had an almost sixfold lower apparent affinity
for K
+
(EC
50
¼ 21.95 ± 1.41 m
M
). Mutation of Asp893
or Gly896 produced inactive proteins. When an anti-b1
subunit immunoglobulin was used to co-immunoprecipitate
the a1 subunit, neither the Gly896fiArg nor the
Gly896fiIle mutant could be visualized by subsequent
probing with an anti-a1 subunit immunoglobulin. On the
other hand, co-immunoprecipitation was obtained with the
inactive Asp893fiArg and Asp893fiGlu mutants. Thus, it
might be that Asp893 is involved in enzyme conformational
transitions required for ATP hydrolysis and/or ion translo-
cation. The results obtained h ere demonstrate the import-
ance of the highly conserved peptide DSYG(893–896) for
the function of a/b heterodimeric P-type ATPases.
Keywords:Na

+
/K
+
-ATPase; a/b subunit interactions;
immunoprecipitation; ouabain binding; thermal stability.
The sod ium pump (Na
+
/K
+
-ATPase, EC 3.6.3.9) is an
a/b-oligomeric enzyme embedded in the plasma membrane
of animal cells. The enzyme hydrolyzes ATP to transport
three Na
+
ions out of the cell and two K
+
ions into the cell.
Although ATP binding and ion occlusion seem to be tightly
connected to the a subunit, the o verall catalytic activity,
defined as ATP-driven ion transport, requires the inter-
action of both a and b subunits of the enzyme.
The subunits are known to interact with each other at
extracellular s ites. On the a subunit, a stretch of 26 amino
acids localized within the peptide loop that connects M7 and
M8 membrane-spanning domains (hereafter denoted L7/8)
was first identified as being important for interactions with
the b subunit [1]. In a more detailed study, using a yeast
two-hybrid system, the SYGQ(894–897) sequence from
the 26-amino acid peptide was identified as an essential
component for a/b subunit interactions following

replacement with four alanine residues: SYGQ(894–
897)fiAAAA(894–897) [2]. (Note that the peptide number-
ing corresponds to the sheep Na
+
/K
+
-ATPase a1 subunit.)
Using the same system, either Ser894 or Tyr895 were
identified as being essential for a/b interactions [3].
Besides being important for interactions with the b
subunit, the L7/8 region of the a subunit also seems to
be involved in ion translocation or rec ognition. This is
supported by the results of various investigations involving
either enzymatic analysis of a subunit mutants [4] or metal
ion-catalysed oxidative cleavage of a and b subunits,
resulting in the loss of Rb
+
occlusion [5].
Recognition of K
+
or Na
+
was also found to depend on
the b subunit structure, a finding that was based on subunit-
substitution studies involving various chimeras between the
sodium pump b subunit and the gastric proton pump b
subunit [6,7]. Thus, besides its role in stabilization of the
a subunit and its function as a vehicle for bringing the a
subunit from the en doplasmic reticulum to the p lasma
membrane [8], the b subunit may be directly involved in ion

recognition or transport.
To better understand a/b interactions and their involve-
ment in ion transport, we introduced mutations within the
DSYG(893–896) sequence of the L7/8 peptide o f the sheep
a1 subunit, which is highly conserved among the hetero-
dimeric P-type ATPases. The mutant a1 subunits were
expressed in yeast and investigated with respect to their
enzymatic properties and their interaction with the coex-
pressed b subunit. The results described here demonstrate
Correspondence to G. Scheiner-Bobis, Institut fu
¨
r Biochemie und
Endokrinologie, Fachbereich Veterina
¨
rmedizin, Justus-Liebig-Uni-
versita
¨
t Giessen, Frankfurter Str. 100, D-35392 Giessen, Germany.
Fax: +49 641 9938189, Tel.: +49 641 9938180,
E-mail:
Abbreviations:Na
+
/K
+
-ATPase, sodium- and potassium-activated
adenosine triphosphatase; NaCl/P
i
-T, phosphate-buffered saline
containing 0.1% (v/v)Tween
TM

20.
Enzymes:Na
+
/K
+
-ATPase (EC 3.6.3.9).
*Note: Both authors contributed equally to the scientific work
presented here.
(Received 9 June 2004, revised 21 July 2004, accepted 26 July 2004)
Eur. J. Biochem. 271, 3821–3831 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04305.x
that single mutations within the highly conserved
DSYG(893–896) peptide influence enzyme activity, enzyme
stability, interac tions with K
+
, a nd assembly of the a and
b subunits.
Experimental procedures
Vectors and strains
The shuttle vectors YhNa1, GhNb1 and pCGY1406ab,
used for the expression of a1andb1 subunits of Na
+
/K
+
-
ATPase in the yeast Saccharomyces cerevisiae, have been
described previously [9,10]. The pBluescriptÒ
TM
KS
II+ (Stratagene, La Jolla, CA, USA) was used for the
introduction of mutations and the amplification of DNA in

Escherichia c oli strain DH5aF¢ (Life Technologies, Eggen-
stein, Germany). Conditions for cell growth and media
compositions have been described previously [9].
Introduction of mutations
Mutations of Asp893, Ser894 and Gly896 were introduced
by inverse PCR [11] using, as a template, the plasmid
LPSKH5-7 [4] (a derivative of the pBluescriptÒII KS
+
)and
appropriate amplification primers (Roth, Karlsruhe, Ger-
many) shown in Table 1. PCR reaction mixtures o f 100 lL
contained 1 l
M
mutation primer, 1 l
M
reverse primer, 1 ng
of the plasmid LPSKH5-7, 1.5 m
M
MgCl
2
,2UTfl DNA
polymerase (Promega, Madison, WI, USA), 0.2 m
M
each
dNTP, a nd the appropriate amount of buffer provided by
the supplier. After 20 PCR cycles, the amplification product
was isolated by agarose gel electrophoresis, treated with T4
DNA polymerase (Promega) to remove the dA overhang
produced by the Tfl DNA polymerase, and recircularized by
the use of T4 DNA ligase (MBI Fermentas, Vilnius,

Lithuania). After amplification in E. coli, the plasmids were
tested by restriction analysis with AseI(allrestriction
enzymes purchased from MBI Fermentas), and DNA
sequencing was carried out according to Sanger et al.[12]
using T7 DNA polymerase (Amersham Life Science, Little
Chalfont, Bucks., UK) and [
35
S]dATP (ICN Radiochemi-
cals, Irvine, CA, USA). A 589 bp MunI/BglII fragment,
fully sequenced to exclude additional unintended mutations,
was removed from the LPSKH5-7 plasmid and inserted into
the MunI/BglII site of the yeast exp ression vector,
pCGY1406a. T he pCGY1406ab vectors now carrying t he
desired mutations in the a1 subunit cDNA (Table 1) and
the wild-type cDNA for the sodium pump b1 subunit [9]
were used to transform yeast [13].
Mutations of Tyr895 were introduced by PCR using the
QuikChange
TM
Site-Directed Mutagenesis Kit (Stratagene
Europe, Amsterdam, the Netherlands) and the primers
shown in Table 1. The template plasmid was pBluescript
TM
KS II+ containing, in the multiple cloning site, a 1528 bp
BglII/AflII fragment of the a1 subunit cDNA. The protocol
of the provider was used for the amplification of the mutant
cDNA. After complete automatic sequencing, the 1528 bp
BglII/AflII fragments carrying the desired mutations were
ligated back into the yeast vector and used for yeast
transformations.

Isolation of membranes containing native or mutant
sodium pumps
The methods involved in the isolation of membranes
from yeast cells, and for the preparation of SDS-treated
microsomes enriched in the sodium pump, have been
described previously in great detail [14,15]. The Na
+
/
K
+
-ATPase activity in the i solated fractions was d eter-
mined by a coupled spectrophotometric assay in the
presence or absence of 1 m
M
ouabain [9,16]. The protein
concentration of the microsomal preparations was deter-
mined by the method of Lowry [17] using BSA as a
standard.
Table 1. Primers used for mutations.
Mutation Oligonucleotide
Primers used for mutations by inverse PCR
Wild type
5¢-GTGGAGGACAGCTATGGGCAGCAG-3¢
Asp893fiArg
5¢-GTGGAGCGCAGCTATGGGCAGCAG-3¢
Asp893fiGlu
5¢-GTGGAGGAGAGCTATGGGCAGCAG-3¢
Asp893fiAla
5¢-GTGGAGGCCAGCTATGGGCAGCAG-3¢
Ser894fiAsp

5¢-GTGGAGGACGACTATGGGCAGCAG-3¢
Ser894fiIle
5¢-GTGGAGGACATCTATGGGCAGCAG-3
Gly896fiArg 5¢-GTGGAGGACAGCTATAGGCAGCAG-3¢
Gly896fiIle
5¢-GTGGAGGACAGCTATATCCAGCAG-3¢
Second primer for all of the above
5¢-GTCATTAATCCAACGGTCATCCCA-3¢
a
Primers used for mutations by PCR and the QuikChange
TM
Site-Directed Mutagenesis Kit
Wild type
5¢-GTGGAGGACAGCTATGGGCAGCAGTGG-3 ¢
Tyr895fiCys 5¢-GTGGAGGACAGCTGTGGGCAGCAGTGG-3¢
Second primer
5¢-CCACTGCTGCCCACAGCTGTCCTCCAC-3¢
Tyr895fiPhe
5¢-CGATGTGGAGGACAGCTTTGGCCAGCAGTGGACCTATG-3¢
Second primer
5¢-CATAGGTCCACTGCTG
G
CCAAAGCTGTCCTCCACATCG-3¢
b
Tyr895fiTrp 5¢-CGATGTGGAGGACAGCTGGGGGCAGCAGTGGACC-3¢
Second primer
5¢-GGTCCACTGCTGCCCCCAGCTGTCCTCCACATCG-3¢
a
Silent mutations produce a diagnostic restriction site for AseI.
b

The silent mutation produces a diagnostic restriction site for EaeI.
3822 S. Becker et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Co-immunoprecipitation of a1 and b1 subunits
by an antibody against b1 subunits
Unless otherwise specified, all of the following steps were
carried out at 4 °C. A total of 1 mg of microsomes enriched
in Na
+
/K
+
-ATPase [9] was centrifuged for 20 min at
13 000 g and subsequently suspended in 1.5 mL of a buffer
consisting of 50 m
M
Tris/HCl, pH 7 .5, 150 m
M
NaCl, 1 m
M
Na
2
EDTA, 0.2% BSA (w/v), 1% Triton X-100 (w/v),
2m
M
dithiothreitol, 0.2 m
M
phenylmethanesulfonyl fluor-
ide, 0.5 mgÆmL
)1
leupeptin and 0.7 mg ÆmL
)1

pepstatin.
Thereafter, 0.5 lLoftheanti-b1 immunoglobulin (Alexis
Corporation, Gru
¨
nberg, Germany) w as added t o the solu-
tion and the reaction was allowed to proceed for 5 h under
continuous, g entle shaking. Then, 20 lLofproteinG–
SepharoseÒ 4B (Sigma, Deisenhofen, Germany) was added
and the incubation extended for an additional 16 h. The
solution was then c entrifuged at 900 g for5sandthe
supernatant removed by aspiration. The pellet w as then
subjected to a washing routine involving alternating steps of
careful suspension of the complex i n the extraction buffer
followed b y centrifugation at 900 g for 5 s a t 4 °C, as
described i n t he protocol of Tamkun & Fambrough [18]. The
Sepharose beads were first washed three times in 1.5 mL of
buffer A [150 m
M
NaCl, 50 m
M
Tris/HCl, pH 7 .5, 1 m
M
Na
2
EDTA, 0.5% Triton X-100 (w/v)], then once with
1.5 m L of a solution consisting of 300 m
M
NaCl, 50 m
M
Tris/HCl, pH 7.5, 0.1% SDS (w/v), 0.1% Triton X-100

(w/v), th en once with 1.5 mL of 1
M
NaCl, 50 m
M
Tris/HCl,
pH 7 .5, 0.5% Triton X-100 (w /v), twice with 1.5 mL of
buffer A, and finally with 1.5 mL of 1% Triton X-100 (w/v).
Thereafter, the antigen/antibody/Sepharose bead com-
plex was suspended in 20 lL of sample buffer consisting
of 250 m
M
Tris/HCl, pH 6.8, 10% SDS (w/v), 10%
2-mercaptoethanol (v/v), 1 m gÆmL
)1
Coomassie Brilliant
BlueÒ and 25% glycerol (v/v), and heated for 2 min at
100 °C. The Sepharose beads were pelleted by centrifuga-
tion and 20 lL of the supernatant was mixed with 20 lLof
8
M
urea and heated for 5 min at 70 °C. Proteins in 20 lL
of this mixture were separated by electrophoresis on a
polyacrylamide gel containing 10% polyacryla mide and
0.3% N,N¢-methylene-bisacrylamide [19]. T he gel was then
equilibrated for 30 min in 0.1% S DS (w/v), 12.5 m
M
Tris,
96 m
M
glycine, 20% methanol (v/v), pH 8.4, and blotted

onto a nitrocellulose membrane (Schleicher & Schuell,
Dassel, Germany) for 2 h at 2.5 AÆcm
)2
[20].
The nitrocellulose membrane was first blocked overnight
in NaCl/P
i
(PBS) containing 0.1% Tween 20 (v/v) ( NaCl/
P
i
-T) and 5% (w/v) nonfat dried milk, then washed three
times (10 min each wash) with NaCl/P
i
-T, and subsequently
incubated for 2 h with either an anti-a1mAb(diluted
1 : 300 in NaCl/P
i
-T) o r a n anti-b1 m Ab (diluted 1 : 1000
in NaCl/P
i
-T), bo th raised in mouse (Alexis Cor poration).
The detection of antibody-bound a1orb1 subunits was
carried out by incubating the nitrocellulose membrane for
90 m in with a n alkaline phosphatase-conjugated anti-
mouse IgG (SeroTec, Oxford, UK; diluted 1 : 2500 in
NaCl/P
i
-T) and subsequently adding the alkaline phos-
phatase substrates 5-bromo-4-chloro-3-indoyl-phosphate
(Molecular Probes, Eugene, OR, USA) for the detection

of a1 subunits, or Nitro Blue tetrazolium (Serva, Heidel-
berg, Germany) f or the detection of b1 subunits, according
to the corresponding protocols of the providers. The
chromogenic reaction was interrupted by adding 20 m
M
EDTA in NaCl/P
i
-T.
Metabolic labelling and immunoprecipitation
of a1 subunits
Single yeast colonies were grown in 5 mL of selective
minimal medium for 16 h at 30 °C to mid-logarithmic
phase [i.e. an attenuance (D), at 600 n m, of 0.5–1]. A total of
2.5 · 10
6
cells, corresponding to a D
600
of 2.5, were then
centrifuged at room temperature for 5 min at 1800 g and
washed with sterile H
2
O. T his procedure was repeated.
Thereafter, cells were suspended i n 1.25 mL of minimal
medium and incubated at 3 0 °C for 60 min with continu-
ous, gentle shaking. Cells were pelleted again, a s described
above, and subsequently suspended in 1.25 m L of the
minimal medium now containing 100 lCiÆmL
)1
of
[

35
S]methionine. Incubation was allowed to proceed with
gentle shaking at 30 °C for a further 30 min.
Labelled c ells we re centrifuged, as described above, and
washed with 1 mL o f ice-cold water. After a second w ash,
the pelleted cells were suspended in 100 lLofanextraction
buffer comprising 50 m
M
Tris/HCl, pH 7.4, 150 m
M
NaCl,
10 m
M
MgCl
2
,1m
M
EDTA, 10% glycerol (w/v), 0.2%
BSA (w/v), 2 m
M
dithiothreitol, 0.2 m
M
phenylmethane-
sulfonyl fluoride, 0.5 mgÆmL
)1
and 0.7 mgÆmL
)1
pepstatin
(phenylmethanesulfonyl fluoride, leupeptin and pepstatin
were obtained from Boehringer Ingelheim, Heidelberg,

Germany). Then,  100 lL of glass beads (0.25–0.3 mm in
diameter) were added and the cells were broken by 10 bursts
of 20 s of vigorous mixing at the highest speed in a vortex
mixer, each time followed by a 40 s cooling phase on ice.
The supernatant was then transferred to a new vial and the
glass beads washed twice with 100 lL of the extraction
buffer. The combined supernatants ( 300 lL) were cen-
trifuged at 7500 g for 20 min at 4 °Ctoremovedebris.
After adjusting the radioactivity in the supernatants with
extraction buffer to be the same in a final volume of 750 lL,
0.5 lLofananti-a1 immunoglobulin (Alexis Corporation)
was added and the s olution w as incub ated w ith g entle
shaking for 16 h at 4 °C. Subsequently, 25 lL of a protein
G + agarose suspension ( Sigma) were added and incuba-
tion was continued for another 4 h.
Thereafter, the antigen/antibody/protein-G + agarose
complex was sedimented at 4 °C by centrifugation for 5 s
at 900 g, and the supernatant was removed b y aspiration.
The pellet was then washed, essentially as described above,
for the immunoprecipitation using the anti-b immuno-
globulin.
After the last wash, the antigen/antibody/protein
G + agarose complex was equilibrated with 20 lLofa
buffer containing 125 m
M
Tris/HCl, pH 6.8, 4
M
urea, 5%
SDS (w/v), 5% 2-mercaptoethanol (v/v), 12.5% glycerol (v/
v), 0.5% of a solution of 0.1% eth anol saturated w ith

bromophenol blue solution (v/v), and heated at 70 °Cfor
15 min . Then, the protein G + agarose was removed by
centrifugation at 900 g for5sat4°C and solubilized
proteins in the supernatant were separated by SDS/PAGE
on gels containing 10% polyacrylamide and 0.3% N,N¢-
methylene-bisacrylamide, prepared according to Laemmli
[19]. After electrophoresis, proteins in t he gel were stained
Ó FEBS 2004 DSYG(893–896) in the Na
+
/K
+
-ATPase a1 subunit (Eur. J. Biochem. 271) 3823
with Coomassie Brilliant BlueÒ (Serva) and, after drying,
exposed for 2 days at )80 °CtoaKodakX-OmatX-ray
film.
Immunodetection of wild-type and Tyr895 mutant
a1 subunits by Western blotting
A total of 50 lg of SDS-extrac ted yeast membrane proteins
[9], containing either native or mutant Na
+
/K
+
-ATPase,
was suspended in 10 lL of load ing buffer a nd separated by
SDS/PAGE following established protocols [19]. Mem-
brane extracts from untransformed yeast served as the
negative control. Protein was then transferred onto nitro-
cellulose membranes followin g the instructions provided by
the commercially available ECL Western blotting system
PRN 2180 kit (Amersham Pharmacia Biotech, Freiburg,

Germany). Following the same protocol, the a1orb1
subunit of the Na
+
/K
+
-ATPase was detected using specific
antibodies (Alexis Corporation) raised in mice, each used
at a dilution of 1 : 2500. The s econdary antibody was a
horseradish peroxidase-coupled anti-mouse IgG provided
by the kit.
Binding of [
3
H]ouabain under various conditions
To obt ain a relative af finity f or ATP, a tot al o f 2 50 lgof
microsomal protein isolated from yeast cells expressing
either wild-type or mutant sodium pumps was incubated a t
30 °C for 5 min in a mixture containing 10 m
M
Tris/HCl,
pH 7.4, 50 n
M
[
3
H]ouabain, 50 m
M
NaCl, 5 m
M
MgCl
2
and various concentrations of ATP (Tris salt). The total

volume of each sample was 250 lL. Thereafter, the protein
was pelleted by centrifugation at 13 000 g for 2 min,
washed twice with H
2
Oat4°C, and dissolved in 200 lL
of 1
M
NaOH by incubation at 80 °C for 10 min. After the
addition of 200 lLof1
M
HCl, the samples were mixed
with 3.5 m L of scintillation cocktail (Roth) and counted for
radioactivity.
To obtain a relative affinity of the enzyme and its mutants
for Na
+
, this last experiment was performed using a
constant concentration of 100 l
M
ATP (Tris salt) and
varying t he concentration of N a
+
. B efore the m icrosomes
were used, however, they were washed twice in 1 mL of
10 m
M
Tris/HCl, pH 7.4, to remove any Na
+
from the
microsomes storage buffer, which was composed of 25 m

M
imidazole/1 m
M
Na
2
EDTA, pH 7.4 [9,15]. All other con-
ditions were unchanged.
To obtain a relative affinity for K
+
, the enzyme or its
mutants were incubated in 1 0 m
M
Tris/HCl, pH 7 .4, for
60minwith50n
M
[
3
H]ouabain, 5 m
M
phosphate (Tris
salt), 5 m
M
MgCl
2
and various concentrations of KCl. The
other conditions were as described above.
Thermal stability of wild-type Na
+
/K
+

-ATPase and
mutants
This experiment was carried out according to a p reviously
published protocol [21]. Briefly, a total of 125 lgof
microsomal protein w as incubated on ice to serve as a
control. An equivalent amount of protein was heated for
5minat50°C. Then, both samples were incubated for an
additional 15 min on ice followed by 30 min at 30 °Cwith
5m
M
phosphate (Tris salt), 5 m
M
MgCl
2
,10m
M
Tris/HCl,
pH 7.4, and 50 n
M
[
3
H]ouabain. T he total volume w as
500 lL. Bound radioactivity was determined as described
above.
Results
Na
+
/K
+
-ATPase activity

Yeast membrane preparations contain endogenous ATP-
ases. Unlike the mammalian sodium pump, these are
ouabain in sensitive. Therefore, in order to distinguish yeast
endogenous ATPases from heterologously expressed Na
+
/
K
+
-ATPase, ATPase activity was determined in SDS-
extracted membrane preparations in the presence or absence
of 1 m
M
ouabain. In Fig. 1A, it can be seen that no
significant Na
+
/K
+
-ATPase activity was detected in mem-
brane preparations from cells expressing any of the Asp893
or Gly896 mutants. The same applied for membranes from
nontransformed cells. A significant, ouabain-sensitive
ATPase activity was, however, detected in membrane
preparations from cells expressing the wild-type enzyme
Fig. 1. Na
+
/K
+
-ATPase activity in yeast membrane preparations. (A)
Inactive mu tants. (B) Active mutants. Na
+

/K
+
-ATPase activity was
determined, a s de scribed in the Experimental pro cedure s, by a coupled
spectrophotometric assay as the o uabain-sensitive fraction of a ll
ATPase activity that is present in t he preparations. All resu lts r epresent
the mean ± SD of three indep endent experiments. Membran e pre p-
arations from cells expressing either Asp893fiArg or Gly896fiIle did
not display any Na
+
/K
+
-ATPase-specific activity. The unit 1 mU is
defined as the enzymatic activity that hydrolyzes 1 nmol of ATP in
1 min at 37 °C. NT, nontransformed y east cells. *Significantly lower
(P < 0.05) than the activity obtained with the wild-type preparation.
3824 S. Becker et al. (Eur. J. Biochem. 271) Ó FEBS 2004
and all of the Ser894 or Tyr89 5 mutants (Fig. 1B). While the
Ser894 mutants and Tyr895fiCys or Tyr895fiPhe mutants
displayed ATPase activities comparable to that of the wild-
type enzyme, t he activ ity of the Tyr895fiTrp m utant was
significantly reduced.
Coimmunoprecipitation of a1 and b1 subunits
by an anti-b1 immunoglobulin
As the mutations are all localized within a peptide sequence
of the a subunit that has been shown to interact with the b
subunit [ 1,3], i t was im portant to evaluate whether the
observed loss of ATPase activity shown in Fig. 1A was
caused by the loss of a/b interactions.
Following a well-established protocol for co-immuno-

precipitation of a1andb1 b y using an anti-b1
immunoglobulin [18], it was possible to demonstrate
co-immunoprecipitation of t he wild-type a1withtheb1
subunit (Fig. 2A). Co-immunoprecipitation was also
observed with t he inactive Asp893fiArg and Asp893fiGlu
mutants of the a1 subunit (Fig. 2A). In contrast, a1 did not
co-immunoprecipitate with b1 when membrane prepara-
tions from yeast expressing the inactive forms of the a1
subunit (Gly896fiArg or Gly896fiIle) were used (Fig. 2A).
The b1 subunits, however, were found, as expected, in all
immunoprecipitates except in membranes from nontrans-
formed cells (Fig. 2B), verifying that the absence of
Gly896fiArg or Gly896fiIle in Fig. 2A was not c aused
by the lack of b1 s ubunit e xpression or by degradation of
this protein. Nevertheless, the quantities of the b1 subunits
detected varied among the lanes, indicating either different
levels of expression or variations in experimental recovery.
Hence, in order to obtain a value that relates to the
abundance of co-immunoprecipitated a1 subunits to the
precipitated b1 subunits, the protein bands in Fig. 2B were
analysed by densitometry using the image analysis system of
Biostep (Jahnsdorf, Germany). By setting the abundance
of b1 subunits in lane 4 (Asp894fiGlu) of Fig. 1B to
100%, the b1 a bundance in lane 1 (wild type), lane 3
(Asp893fiArg), lane 5 (Gly896fiArg) and l ane 6
(Gly896fiIle) were 91%, 40%, 54% and 51%, respectively.
Lane 2 (nontransformed cells) w as considered to represent
the background signal.
In an analogous way, the a1 subunit detected in lane 1
(wild type) of Fig. 2A was set to represent the 100% value.

In comparison, the relative abundance of the Asp893fiArg
mutant a1 subunit (lane 3) only accounted for 47% of this
value. The equivalent value for the Asp894fiGlu (lane 4)
mutant wa s 9 0%. L ane 2 was set to indicate the back-
ground. No protein bands were detected in lanes 5 and 6,
containing the Gly896 mutants, by the auto-detect function
of the software program.
The values obtained from the densitometric scans were
used to define the stoichiometry between co-immunopre-
cipitated a1andb subunits by forming a quotient between
the relative abundance of these subunits. For all the a1/b
heterodimers that co-immunoprecipitated (wild type,
Asp894fiArg/b subunit, or Asp894fiGlu /b subunit), the
quotient of a1 abundance to b1 abundance was approxi-
mately 1, indicating proportional expression and recovery
levels.
Detection of [
35
S]methionine-labelled wild-type and
mutant a1 subunits expressed in yeast
Our inability to co-immunoprecipitate the Gly896fiArg
and Gly896fiIle mutants of the a1 subunits (Fig. 2A) might
have been caused by a lack of e xpression. Therefore, yeast
cells transformed with plasmids coding for Asp893fiArg,
Asp893fiGlu, Gly896fiArg or Gly896fiIle mutants of the
a1 subunit of the sodium pump were m etabolically labelled
with [
35
S]methionine and subsequently used to isolate
membrane fractions. Mutant or wild-type a1 subunits were

isolated from this mixture by immunoprecipitation with an
anti-a1 immunoglobulin. As shown in Fig. 3, it is apparent
Fig. 2. Coimmunoprecipitation of inactive mutant a1 subunits with an
antibody a ga inst b1 subunits. (A) As w ith t he wild-t ype a1 subunit (lane
1), the inactive Asp893fiArg (lane 3) and Asp893fiGlu (lane 4)
mutants c o-immunoprecipitate w ith the b1 subunits, indicating that
a/b assembly is not affected by these two mu tations. In contrast, the
Gly896fiArg (lane 5) or Gly896fiIle (lane 6) mutants do not co-
immunoprecipitate w ith the b1 subunits. The co-immunoprecipitated
subunits were visualized in a Western blot using an antibody against
the a1 subunit as a primary antibo dy and a n alkaline phosphatase-
conjugated anti-imm unoglobuli n G (IgG) as a secondary a ntibody,
with 5-bromo-4-chloro-3-indoyl-ph osphate as a chromogenic alkaline
phosphatase substrate. (B) All b1 subunits precipitate as an antigen/
antibody/protein G–Sepharose complex and can be detected in the
Western blot using an antibody against b1 subunits as a primary
antibody and the same secondary antibody mentioned above. The
chromogenic substrate of alkaline phosphatase used he re was N itro
Blue tetrazolium. Neither a1norb1 subunits were visualized in
membranes from nontransformed yeast cells (lane 2).
Fig. 3. Immunoprecipitation of metabolically labelled, inactive mutant
a1 subunits. [
35
S]Methionine-labelled proteins were immunoprecipi-
tated by an anti-a1 immunoglobulin, as described in the Experimental
procedures. After SDS/PAGE, labelled proteins were detected by
autoradiography. Wild-type (lane 1) or mutant a1 subu nits
(Asp893fiArg, lane 3; Asp8 93fiGlu, lane 4; Gly896fiArg, lane 5;
Gly896fiIle, lane 6) were present in the membrane preparations from
transformed cells. A labelled protein of  110 kDa was n ot found in

membranes from nontransformed cells (lane 2).
Ó FEBS 2004 DSYG(893–896) in the Na
+
/K
+
-ATPase a1 subunit (Eur. J. Biochem. 271) 3825
that a labelled protein of 110 kDa was found only in
membrane preparations from yeast cells expressing either
the wild-type or the mutant a subunits. As the sodium pump
a1 subunit displays a relative molecular mass of  110 kDa,
and because no similar p rotein was detected i n m embrane
preparations from nontransformed cells (lane 2), it is very
likely that t he precipitated proteins are the wild-type a1
subunits and its mutants. Furthermore, these results dem-
onstrate that the a1 subunits are expressed at approximately
the same level.
Immunodetection of the Tyr895 mutants by Western
blotting
The Tyr895 mutants are all active (Fig. 1 ). Nevertheless,
the Tyr895fiTrp mutant displayed a significantly de-
creased activity when compared to the wild-type enzyme
and to the mutants Tyr895fiCysorTyr895fiPhe. In
order to evaluate whether this difference was caused by
different e xpre ssion levels, SDS-extracted membranes of
cells expressing either of these mutants or the wild-type
enzyme were probed in a Western blot w ith antibodies
against the a1orb subunit of the sodium pump.
As s hown in Fig. 4 , t he abundance of the Tyr895fiTrp
mutant is considerably reduced when compared to the
abundance o f t he wild-type enzyme a nd the other

mutants. Comparison of the signals for the wild-type
and the Ty r895fiCys and Tyr895fiPhe mutants indicates
similar expression of these enzymes, as determined by
optical densitometry using the image analysis s ystem of
Biostep, described above. Using the same system, the
quotient of a1 abundance to b1 abundance was approxi-
mately 1 for all the a1/b heterodimers that were detected
in this Western blot (wild type/b subunit, Tyr895fiCys/b
subunit, T yr895fiPhe/b subunit or Tyr895fiTrp/b sub-
unit), indicating proportional expression and recovery
levels.
Binding of [
3
H]ouabain as a function of ATP
concentration
In the presence of ATP, Na
+
,Mg
2
+
and [
3
H]ouabain, the
sodium pump a1 subunit forms a stable [phosphoen-
zymeÆ[
3
H]ouabain] complex that can be easily measured
[4]. Figure 5 shows the binding of [
3
H]ouabain to yeast

membrane preparations as a function of the ATP concen-
tration in the presence of Na
+
and M g
2
+
.After5minof
incubation, [
3
H]ouabain binding was detectable with mem-
branes containing the wild-type enzyme o r either of the
Ser894 mutants. The EC
50
for ATP was 0.77 ± 0.11 l
M
for the wild-type enzyme and 0.46 ± 0.10 l
M
or
0.94 ± 0.11 l
M
for the Ser894fiAsp and Ser894fiIle
mutant enzymes, respectively.
Similar results were obtained with the Tyr895 mutants
(Table 2). These values and the values obtained with the
Ser894 mutants a re all in g ood agreement with K
D
values
determined for ATP binding to sodium pumps from
mammalian tissues [22].
Binding of [

3
H]ouabain as a function of Na
+
concentration
When [
3
H]ouabain binding was measured as a function of
the Na
+
concentration i n the presence of 100 l
M
ATP,
Na
+
-enhanced [
3
H]ouabain binding to the w ild-type
enzyme, with an EC
50
of 1.26 ± 0.38 m
M
, was observed
(Fig. 6 ). The corresponding values determined with mem-
branes containing either the Ser894fiAsp or Ser894fiIle
mutants were 1.46 ± 0.58 m
M
and 1.56 ± 0.58 m
M
,
respectively, and indicate the action of Na

+
on cytosolic
sites [23,24]. No specific binding was seen under these
conditions with membranes from nontransformed cells. The
Tyr895 mutants displayed s imilar sensitivities towards Na
+
(Table 2).
Fig. 4. Immunodetection of active Tyr895 mutants. The W estern blot
experiment demonstrates reduced expression for the Tyr895fiTrp a1
(A) a nd b1 subunits ( B), t hus e xplaining t he reduced Na
+
/K
+
-ATPase
activity observed with membrane preparations containing this mutant.
The wild-type, T yr895fiCysorTyr895fiPhe a1(A)orb1 subun its ( B)
were expressed at comparable levels. The multiple protein bands r eflect
various glycosylation states of the b1 subunit [9].
Fig. 5. Binding of [
3
H]ouabain as a function of the ATP concentration.
Yeast membranes from cells expressing either the wild-type (h)orthe
Ser894fiAsp (s) and Ser894fiIle ( .) mutants were incubated for
5minwith50n
M
[
3
H]ouabain, 50 m
M
NaCl, 5 m

M
MgCl
2
and var-
ious concent rations o f A TP ( Tris sa lt; s ee th e E xperim ental p rocedures
for details). ATP promotes [
3
H]ouabain binding to wild-type and
mutant enzymes with similar EC
50
values of  1 l
M
.
3826 S. Becker et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Binding of [
3
H]ouabain as a function
of K
+
concentration
In order to obtain a value for the relative affinity for K
+
of
the wild-type enzyme and the S er894fiAsp or Ser894fiIle
mutants, the binding of [
3
H]ouabain to these enzymes in the
presence of phosphate and Mg
2
+

was measured as a
function of the K
+
concentration. Under these conditions,
K
+
caused a reduction in [
3
H]ouabain binding to the wild-
type enzyme, showing an EC
50
of 3.8 ± 0.33 m
M
(Fig. 7 ).
The corresponding value obtained with the Ser894fiAsp
mutant was 1.4 ± 0.06 m
M
. A similar experiment carried
out with the Ser894fiIle mutant yielded an EC
50
of
21.95 ± 1.41 m
M
, and the Tyr895 mutants revealed K
+
sensitivities comparable to that of the wild-type enzyme
(Table 2).
Table 2. Properties of the Tyr895 mutants.
Mutant
EC

50
for ATP
(l
M
)
EC
50
for Na
+
(m
M
)
EC
50
for K
+
(m
M
)
Detection of a and b
subunits in the Western blot
Na
+
/K
+
-ATPase activity
(mUÆmg protein
)1
)
ab

Tyr895fiCys 0.72 ± 0.3 1.44 ± 0.11 3.74 ± 1.14 + + 13.16 ± 1.95
Tyr895fiPhe 1.07 ± 0.15 1.36 ± 0.71 1.75 ± 0.78 + + 13.30 ± 1.61
Tyr895fiTrp 0.8 ± 0.2 1.87 ± 0.31 2.78 ± 0.59 + + 7.60 ± 1.89
Fig. 6. Binding of [
3
H]ouabain as a function of Na
+
concentration. (A)
Yeast membranes from cells expressing the wild-type (h), the
Ser894fiAsp (s) or the Ser894fiIle (.) mutants were incubated for
5minwith50n
M
[
3
H]ouabain , 5 m
M
MgCl
2
, 100 l
M
ATP (Tris salt)
and various concentrations of Na
+
. The latter promotes [
3
H]ouabain
binding to wild-type and mutant enzymes. (B) T he double re ciprocal
plot of the values obtained above reveals that Na
+
promotes ouabain

binding to the wild-type and mutant enzymes with similar EC
50
values
of  1.5 m
M
.
Fig. 7. Inhibition of [
3
H]ouabain binding by K
+
. Yeast membranes
from cells expressing the wild-type (h), the Ser894fiAsp (s)orthe
Ser894fiIle (.) m utants were incubated in 10 m
M
Tris/HCl, pH 7.4,
for 60 min with 50 n
M
[
3
H]ouabain, 5 m
M
phosphate (Tris salt), 5 m
M
MgCl
2
, and various concentrations of KCl. The other conditions were
as described above. In all cases, K
+
leads t o a reduction of ouabain
binding. (B) By plottin g the reciprocal binding of [

3
H]ouabain against
the K
+
concentration, the EC
50
for K
+
can be obtained from the
intercept of the straight lines with the abscissa.
Ó FEBS 2004 DSYG(893–896) in the Na
+
/K
+
-ATPase a1 subunit (Eur. J. Biochem. 271) 3827
Thermal stability of the Tyr895 mutants
The binding of [
3
H]ouabain to membrane preparations that
have been preheated at 50 °C ca n serve as a measu re for the
stability of the a/b heterodimer that forms the catalytically
active enzyme [21]. After preheating the wild-type sodium
pump at 50 °C f or 5 min, the enzyme was c apable of
binding only 62.0 ± 8.0% of the ouabain that was bound
by the same control enzyme that had been incubated for the
same length of time on ice (Fig. 8). The Tyr895 mutants
displayed a similar behaviour. Thus, after preheating, the
Tyr895fiPhe mutant bound 67.6 ± 16.0% of the ouabain
bound by the control, while the corresponding value w ith
the Tyr895fiTrp mutant was 51.4 ± 5.9%. Therefore,

these two conservative mutants did not convert into
more temperature-sensitive forms. The nonconservative
Tyr895fiCys mutant, however, was able to bind only
36.5 ± 6.5% of the ouabain that was bound by the
unheated control, indicating a higher thermal sensitivity
than the wild-type enzyme or its conservative Tyr895
mutants (Fig. 8). This result, which shows a significantly
lower value than that obtained w ith the wild-type enzyme,
points towards an involvement of the aromatic group in
enzyme stabilization (Fig. 8).
Discussion
The extracellularly localized peptide of the sodium pump a
subunit that connects the M7 and M8 membrane-spanning
domains (L7/8) contains 26 amino acids that are important
for assembly with the b subunit [1]. Corresponding peptides
of oth er K
+
-transporting ATPases seem to play a compar-
able role. Results from st udies of chimeric constructs
formed by replacing the 26 amino acids of the r at a3
subunit (Asn886)Ala911) with the corresponding region
from either the gastric (Gln905-Val930) or the distal
(Asn908-Ala933) colon H
+
/K
+
-ATPase of the rat demon-
strated interaction of the chimeras with the H
+
/K-ATPase

b subunits and h elped t o i dentify Val904, Tyr898, and
Cys908 of the sodium pump a3 subunit as important for
assembly with the b subunit [21,25]. In addition, the L 7/8
loop confers sensitivity towards specific inhibitors of P
2
-type
ATPases, as shown u sing chimeric constructs between the
a subunits of Na
+
/K
+
-ATPaseandthegastricH
+
/K
+
-
ATPase [26]. This segment was also demonstrated to be
important for ion conduction, as shown for Asp884 and
Asp885 mutants [4], to affect interactions with Na
+
or K
+
,
as demonstrated with a/b heterohybrids [ 7,27], or to be
associated with the loss of Rb
+
occlusion, as shown by
Cu
2+
-catalysed oxidative cleavage near His875 [5]. Finally,

the i mportance o f t he L7/8 loop is underlined by the high
degree of homology of the 26-amino acid peptide seen in all
K
+
-transporting P
2
-type ATPases (Fig. 9). The sequence
DSYG(893–896) is – with one exception seen in Hydra –
absolutely conserved in all of the K
+
-transporting P
2
-type
ATPases, indicating that some important role for this
peptide was conserved in the course of evolution. Thus, to
investigate the function of this tetrapeptide, single mutations
were introduced within this area of the a1 subunit. The
investigation of these mutants revealed that each of the
altered a mino acids had an impact on the enzyme proper-
ties, although in somewhat different ways. In general,
however, we can distinguish between catalytically inactive
and catalytically active mutants.
Catalytically inactive mutants
All mutants of Asp893 and Gly896 were i nactive (Fig. 1) .
Lack of expression might be one plausible reason for the
lack of measurable activities. Alternatively, for some of the
mutants it could be that a1andb1 subunit interactions were
disturbed, as all of the mutations are within the 26-amino
acid peptide that w as found to be important for a ssembly [1].
A possible c hange in expression caused b y t he mutations was

investigated by applying an immunoprecipitation protocol
after metabolic labelling of the wild-type and mutant a1
subunits. The immunoprecipitation method was preferred
over a standard Western blot to prevent possible degrada-
tion of the mutant a1 subunits, which are known to be a
target for proteases unless they form heterodimers with the b
subunit [28,29]. This experiment, however, verified that all
Asp893 and Gly896 m utants were expressed similarly to that
of the wild-type a1 subunit (Fig. 3). B ased on that result, the
loss of activity is not caused by the lack of expression.
A possible loss of a/b interaction because o f the
mutations was investigated by applying a co-immunopre-
cipitation protocol, described previously [18], which verified
alossina/b interactions when Gly896 is replaced with either
Arg or Ile. This may provide an explanation for the lack of
any detectable enzymatic activity with these two mutants, as
formation of the a/b-heterodimer is a presupposition for
Na
+
/K
+
-ATPase activity [9].
Although rather unusual, single mutations that entirely
change, or e ven obliterate, protein–protein i nteractions are
not that uncommon. An arginine residue was found to
be absolutely essential for oligomerization o f ribulose-1,
5-bisphosphate carboxylase [30]. A similar experience was
Fig. 8. Thermal stability of t he wild-type ATPase and the Tyr895
mutants. When the wild-type sodium pump is heated for 5 min at
50 °C, the enzyme binds only 62.0 ± 8.0% of the [

3
H]ouabain bound
by the unheated control. The Tyr895fiPhe and the Tyr895fiTrp
mutants behave similarly. Ouabain binding to the nonconservative
Tyr895fiCys mutant, however, is only 36.5 ± 6.5% of that obtained
with the unheated control under these conditions. This result is signi-
ficantly lower (*P < 0.05) than that obtained with the wild-type
enzyme. For all measurements n ¼ 3, error bars represent ± SD.
3828 S. Becker et al. (Eur. J. Biochem. 271) Ó FEBS 2004
also observed with s odium pump/proton pump hybrids
where the amino acids Tyr898, Val904, and Cys908 of the
a3 subunit of the sodium pump (corresponding to Tyr901,
Val907 and Cys911 of the sheep a1 subunit u sed in the
current investigation) were found to be important for
assembly with the b subunit [ 21].
On the other hand, co-immunoprecipitation was ob-
tained with the inactive Asp893fiArg and Asp893fiGlu
mutants that w as similar i n relative a mount to t he co-
immunoprecipitated wild-type a1 subunit (Fig. 2). Thus, the
very strong negative effects of the Asp893 mutations on
enzyme activity cannot be explained by a lack of association
of a and b subunits. One possibility is that th e mutation of
this amino acid results in reduced affinity for ouabain, and
therefore binding of [
3
H]ouabain was n ot detectable under
the e xperimental c onditions applied in this investigation.
Although this explanation cannot be excluded, two facts
speak against it: first, in the coupled spectrophotometric
assay, preincubation with 1 m

M
ouabain did not result in
different A TPase a ctivities for the assays performed i n the
presence or absence of t he glycoside. Unless ouab ain
sensitivity is completely abolished by the mutation, 1 m
M
ouabain should be sufficient to detect some inhibition.
Second, comparison of the primary sequen ces reveals that
the aspartic acid investigated is highly conserved in all K
+
-
transporting P-type ATPases, regardless of w hether or not
they bind ouabain (Fig. 8). This latter fact suggests that this
highly conserved a spartic acid i s unlikely to b e directly
involved in ouabain binding. As Asp893 is within an area of
the p rotein that interacts w ith the b subunit [1,2], and
because the b subunit h as been shown not only to influence
enzyme properties [6,26,27] but also to be absolutely
essential for catalytic activity [9,31], it m ight be that
Asp893 is involved in enzyme conformational transitions
required for ATP hydrolysis and/or ion translocation. This
assumption is difficult to investigate further, however,
because in various experiments that were not shown here
it was not possible to detect any partial activities that are
typical for the sodium pump [32] with any of the Asp893
mutants.
Catalytically active mutants
Mutants of S er894 and Tyr895 were all active (Fig. 1 ). This
was a rather unexpected result, because, by using the tw o-
hybrid system, p revious reports had identified these amino

acids as being critical for a/b assembly and for enzyme
activity [3]. In addition, interactions of the Tyr895 mutants
with cations or ATP were not altered when compared w ith
the properties o f the wild-type enzyme (Table 2 ). The fact,
however, that in Hydra the amino acid corresponding to
Tyr895 is a phenylalanine (Phe910; Fig. 9) indicates that
although Tyr895 might not be a critical amino acid, the
presence of an aromatic group might be important for
enzyme stability. This seems to be the case, as the
Tyr895fiCys mutant displayed a significantly higher
thermal sensitivity than the wild-type enzyme or the
Tyr895fiPhe and Tyr895fiTrp mutants (Fig. 8). The
Ser894 mutants were all active and d isplayed, in most
cases, properties similar to th ose of the w ild-type enzyme.
Their interactions with ATP or Na
+
were essentially
unaffected (Figs 5 and 6). Nevertheless, the interactions of
Fig. 9. Comparison of primary structures. The 26-amino acid p eptide of the sodium p um p a1 s u bunit that is known t o be important for th e assembly
with b subunits is c ompared with equivalent a reas of other K
+
-transporting P-type ATPases. The A sp893, Ser894 and Gly896 residues inv estigated
here (underlined) correspond to highly conserved aspartic acid, serine and glycine residues present in all K
+
-transporting P-type ATPases. Similar
aminoacidsarenotfoundintheCa
2+
ATPases or in the Na
+
ATPases. The Tyr895 is replaced with a phenylalanine (Phe910) in Hydra.*The

numbering here takes into consid eration the deduction of a 5-amino acid propeptide.
Ó FEBS 2004 DSYG(893–896) in the Na
+
/K
+
-ATPase a1 subunit (Eur. J. Biochem. 271) 3829
the mutants Ser894fiAsp and Ser894fiIle with K
+
were
clearly different from these of the wild-type enzyme. As
showninFig.6,K
+
reduces [
3
H]ouabain binding to
membranes containing the wild-type enzyme, with an EC
50
of 3.8 ± 0.33 m
M
. The membranes containing the
Ser894fiAsp mutants, however, display an EC
50
of
1.4 ± 0.06 m
M
, an almost threefold-higher relative affinity
towards K
+
than that of the wild-t ype enzyme (Fig. 6). In
thecaseoftheSer894fiIle mutant, the relative affinity for

K
+
was 21.95 ± 1 .41 m
M
, which is about six t imes lower
than the a ffinity of the wild-type e nzyme.
How can mutation of Ser894 affect the interactions of the
enzyme with K
+
? Here, too, one can only assume that this
highly conserved s erine stabilizes a s tructure of the p rotein
important for enzyme interactions with K
+
on the extra-
cellular surface. The fact that replacement o f the serine
hydroxyl group by the aspartic acid carboxyl group, with its
higher dipole character, results in an enzyme with higher
apparent affinity for K
+
, whereas replacement by the
nonpolar isoleucine results in an enzyme with lower affinity
for t he cation, could indicate (but does not necessarily
require) a direct involvement of Ser894 in the process
involved in the uptake of K
+
from the extracellular milieu.
A different explanation should also be considered: because
the b subunit is known to influence the interactions of the
enzyme w ith K
+

and because Ser894 is within a sequence
known to interact with the b subunits, it is possible that
mutations of this amino acid influence i nteraction between
the subunits sufficiently enough to affect K
+
recognition
without being absolutely essential for activity. Based on the
present data, it is not possible to discern between these two
possibilities.
The results obtained h ere demonstrate a variety of
functional implications associated with the highly conserved
peptide DSYG(893–896). While Tyr895 appears to be
rather neutral for the enzyme properties, Ser894 is involved
– directly or indirectly – in enzyme interactions with K
+
.
Also, Asp893 and Gly896 were proven to be absolutely
essential for activity. Nevertheless, while mutations of
Gly896 demonstrate that t his a mino acid is critical for
assembly between a and b subunits, the results obtained
with the Asp893 mutants demonstrate that loss of a ctivity
cannot be entirely explained on t he basis of structural
disturbances leading to the loss of a/b subunit interactions.
Together with the results of previous investigations, show-
ing the involvement of amino acids from within the L7/8
loop in Na
+
conduction [4] and studies showing the loss of
Rb
+

occlusion after Cu
2+
-catalysed cleavage of the L7/8
peptide [5], the study presented here supports and underlines
the importance of the L7/8 peptide for the function of a/b
heterodimeric P-type ATPases by addressing the functional
role of single amino acids from the highly conserved peptide
DSYG(893–896).
Acknowledgements
The authors thank E. A. Martin son for reading the manuscript and
R. A. Farle y for t he generous gift of the vectors YhNa1 and GhNb1.
This work was supported b y t he Deut sche Forschungsgemeinschaft,
Sche 397/5-1 a nd 397/5-2. S. B. was supported throu gh the Gradu-
iertenkolleg ÔMolekulare Biologie und Pharmakolo gieÕ of the Justus-
Liebig-Univ ersit y Giessen.
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Ó FEBS 2004 DSYG(893–896) in the Na
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