The
sodium ALICIA
pump
needs
A. McDONOUGH,*I
*Department 90033,
of Physiology
USA;
KATHI
and Biophysics,
and tlnstitut
GEERING,1 University
de Pharmacologie,
AND
of Southern
L ‘Universite
The
sodium
plasma
pump
membrane
a family
of all
has
of catalytic
(a) and
subunit port
been
or enzymatic
function
has
of j3
and summarize j3 subunits
can
of both
that
lead
to
increased
These findings are that the /3 subunit
puzzle.
about
causes
of
consistent
regulates,
to the its
Key tein
/3
K.;
FARLEY,
FASEBJ.
subunit.
R. A. The sodium 4:
Words: Na,K-ATPose regulation
1598-1605;
oligomer
pumps. hypothesis of a(3 transported A. A.;
pump
needs
1990.
assembly
glycopro-
SODIUM PUMP, NA,K-ATPAsE, is an intrinsic and vital plasma membrane-bound oligomer. This pump maintains cellular ionic gradients, osmotic balance, and membrane electrical potentials through enzymatic hydrolysis ATP coupled to the counter-transport of Na (out) for K (in) across the plasma membrane. This oligomer is composed of two dissimilar protein subunits, a and /3, and possibly a third proteolipid component. The a subunit (112 kDa) is referred to as catalytic because it contains binding sites for ATP and cardiac THE
glycosides,
There
which
are three
specifically
isoforms
inhibit
of the cx subunit
a3) that display distinct sodium sitivity, and tissue distribution
affinities, (reviewed
Na,K-ATPase.
(a!,
a2,
and
ouabain senin ref 1). The
/3 glycoprotein subunit (35 kDa) has not been directly associated with enzymatic or transport functions of the sodium pump; however, separation of the /3 subunit
1598
ATPase
a subunit
results
in irreversible
transport
other
activity.
is highly
homologous
members
of
inactivation
Although the
the
of
Na,K-
to the catalytic family
of
cation-
translocating ATPases, including Ca-ATPases and H,K-ATPase, these ATPases have not yet been shown to require a subunit homologous to /3. Why then does Na,K-ATPase need its /3 subunit? The aim of this report is to review the recent data supporting the crucial role of the /3 subunit in the synthesis, expression, and regulation of abundance of Na,K-ATPase.
Primary
provide
assembly
the number of sodium pumps plasma membrane.-McDoNouGH,
and
STRUCTURE,
PROPERTIES,
AND
ISOFORMS
of j3 alone the
Ca4fornia
in ac-
we
sodium
with through
the a subunit
3 SUBUNIT
of both activity,
up-regulation
Los Angeles,
Switzerland
enzymatic of
A. FARLEY*
School of Medicine,
this
structure
a decrease
heterodirners, GEERING,
In
the
and
abundance all
trans-
for Na,K-ATPase
pretranslational
/3
The
in Na,K-ATPase a
/3 subunits,
a and
only
that expression
of glycosylation
cumulation
evidence
evidence
highly
subunits.
ROBERT Caifornia
subunits
of
family,
the pump’s
its role
is known
is required
inhibition
(/3)
recently,
what
share
this
with
and
until
the
to be a heterodimer
associated
activity,
we describe
that
established
in
is a member
In
glycoprotein
been,
review
a and
subunits.
not been
has
cells,
ATPases
catalytic
Na,K-ATPase
located
animal
of ion-translocating
homologous
that
Na,K-ATPase,
subunit
de Lausanne,
from ABSTRACT
1
its
structure
The /3 subunit of Na,K-ATPase is composed of 302-304 amino acids, depending of the species or tissue location. The polypeptide passes through the cell membrane once (2, 3), approximately 30-50 amino acids from its amino terminus, which is located within the cytoplasm of the cell. The /3-subunit glycoprotein has three N-linked carbohydrate groups exposed on the noncytoplasmic surface of the membrane (4). Comparison aligned
of individual sequences of
amino acid residues 13-subunit polypeptides
in
the from
several mammalian tissues indicates that approximately 90% of the amino acids are identical. The sequences of /3 subunits from Torpedo, Xenopus, or avian species are approximately 60% identical to those of the mammalian sequences. Amino acid substitutions in the /3-subunit sequences are nearly always conservative, preserving the ionic, hydrophilic, or hydrophobic character of the replaced residue. The distribution of charges throughout the sequences is highly conserved, with the nonconservative substitution of charged amino acids occurring at only nine positions, most often within avian or Torpedo sequences. The locations of seven cysteine residues are 100% conserved within the different sequences, and participate in three extracellular disulfide bonds (5, 6). Only one cysteine, predicted to be located within the membrane-spanning region of the polypeptide, is not found in a disulfide bond. The mature /3 subunit is resistant to proteolysis in either the
‘To whom
correspondence
should
be sent.
0892.6638/90/0004-1598/$01.50.
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membrane-bound holoenzyme or as a detergent-purified polypeptide, suggesting that the polypeptide retains a compact tertiary structure even after denaturation. The three disulfide bonds may be important in maintaining the stable tertiary structure of this polypeptide.
Secondary
structure
As expected for polypeptides of similar primary structure, all 13-subunit sequences exhibit the same predicted secondary structure pattern. The protein appears to be primarily a helical from the amino terminus until just before the first glycosylation site at Asn157 (numbering for dog, ref 7). The carboxyl-terminal half of the polypeptide appears to be organized predominantly into regions of 13-sheet or extended conformation, with two short helical regions. The transmembrane segment is thought to be helical although the predictive algorithms do not identify it as helical. Three helical regions of strong amphipathic character between amino acids 95-110, 120-130, and 140-150 are identified by hydrophobic moment analysis. These regions may represent three helical segments clustered so that a common hydrophobic face is shared. The presence of a disulfide bond in this region between cysteines 125 and 148 indicates that the domain must maintain a compact tertiary structure, and a folding pattern in which the hydrophobic residues are sequestered from the aqueous environment would be thermodynamically favored.
Tertiary
COOH
structure
No data support definitive statements about the tertiary structure of the /3 subunit. A schematic diagram intended to represent possible structural features that are consistent with hydropathy analysis, secondary structure, and hydrophobic moment analysis, and the experimentally determined transmembrane orientation, glycosylation sites, and disulfide bond arrangement of the /3 subunit appears in Fig. 1. NH2
Isoforms
of /3
The /3 subunit is encoded by multiple mRNAs that differ in the lengths of their 3’ and 5 untranslated regions. The variations appear to arise from differences in transcription from multiple initiation sites as well as the use of different polyadenylation sites (8). However, these mRNAs all code for a single /3 peptide, and evidence suggests that they are transcribed from a single gene (9, 10). Some tissues (liver, lung, spleen, thymus, mammary gland) have low to undetectable levels of this /3-mRNA and subunit, which suggests that isoforms of this subunit may also exist (11). cDNA clones indicating polypeptides with approximately 53% amino acid homology to other mammalian /3-subunit sequences have recently been isolated from a human fetal liver library and a rat brain cDNA library (12). The /3 subunit of Na,K-ATPase is the only protein
Figure 1. Model of the Na,K-ATPase (3 subunit tertiary structure. A model of the 13-subunit tertiary structure was constructed by combining the experimentally deduced information on transmembrane orientation (2), glycosylation sites (4), and disulfide bond arrangement (5, 6) with structural features consistent with results of hydropathy, hydrophobic moment, and secondary structure analyses of dog kidney. 13-Subunit tertiary structure was constructed by combining the experimentally deduced information on transmembrane orientation (2), glycosylation sites (4), and disulfide bond arrangement (5, 6) with structural features consistent with results of hydropathy, hydrophobic moment, and secondary structure analyses of dog kidney 13-subunit amino acid sequence (7). The model is intended to represent possible structural domains of the /3 subunit rather than the actual folded glycoprotein. Except for the location of the disulfide bonds (S - S), free sulfhydryl (SH), and oligosaccharide modifications (branched structures), references to individual amino acids have been omitted, as the terminal ends of the secondary structure domains cannot be precisely identified by the predictive methods used. Coils represent predicted a-helix regions, arrows
represent
represents
predicted
the approximate
13-sheet
position
regions,
and
the
shaded
area
of the cell membrane.
1599 SODIUM PUMP NEEDS ITS (3SUBUNIT ww.fasebj.org by Univ Louisiana Dupre Library/Serials Dept (130.70.8.131) on November 02, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumbe
transcription
alpha
in the National Biomedical Research Foundation2 (NBRF) data base that shows significant similarity to the sequence of these clones, and it was suggested that these clones represent an additional isoform of the /3 subunit called /32. Both the N-linked glycosylation sites and the locations of the extracellular cysteine residues are conserved between the /31 and /32 structures, suggesting that the tertiary structures of these polypeptides are similar. The mRNA for the /32 polypeptide is also expressed in cell lines derived from rat central nervous system that do not express mRNA for /31 polypeptide, consistent with the hypothesis that /32 is a structural variant of the Na,K-ATPase /31 subunit. However, the possibility has not been excluded that this polypeptide represents a 13-like subunit for some other ion pump.
MODEL OF Na,K-ATPase AND ASSEMBLY
ROLE
OF /3 SUBUNIT INTRACELLULAR
beta
gene
beta
mRNA
processing alpha
mRNA translation
bIy
SYNTHESIS
Synthesis of sodium pumps starts with transcription of a and 3 genes. Chromosomal localization studies (9, 10) indicate that it is unlikely that the genes are under the control of common th-acting regulatory elements. In fact, the a isoforms are not all found on the same chromosome as /3. Thus, in generating a model of Na,K-ATPase synthesis and assembly, as shown in Fig. 2, we must assume that the transcription of the a and /3 genes, followed by the processing and translation of aand /3-mRNAs, are separate pathways and thus potentially subject to unique regulation. Once the a- and /3mRNAs are translated into peptides with accompanying membrane integration and /3 core glycosylation, they presumably acquire the potential for a/3 subunit complex formation. Little if anything is understood about how this process occurs. Once af3 subunit complexes are formed they can exit the endoplasmic reticulum (ER) and progress through the Golgi, where the /3subunit glycosylation sites are modified to their final structures. Finally, the a/3 subunit complexes move, via unknown signals, to their target location in the plasma membrane. This review will provide additional details to the scaffolding of this model and demonstrate the importance of 3 core glycosylation, the a/3 subunit oligomer formation, and the independent regulation of a vs. /3 subunit expression in determining the levels of expression of the sodium pump.
AND
gene
IN THE MATURATION TRANSPORT
export
from
processing
ER In Golgi
routing recycling
Na
K
degradation
Figure 2. Model of Na,K-ATPase synthesis and assembly. ER, endoplasmic reticulum; PM, plasma membrane. The a and 13 subunits (glycosylation sites on 13 represented by the branched Structures) are cotranslationally inserted into the ER. After afi oligomer formation, the unit leaves the ER and proceeds through the Golgi network (where the 13glycosylation sites are modified) to the plasma membrane. In the plasma membrane, the functional sodium pumps translocate Na out of the cell and K into the cell coupled to the hydrolysis of ATP.
ing data on the functional relationship between the two Na,K-ATPase subunits. Thus, since the study by Noguchi Ct al. (13) of the Xenopus oocyte, it has become clear that both a- and j3-mRNA are necessary for expression of functional pumps at the plasma membrane. In addition, studies of transfected cells (which take advantage of the production of hybrid af3 complexes from exogenous and endogenous Na,K-ATPase subunits)
OF Na,K-ATPase To approach the question of the /3 subunit role, in vivo reconstitution of Na,K-ATPase has become a useful tool. Even though cells of higher eukaryotes are not ideal experimental systems because they all express Na,K-ATPase themselves, the use of Xenopus oocytes microinjected with a and /3 cRNA, and somatic cells transfected with a and 3 cDNA, have yielded interest1600
Vol. 4
April
1990
2Abbreviations: Na, K-ATPase, sodiumand potassium-activated adenosine triphosphatase; NBRF, National Biomedical Research Foundation; ER, endoplasmic reticulum; BIP, immunoglobulin binding protein; MDCK, Madin-Darby canine kidney cells; T3, triiodothyronine; LLC-PK1, porcine kidney cell line; BLM, basolateral membranes; 1CM, intracellular membranes; SH, sulfhydryl; PM, plasma membrane; BLM, basolateral membrane.
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have revealed that at least some of the functional properties of the enzyme appear to be solely determined by the a subunit. For instance, ouabain affinity of a/3 complexes produced in transfected cells depends on the origin of the a subunit, and not on whether the /3 subunit was derived from a ouabain-sensitive or ouabain-resistant species (14, 15). What then is the role of the 3 subunit? One major issue emerging from an analysis of other multimeric proteins is that subunit assembly is essential for most multimers to rapidly achieve a correct folding after synthesis. Correct folding is essential for stability, functional maturation, and/or exit from the ER (for review, see ref 16). In view of this postulate, we will focus on available data in support of a potential role for the /3 subunit in the early life cycle of the Na,K-ATPase (for a more extensive review, see ref 17). The a and /3 subunits rapidly assemble into stoichiometric complexes during or soon after synthesis (18). Even though a and /3 subunits can be inserted into the
ER membranes
independently
of each other
in vitro
(19), recent data suggest that a rapid interplay between the two subunits is necessary for an efficient cellular expression of the enzyme. The first observation supporting this hypothesis was made in cultured epithelial cells treated with tunicamycin, an inhibitor of protein glycosylation. At appropriate concentrations, this drug has no measurable effects on total or specific protein synthesis, and it leads to the cellular expression of nonglycosylated j3 subunit of Na,K-ATPase (20, 21). Inhibition of/3 subunit glycosylation does not affect formation of a/3 complexes, their degradation rate, their expression on the cell surface, or their transport properties (reviewed in ref 17). Interestingly, however, inhibition of core glycosylation by tunicamycin leads to a significant decrease in the cellular abundance not only of newly synthesized /3 subunits, but also a parallel decrease in the abundance of a subunits that are not glycoproteins (20, 21). A plausible explanation of this is that: 1) the synthesis rate and/or the stability of the nascent /3 subunit is influenced by inhibition of its glycosylation, resulting in decreased cellular expression, and 2) the concomitantly synthesized a subunit is not able to adopt a stable membrane organization for lack of association to the limited /3 subunit, and is in turn rapidly degraded. That the a subunit indeed undergoes a structural reorganization soon after its synthesis is evidenced from the fact that it is converted from a highly trypsinsensitive to a trypsin-resistant form within 20 mm after synthesis in cultured epithelial cells (22). In parallel, a subunit acquires the ability to change configuration in response to Na and K, and to bind ouabain in an ATPdependent manner (reviewed in ref 17). Recent data obtained from the Xenopus oocyte provide further direct evidence that a-/3 subunit association might be responsible for the important posttranslational modifications of the catalytic a subunit. Xenopus oocytes express functional Na,K-ATPase in the plasma membrane as other animal cells. In radioiodinated oocytes, immunoreactive a subunit material is
SODIUM
PUMP
NEEDS
ITS (3SUBUNIT
detected in a plasma membrane-enriched fraction along with a /3 subunit that differs in molecular mass from the Xenopws kidney /3 subunit (23). However, these oocytes synthesize much less /3 subunit than a subunit, as judged by the lack of detection of /3 subunits in metabolically labeled oocytes. The a subunit synthesized in excess over the /3 subunit is highly trypsin sensitive, and in this respect resembles the immature a subunit population in the ER of differentiated cells. In contrast to the a subunits in somatic cells, which are coexpressed with /3 subunits, the overproduced Pocyte a subunits never become trypsin resistant. Significantly, however, the oocyte a subunit acquires trypsin resistance after /3 RNA (from Xenopus) is injected into the oocytes (23). This indicates that association of exogenous /3 subunit to the endogenous a subunit provokes a structural stabilization of the catalytic a subunit that may be essential for its functional maturation. Although the general conclusions on the potential role of the /3 subunit are not compromised, an interesting alternative might be envisaged to explain the experimental observations in the oocyte. It is conceivable that Xenopus oocytes synthesize /3 isoforms, which may be assembled to a population of oocyte a subunits but cannot be detected with the antisera against Xenopus kidney /3 subunits. In injected oocytes, the overexpressed /31 subunit may compete with the oocyte /3 iso(prm for a limited number of oocyte a subunits. In this scenario, trypsin sensitivity and trypsin resistance of th oocyte a subunit in noninjected and /31 cRNAinjected oocytes, respectively, might reflect differences in intrinsic functional properties of various /3 isoforms. Although the process of subunit assembly and the establishment of the stoichiometric organization of multimeric proteins are still poorly understood, increasing experimental evidence supports the idea that correct oligomerization is not only essential for correct functional maturation but also a necessary and sufficient signal for many multimeric proteins to exit the ER (for review, see ref 16). Unassembled and thus misfolded proteins often accumulate in the ER in association with resident ER proteins such as the immunoglobulin binding protein known as BIP and/or are rapidly degraded. Although not yet established in detail, several observations indicate that subunit assembly is also required for Na,K-ATPase to be transported from the ER. First, in mouse cells transfected with an avian cDNA, a significant amount of avian a subunits remains in an intracellular compartment (most likely the ER), and only a limited number of hybrid avian a-mouse /3 complexes are expressed at the plasma membrane (15). This indicates that only a limited pool of mouse /3 subunits is available for the overexpressed avian a subunit to form transport-competent complexes. Furthermore, in Xenopus oocytes injected with /3-cRNA, more ouabain binding sites are detected at the plasma membrane than in noninjected controls (23). If oocytes indeed overproduce a subunits, this observation may indicate that association of oocyte a subunits to exogenous /3 subunits causes their surface expression.
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If the a subunit needs to be associated with /3 subunit to leave the ER, does /3 subunit, in its turn, need to be complexed with a subunit to exit the ER? This question has been addressed in a canine renal cell line (MDCK). Cells were metabolically pulse labeled with [35S]methionine, chased for up to 2 h in cold methionine (a period sufficient for sodium pumps to reach the plasma membrane in this cell line), fractionated into intracellular and plasma membranes, and subjected to immunoprecipitation with saturating amounts of anticanine Na,K-ATPase antiserum (24). A fluorogram of samples after a 2-h chase is shown in Fig. 3. The signal generated by the immunoprecipitated a and /3 subunits is proportional to the [35S]methionine content and thus the abundance of the subunits. The immunoprecipitated sodium pumps of the plasma membranes exhibit a much lower ratio of a subunit to /3 subunit compared with that seen in the intracellular membranes. This can be explained as either an excess of a in the intracellular membranes or an excess of/3 in the plasma membranes. The latter explanation is favored in this study based on two lines of evidence. First, there is a better parallel between Na,K-ATPase catalytic activity and a subunit abundance than there is with /3 subunit abundance. Assuming that catalytic activity is a function of afl subunit complexes in a 1:1 stoichiometry, this supports the idea that the a subunits are largely complexed with /3 subunits. Second, the a and j3 subunit contents of methionines would predict, assuming a 1:1 a to /3 subunit stoichiometry, a fivefold greater autoradiographic signal from total a subunits compared with total /3 subunits (including immature and mature /3 subunits), but the observed ratio of total a subunit to total /3 subunit signal was far less, suggesting an excess biosynthesis of 3 over a subunits. Perhaps this excess /3 subunit synthesis is necessary in these cells to effect efficient assembly of a/3 heterodimers. Questions that need to be addressed regarding this apparent excess plasma membrane-associated /3 subunit include whether it is complexed with another non-a subunit peptide or complexed with the a subunit in a greater than 1:1 stoichiometry, and whether the turnover time is different from that of the a subunit.
ROLE
OF THE
HETEROLOGOUS FUNCTIONAL
/3 SUBUNIT
IN THE
EXPRESSION OF Na,K-ATPase IN YEAST
Unlike animal cells, yeast cells do not contain an endogenous ouabain-sensitive ATPase activity, and thus represent an ideal protein expression system in which to study the role of the /3 subunit in Na,K-ATPase assembly or function. Using this expression system, it has been shown that the a subunit alone is insufficient to endow yeast cells with ouabain-sensitive ATPase activity or high-affinity ouabain binding sites (25). In experiments to test whether the /3 subunit was required for the expression of functional Na,K-ATPase, yeast cells were transformed with expression plasmids containing DNA encoding either the sheep kidney Na,KATPase a subunit, the dog kidney Na,K-ATPase 3 1602
Vol. 4
April
1990
fractIon
1
2
3
4
7
8
9
10
11
12
4
a
s.*
I3i BLM
%pi+p
a/pi+Pa
% Na,K-ATPase activity Na,K-ATPase enrichment
CM
40.5
47.8
61.4
37.3
0.8
1.6
34.1
57.7
4.1X
1.5X
Figure 3. Density distribution of immunoprecipitated Na,KATPase peptides in MDCK cells. Confluent MDCK cells were pulse labeled with [35S]methionine for 15 mm, then chased for 105 mm in medium with cold methionine. Harvested cells were disrupied, and postnuclear supernatants were analyzed on sorbitol density gradients. The membranes (density increasing from fraction 1 to 12) were assayed for Na,K-ATPase catalytic activity and immunoprecipitated with antiserum that recognizes both a and (3 subunits. Based on membrane markers, fractions 1-4 were designated basolateral membranes (BLM) and fractions 7-12, intracellular membranes (1CM). Immunoprecipitates were analyzed by SDSPAGE and fluorography. a and (3 (both immature flu, and mature (3m) subunits were quantitated by scanning densitometry in arbitrary units. Data shown are expressed as percentages of the total gradient absorbance units, or activity.
subunit, subunits.
or DNA encoding Both [3H]ouabain
sensitive
ATP hydrolysis
both of the Na,K-ATPase binding and ouabain-
were assayed
in membranes
prepared from the transformed cells, and [3H]ouabain binding to whole yeast cells was also assayed for each preparation. Ouabain, as a specific inhibitor of Na,KATPase, serves to distinguish Na,K-ATPase from other ATPases in the cells. Only membranes prepared from yeast cells that had been transformed with DNA encoding both the a subunit and the /3 subunit of Na,KATPase were found to contain an ouabain-inhibitable ATPase activity or to possess high-affinity binding sites for [3H]ouabain. The ATPase activity of membranes prepared from yeast cells containing only the a subunit was the same as that for membranes from untransformed yeast cells, and these membranes did not bind ouabain. These observations indicate that the expression of functional Na,K-ATPase molecules in yeast cells requires both the a subunit and the /3 subunit. Noguchi Ct al. (13) also concluded that the /3 subunit was essential for expression of functional Na,K-ATPase after injection of rat brain mRNA into Xenopus oocytes. Unlike the frog oocyte, in which it appears that rat a subunits
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cannot combine with Xenopus /3 subunits, the results obtained from yeast demonstrate that functional Na,KATPase molecules can be formed between mammalian a subunits and /3 subunits from different species. This result was expected, however, on the basis of the similar amino acid sequences of the mammalian /3 subunits. These observations also indicate that yeast cells do not have an endogenous protein that can substitute for the Na,K-ATPase 3 subunit in the assembly of the functional enzyme. In neither yeast nor the oocytes, however, was the role of the /3 subunit determined. As indicated above for the case of epithelial cells (22), proteolytic digestion experiments support the suggestion that the /3 subunit and the a subunit associate in the ER to form a complex that is subsequently exported from the ER. It will be interesting to see whether the putative /32 isoform of Na,K-ATPase can assemble with an a subunit in yeast cells to generate functional enzyme, considering the low degree of amino acid identity of this polypeptide with the /31 subunit. Perhaps the amino acid sequence differences between /31 and /32 subunits define interactions that are compatible with specific a isoform-/3 isoform interactions. Alternatively, if the a subunit of the Na,K-ATPase is normally in excess relative to the /3 subunit, as suggested by data from Xenopus oocytes discussed in the previous section, and the number of functional pumps that mature to the plasma membrane depends on the efficiency with which the two polypeptides associate, then the different isoforms of the /3 subunit may represent another mechanism to regulate the number of pumps that are assembled. Regions of sequence similarity between /31 and /32 subunits may provide useful targets for mutagenesis in order to define regions that interact directly with the a subunit or otherwise play a functional role in pump activity. To address these issues, the yeast expression system appears well suited for the investigation of the functional consequences of expression of different isoforms of the a subunit together with different isoforms of the /3 subunit. ROLE OF Na,K-ATPase
/3 SUBUNIT
IN THE REGULATION EXPRESSION AND ABUNDANCE
PUMP NEEDS ITS
(3SUBUNIT
3 5
6 #{149}5
9 II
12
18 II
24hinJK It
a
OF
Sodium pump abundance in the cell has been shown to be regulated by changes in ionic environment that necessitate increased sodium pumping for cell survival (26, 27), and by hormones such as aldosterone and thyroid hormone (T3) that up-regulate pump expression to maintain ionic or metabolic homeostasis of the whole animal (28). An increased sodium pump abundance dictates a parallel and stoichiometric increase in the a and j3 subunits of mature Na,K-ATPase molecules. Mechanisms reported to account for the increased abundance include increased synthesis rate (27-29), decreased degradation rate (30, 31), and both processes working in concert (31). With regard to upregulation via pretranslational mechanisms, the changes in a- and j3-mRNA levels are similar in magnitude in cases such as regulation by T3 in kidney (29), and quite SODIUM
different in magnitude in cases such as regulation by T3 in heart and liver (32, 33) and regulation of K depletion in LLC-PK1 cells (34). The consequences of differential induction of a- and /3-mRNA and subunit levels will be discussed below. When LLC-PKI cells, an epithelial pig kidney cell line, are incubated in medium containing only nominal K, they respond by increasing Na,K-ATPase abundance and activity (34). Typically, after 24 h of K depletion, the a and /3 subunits’ abundance increases coordinately to twofold over control, and the Na,KATPase activity increases to 1.4-fold over control. These increases are preceded by a twofold increase in the level of f3-mRNA with no significant accompanying change in a-mRNA level, as illustrated in the Northern blot shown in Fig. 4. How is the abundance of a subunits increased without an increase in a-mRNA? A decrease in pump turnover rate is observed in these cells only after 16 h in low K, so it cannot account for the increase in pump activity and abundance seen as early as 8 h. Thus, an increase in a-mRNA translatability and/or stabilization of newly synthesized a peptides are implicated as potential mechanisms to increase a abundance. Results supporting changes in stability of the newly synthesized a are obtained from the synthesis experiments. Subunit synthesis rate was assayed by immunoprecipitating subunits from cells pulse-labeled with [35S]methionine. The rate of a synthesis appeared to nearly double by 24 h in low K, compared with controls, when the cells were labeled for 30 mm. However, the magnitude of the increase diminished when the cells were pulsed for shorter periods. These findings are consistent with the hypothesis that the apparent increase in a synthesis is due to increased stability of newly synthesized a subunits, perhaps a consequence of association with the larger pool of new-
13 I
C .JKCIK
CJK CJK CJK CJK
4. Northern analysis of a and (3 mRNA levels in LLC-PK1 cells incubated in low K medium. Confluent monolayers of LLCPKI cells were incubated in 0.25 mM K-containing medium (K) or control 5.5 mM K medium (C) for the times indicated. Total cytoplasmic RNA was extracted, resolved by electrophoresis in agarose/formaldehyde gels, blotted onto nitrocellulose, and probed with 32P-labeled canine al- and 13-cDNAs. The films shown are from the same blot: sequentially probed with subunits and a and then (3, and exposed for 6 and 24 h, respectively.
Figure
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ly synthesized /3 subunits. These findings are compatible with the finding, discussed earlier, that inhibiting glycosylation of the /3 subunit in A6 cells results in a decreased abundance of both a and j3, presumably a consequence of less efficient a/3 oligomer formation. Both of these findings support the hypothesis that /3 peptides are rate limiting for a/3 oligomer formation
a
(20, 21). In the rat, thyroid status affects Na,K-ATPase abundance and activity in various T3-responsive tissues. In the kidneys of hyperthyroid rats, the abundance of both a- and /3-mRNAs and a subunits increases to about 1.5-fold over euthyroid levels, giving rise to a 1.5-fold increase in maximal enzymatic activity (29). The situation in the hypothyroid rat is more puzzling. Although the enzymatic activity is decreased to 0.7 of euthyroid values, no differences are seen between the euthyroid and hypothyroid a- or /3-mRNA levels, or a abundance. In more recent experiments, a and 3 subunit abundance was measured in kidney homogenates of these two populations, and /3 subunit was found to decrease in the hypothyroid samples to 0.4 of euthyroid levels whereas abundance of a remained unaltered, as shown previously (Fig. 5). The results of this Western blot suggest that a decreased ratio of a to /3 subunit abundance in these samples is associated with decreased enzymatic activity. The change in the ratio suggests that either the oligomer stoichiometry is not fixed at 1:1 a./3, but increases in hypothyroid kidneys; that there are spare, uncomplexed a subunits in hypothyroid kidney; or alternatively, that the antigenic epitopes on /3 subunits are altered, which leads to a decrease in its autoradiographic signal in hypothyroid kidney. In any case, the alteration may be responsible for the change in enzymatic activity. As the kidney /3-mRNA levels are not
different
between
hypothyroid
and
euthyroid
animals, the decrease in apparent abundance in the hypothyroids is most likely a consequence of a translational or posttranslational modification (32).
CONCLUSIONS
AND
GAPS
Studies summarized here have shown that low levels of expression of the /3 subunit as seen with the glycosylation inhibitor, or as seen in the oocyte, are associated with low levels of Na,K-ATPase abundance. Alternatively, pretranslational increases in the levels of /3 subunit expression relative to a subunit expression, either by transfection as seen in the oocyte and yeast systems, or by ionic up-regulation as seen in the LLC-PKI cells, are associated with increases in Na,K-ATPase abundance. These findings support the idea that the /3 subunit regulates, through a/3 assembly, the number of pumps transported to the plasma membrane. These findings are consistent with those for other multimeric membrane proteins (16), but do not address the question of how other members of the cation translocating ATPase family are processed without an analogous /3 subunit.
1604
Vol. 4
April 1990
p
Na,K-ATPase activity ± S.E.M
EU
HYPO
12.3 ±0.5
7.8 ±0.8
Figure 5. Immunoblot analysis of Na,K-ATPase from euthyroid (EU) and hypothyroid (HYPO) rat kidney cortex. A constant amount of homogenate (10 jtg protein . sampl&’ . lane’) from three euthyroids and four hypothyroids was resolved by SDS-PAGE, transferred to diazotized paper, incubated with anti-rat kidney Na,K-ATPase antiserum, and labeled protein A for the detection of
antibody-antigen
complexes.
The
resultant
autoradiogram
is
shown. For quantitation of a and 13signals in the linear range of the film, multiple exposure times were necessary. The films shown are from the same blot: a and 13subunits exposed for 2 and 14 h, respectively. Na,K-ATPase activity was also measured in homogenates, and activity is expressed as mol Pi mg protein’. h’.
DO OTHER a$
ION
PUMPS
ALSO
REQUIRE
SUBUNIT?
At this time only the Na,K-ATPase has been shown to require the participation of two polypeptide subunits for expression of functional enzyme. Although it had been suggested that a 53-kDa glycoprotein found in high abundance in sarcoplasmic reticulum may serve the same function for the Ca-transport ATPase that the /3 subunit serves for Na,K-ATPase (35), the Ca transport activity of microsomes prepared from COS-1 cells transfected with cDNAs encoding the Ca-transport ATPase either alone or with the 53-kDa glycoprotein were identical (36). This observation suggests that this glycoprotein is not an essential component of the Catransport ATPase of sarcoplasmic reticulum. Perhaps the lack of /3 subunits leads to the appropriate retention of this ATPase in the sarcoplasmic reticulum. In contrast to the sarcoplasmic reticulum Ca-ATPase, the gastric H,K-ATPase, like Na,K-ATPase, is found in the plasma membrane. Recently, a photoactivated radiolabeled derivative of a specific inhibitor of the H,KATPase of gastric parietal cells was used to label this ion pump, and two peptides were obtained from a tryptic digest of the labeled protein (G. Sachs, personal communication). One peptide was identified within the sequence of the 100-kDa a catalytic subunit of H,KATPase, in a region predicted by hydropathy analysis to be located at the extracytoplasmic surface of the cell
membrane.
The second
peptide
sequence,
however,
is
unique, and has approximately 40% homology with the amino acid sequences of Na,K-ATPase /31 or /32 subunits. Furthermore, the region of the Na,K-ATPase
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ET AL.
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/3 subunit that is analogous to the labeled peptide is also on the extracytoplasmic surface of the cell membrane. Although it is not known that this peptide is derived from a /3-like subunit of the gastric W,K-ATPase, these observations indicate that exciting work will soon be forthcoming to answer this question.
16.
17. 18.
Work grants
on this topic from
the
in the authors’
National
Institutes
laboratories of
Health
was supported (DK
34316,
by HL
19.
39295, GM28673), National Science Foundation (DMB 8613999), and from Swiss National Science Foundation (3399086). R. A. E is an Established Investigator of the American Heart Association.
20.
21.
REFERENCES J.
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SODIUM
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alpha
22. Geering, K., Kraehenb#{252}hl, and Rossier, B. C. (1987) Maturation of the catalytic alpha subunit of Na,K-ATPase during intracellular transport. j Cell Biol. 105, 2613-2619 23. Geering, K., Theulaz, I., Verrey, E, Hauptle, M. T., and Rossier, B. C. A role for beta subunit in the expression of functional Na,K-ATPase in Xenopus oocytes. Am. j Physiol. In press 24. Mircheff, A. K., Bowen, J. W., Yiu, S. C., and McDonough, A. A. (1989) Subcellular fraction study of Na,K-ATPase assembly in MDCK cells. FASEBJ 3, A874 (abstr.) 25. Horowitz, B., Eakle, K. A., Scheiner-Bobis, G., Randolph, G. R., Chen, C. Y., Hitzeman, R. A., and Farley, R. A. Synthesis and assembly of functional mammalian Na,K-ATPase in yeast. j Biol. Chem. In press. 26. Pressley, T. A. (1988) Ion concentration-dependent regulation of Na,K-pump abundance. j Membr. Biol. 105, 187-195 27. Bowen, J. W., and McDonough, A. (1987) Pretranslational regulation of Na-K-ATPase in cultured canine kidney cells by low K. Am. j Physiol. 252, C179-C189 28. Rossier, B. C., Geering, K., and Kraehenbuhl, J. P. (1987) Regulation of sodium pump: how and why? TIBS 12, 483-487 29. McDonough, A. A., Brown, T. A., Horowitz, B., Chiu, R., Schlotterbeck, J., Bowen, J., and Schmitt, C. A. (1988) Thyroid hormone coordinately regulates Na,K-ATPase alpha and beta subunit mRNA levels in kidney. Am. J. Physiol. 254, C323-C329 30. Karin, N. J., and Cook, J. 5. (1983) Regulation of Na,K-ATPase by its biosynthesis and turnover. Curi. Top. Membr Trans. 19, 713-751 31. Wolitsky, B. A., and Fambrough, D. M. (1986) Regulation of the (Na + K)-ATPase in cultured chick skeletal muscle.]. Biol. Chem. 261, 9990-9999 32. Horowitz, B., Quintero, M., and McDonough, A. A. (1988) Thyroid hormone regulates mRNA levels of Na,K-ATPase alpha,, alpha2, and beta subunits in a tissue specific manner.] Cell Biol. 107, 126a 33. Gick, G. G., Ismail-Beigi, E, and Edelman, I. S. (1988) Thyroidal regulation of rat renal and hepatic Na,K-ATPase gene expression. j BioL Chem. 263, 16610-16618 34. McDonough, A. A., Putnam, D., and Lescale-Matys, L. Ionic regulation of Na,K-ATPase expression in cultured kidney cells. In Regulation of Potassium Transport Across Biological Membranes (Reuss, L., and Russell, J., eds) University of Texas Press In press 35. Campbell, K. P., and MacLennan, D. H. (1981) Purification and characterization of the 53,000-dalton glycoprotein from the sarcoplasmic reticulum. J. Biol. Chem. 256, 4626-4632 36. Leberer, E., Charuk, J. M. H., Clarke, D. M., Green, N. M., Zubrzycka-Gaarn, E., and MacLennan, D. H. (1989) Molecular cloning and expression of a eDNA encoding the 53,000dalton glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum. j Biol. Chem. 264, 3484-3493
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