Lisa A. Dunbar, Paul Aronson, and Michael J. Caplan. 2000. J. Cell Biol. 148: 769-778.
Paper review by: Carrie L. Smith
The H, K-adenosine triphosphatase (ATPase) of gastric
parietal cells is a protein that pumps protons into the lumen of the stomach
in exchange for potassium. Previous experiments have found that the gastric
H, K-ATPase is a non-GPI (glycophosphatidyl inositol) linked protein that
is targeted to the apical plasma membrane in epithelial cells, and it consists
of an alpha and beta subunit. The 110-kD alpha-subunit that spans the membrane
ten times contains the sorting signal responsible for the H, K-ATPase’s
apical localization, while the 50-60 kD beta-subunit encodes a signal that
stops the secretion of acid and may modulate ion affinity. Research has
shown that tyrosine-based signals target epithelial membrane proteins to
the basolateral surface while GPI-linked membrane proteins are directed
to the apical membrane surface. However, the signal by which non-GPI linked
proteins, such as the gastric H, K-ATPase, are targeted to the apical surface
is not understood. The purpose of this experiment was to determine the
portion of the gastric H, K-ATPase alpha-subunit that is sufficient to
direct the pump to the apical membrane of epithelial cells.
Chimeric proteins composed of
complementary portions of two rat ATPases, the H, K-ATPase and the Na,
K-ATPase, were constructed and transfected into LLC-PK1 cells to identify
the sorting signal responsible for the apical localization of the gastric
H, K-ATPase. The Na, K-ATPase is normally targeted to the basolateral membrane
surface, and it shares 63% amino acid sequence similarity with the H, K-ATPase.
Previous studies have shown that the first 519 amino acids of the H, K-ATPase
are sufficient for the apical localization of a chimera, H519N, which is
composed of the first 519 amino acids of the H, K-ATPase and the –COOH
terminal half of the Na, K-ATPase. This paper reports the results of functional
tests performed on the chimeric proteins to more narrowly define the apical
localization signal within the first 519 amino acids of the H, K-ATPase.
Immunofluorescence was performed
on LLC-PK1 cells expressing three different chimeras. Two antibodies were
used on each chimera; the polyclonal antibody HK9 recognizes the chimera,
while the mAb- 6H recognizes the endogenous Na, K-ATPase alpha subunit.
Figure 1 shows the results of the immunofluorescence and the localization
of chimeras I-III in the LLC-PK1 cells. The HK9Ab was used in figures A,
C, E, G, I, and K, while the H9Ab was used in figures B, D, F, H, J, and
L. The structure of chimeras I-III are shown to the left of the figures-the
black regions are the H, K-ATPase portions while the Na, K-ATPase portions
are shown in gray. Chimera I, which is composed of the first 85 amino acids
of the H, K-ATPase fused to the complementary portion of the Na, K-ATPase,
is a positive control for the chimeric localization in the basolateral
membrane. The first 85 amino acids serve as an epitope for the polyclonal
HK9Ab, and thus allow the discrimination of the chimeric alpha subunits
and the endogenous Na, K-ATPase using the two antibodies. The results shown
in figure 1 indicate that all of the endogenous Na, K-ATPases localized
in the basolateral membrane when viewed en face (figures B, F, and J) and
in xz cross-section (D, H, and L) as expected. In addition, chimeras I
and II were located in the basolateral membrane when viewed en face (A,
E) and in xz cross-section (C, G). However, chimera III localized in the
apical membrane in LLC-PK1 cells (I, K). Thus, it was concluded that the
sorting signal sufficient for the apical localization of the H, K-ATPase
must be located between amino acids 324 and 519 of the H, K-ATPase, which
constitutes the second domain loop and the fourth transmembrane domain
of the ATPase (see chimera III). Talk about no positive control for apical
localization.
To more narrowly define the
apical sorting signal within amino acids 324-519 of the H, K-ATPase alpha
subunit, the research team constructed four additional chimeric proteins
and transfected the chimeras in LLC-PK1 cells. Immunofluorescence
was then performed on the cells using two antibodies, HK9 and mAb-6H, that
recognize the chimeras and the endogenous Na, K-ATPase alpha subunit respectively.
The immunofluorescence results are shown in figure 2. Figure 2 is set up
in the same fashion as figure 1; the structures of chimeras IV-VIII are
displayed to the left of the panels, and the polyclonal antibody HK9 was
used in the panels on the left (A, C, E, G, K, M, and O) while the mAb-6H
was used in the panels on the right (B, D, F, H, J, L, N, and P).
Like figure 1, the results in figure 2 indicate that the Na, K-ATPase localized
to the basolateral membrane in all cell lines as expected (B, D, F, H,
J, L, N, and P). Chimeras IV and VI, which include the second cytoplasmic
loop and the second ectodomain of the H, K-ATPase respectively, localized
at the basolateral membrane when seen en face (A and I) and xz cross-sections
(C and M). However, chimera V, consisting of the second ectodomain and
TM4 of the H, K-ATPase, and chimera VII, consisting of just the TM4 of
the H, K-ATPase, localized at the apical membrane (E, G, M, and O). These
results indicate that the signal sufficient for the apical localization
of the H, K-ATPase is contained in the fourth transmembrane domain of the
gastric H, K-ATPase.
When reading this paper,
I felt that the immunofluorescence results presented in figures 1 and 2
did support the authors’ claim that the signal sufficient for the
apical localization of the rat H, K-ATPase is contained in the TM4 of the
alpha subunit. However, I did observe a few problems in the experimental
set-up. First, I noticed that the authors included a positive control for
the localization of basolateral membranes ( the endogenous Na, K-ATPase),
but they did not include a positive control for the distribution of apically
localized proteins. A difference in localization pattern between the basolateral
and apically targeted proteins was clear in the figures. However, the authors
did not adequately show that the apically localized chimeras were actually
in the apical membrane-a positive control, such as immunofluorescence on
the endogenous H, K-ATPase, would have helped me to more strongly believe
the authors’ claims. In addition, the authors state that hydropathy plots
did predict that amino acids 329-356 of the H, K-ATPase do compose the
fourth transmembrane region(data not shown). However, I think that the
paper would be strengthened if the actual hydropathy plot was included
so that the reader could clearly see that the authors’ claims that the
hypothesized apical sorting region actually passes through the lipid bilayer
of the LLC-PK1 cells’ membranes.
Next, the authors performed
a functional test to prove that the constructed chimeras assembled with
the Na, K-ATPase beta- subunit. In figure 3, immunofluorescence was performed
on an apically localized chimera, chimera V; the LLC-PK1 cells were stained
for either the presence of the chimeric or the Na, K-ATPase beta-subunits.
The authors claim that the figure shows that the chimeric Na, K-ATPase
beta-subunit was localized only at the apical surface (A en face, and C,
xz section), while the endogenous Na, K-ATPase beta subunit localized both
at the basolateral and apical surfaces (B, en face, and D, xz section)
in LLC-PK1 cells expressing chimera V. These results indicate that
the chimera assembled with the endogenous Na, K-ATPase beta subunit and
redirected this normally basolateral protein to the apical surface. In
addition, the endogenous Na, K-ATPase beta subunit also assembled with
the Na, K-ATPase, for it was also located in the basolateral surface.
When analyzing figure 3, I was
not satisfied with the authors’ explanation of how this immunofluorescence
experiment was performed. The authors do not indicate what antibodies were
used to differentiate between the chimeric and endogenous Na, K-ATPase
beta-subunits. Thus, it is not clear whether or not the cells were properly
stained for the chimeric and endogenous Na, K-ATPase beta subunits.
In addition, neither positive controls for basolateral membrane proteins
nor positive controls for apical membrane proteins were presented in the
figure. Overall, figure 3 did not convince me that chimera V definitely
assembled with the endogenous Na, K-ATPase beta subunit. Thus, I am not
completely convinced without question that the chimera localizes at the
apical membrane because it contains a sorting signal rather than because
it is a mal-functional protein. In addition, if the authors wanted
to show that all of the chimeras were functional proteins, they should
have performed this particular immunofluorescence on all of the chimeras
rather than just one.
Knowing that the signal that
is sufficient for the apical distribution of the rat H, K-ATPase lies within
the TM4 region of the alpha subunit, the researchers then compared the
TM4s of the H, K-ATPase and the Na, K-ATPase alpha subunits. They did this
comparison to see if the different membrane distributions of these pumps
could be explained by a high degree of non-homologous amino acid sequences.
The amino acid sequence comparison for the TM4s of the H, K-ATPase and
Na, K-ATPase alpha subunits is shown in figure 4. The boxes in figure 4,
A represent the non-homologous sequences between the two pumps in the TM3/TM4
ectodomain and the TM4 regions. The arrowheads indicate the junction points
of the two chimeras. The results of figure 4 indicate that there is little
homology in the TM4 regions between these two pumps-only 8 out of 28 amino
acids in the TM4 domain are not identical. Figure 4, B is a visual representation
of where the non-identical amino acids are located within the TM4 of the
alpha subunit. Figure 4, B indicates that the area of non-homology is predicted
to be in the outer leaflet of the lipid bilayer. The researchers state
that previous research has shown that this particular region of the apical
membrane is enriched with glycosphinolipids (GSL’s). Thus, the next step
in the authors’ research was to determine whether or not the gastric H,K-ATPase
localizes at the apical surface of gastric epithelial cells because it
associates with GSL-rich domains.
The authors performed
a detergent solubility assay on an apical pump chimera (H519N) consisting
of the TM4 portion of the gastric H, K-ATPase alpha subunit. This was done
to see if the possibility existed that H519N is targeted to the apical
surface because it interacts with glycolipid-rich membrane domains. In
figure 5, LLC-PK1 cells were lysed with Triton X-100 and loaded onto a
sucrose floatation gradient. Figure 5A is a graph that shows the presence
of alkaline phosphatase activity, the chimera, and the Na, K-ATPase within
each of the fractions of the gradient. The presence of alkaline phosphatase
activity serves as a positive control for the location of insoluble, GPI-linked
proteins within the fractions. Because the most alkaline phosphatase activity
was observed within the lighter fractions (2-4), the authors assumed that
GPI-linked proteins would be restricted to these lighter fractions when
the detergent solubility assay was performed. Figure 5B is a western blot
showing that both the chimera and the Na, K-ATPase appear in heavier fractions.
The lower band for the chimera alpha subunit represents the monomer, while
the upper band represents the alpha/beta dimer. More specifically, figure
5A is densitometric quantification of the western blots in 5B. Figure 5A
further shows that both the apically located chimera H519N and the Na,
K-ATPase were present in the heavier fractions (6-10). Because it has been
previously determined that the Na, K-ATPase does not associate with GPI,
the Na, K-ATPase was used in the assay as a positive control to show the
location of non-GPI linked, soluble proteins within the fractions.
Based on the results showing
that the chimera H519N partitioned into the heavier fractions along with
the Na, K-ATPase, the authors concluded that the gastric, H, K-ATPase alpha
subunit is not a GPI-linked protein and thus is not targeted to the apical
surface because it associates with GPI. I believe that the results in figure
5 adequately support the authors’ claim that the chimera is not a GPI-linked
protein. The positive controls for the location of soluble and insoluble
proteins were clearly established, and the results are easy to read and
displayed well. However, a few problems must be mentioned. First of all,
positive controls for the antibodies used to detect the chimera and Na,
K-ATPase in the western blots are not present in 5B. Thus, it is not clear
whether or not the bands in the western blot represent the chimera and
Na, K-ATPase. In addition, because I was not convinced in figure
3 that the chimeras are functional pumps, the results in figure 5 may have
been due to a malfunctioning chimera. I think the authors could have strengthened
their arguments by performing detergent solubility assays on all of the
apical pump chimeras.
The results obtained in figures 1-3
convinced the authors that the TM4 region of the gastric H, K-ATPase alpha
subunit was sufficient to direct the pump to the apical membrane surface.
Next, the authors wanted to know whether or not the TM4 region was necessary
for the apical localization of the gastric H, K-ATPase pump. To answer
this question, a chimera was made that lacked the TM4 region of the H,
K-ATPase but contained the large cytoplasmic loop and second ectodomain
surrounding the TM4 region of the H, K-ATPase, chimera VIII. Immunofluorescence
was performed on LLC-PK1 cells expressing chimera VIII using the same antibodies
as were used in figures 1 and 2-the polyclonal antibody HK9A and mAb-6H.
The results of the immuofluorescence are shown in figure 6. The structure
of chimera VIII is displayed to the left of the figure. The panels in figure
6 show that the Na, K-ATPase was localized in the basolateral membrane
as was expected (B and D). However, chimera VIII was found predominately
at the apical surface of the membrane (A and C). Based on these results,
the authors concluded that the TM4 region of the gastric H, K-ATPase alpha
subunit is sufficient but not necessary for the apical localization of
the pump in gastric epithelial cells. In addition, it was discovered that
the sequences flanking the TM4 region must somehow collaborate to direct
the H, K-ATPase to the apical surface. I think that the data presented
in figure 6 is adequate to support the authors’ claims. However, like in
figures 1 and 2, I would be more likely to believe that chimera VIII actually
was directed to the apical surface if a positive control for apically targeted
proteins was included in the figure.
Finally, the authors wanted to determine
whether or not the steady-state localization of the chimeras correlated
with their enzymatic activities. The data in figure 7 addresses this question.
In figure 7A, the authors tested whether or not chimeras I-VIII could function
as sodium pumps and thus could survive in the presence of 10 micro-molars
of ouabain over a period of five days. Ouabain is a known inhibitor of
the endogenous Na, K-ATPase. The concentration of ouabain used (10 micro-molar)
was found to be lethal in non-transfected LLC-PK1 cells. The left
column in figure 7A shows the structure of chimeras I-VIII, the middle
column indicates whether or not the chimera displayed ouabain resistance,
and the last column states whether the chimera localized at the basolateral
or apical membrane surface. The results in this figure show that all the
basolateral chimeras (I, II, IV, and V) were ouabain resistant, while the
apical chimeras (III, V, and VII) were not ouabain resistant. However,
chimera VIII both localized at the apical surface and was resistant to
ouabain. From the results in figure 7A, the authors concluded that all
of the basolateral chimeras, with the addition of chimera VIII, were enzymatically
active and thus capable of mediating sodium efflux. However, the
apical chimeras, with the exception of chimera VIII, were not enzymatically
active and thus were either inactive or their hydrogen ion efflux activities
could not substitute for sodium efflux.
Because chimera VIII was both enzymatically
active and localized at the apical surface, the authors suggested that
a pump’s location is not correlated to its cation specificity. I thought
that the authors did not have enough data to support this conclusion. In
figure 7A it appears that a correlation between cation specificity and
localization does exist except for one chimera. It could have been the
case that chimera VIII was not properly constructed or functional for that
particular experiment. I think it would have been helpful for the authors
to perform the ouabain resistance experiment multiple times to make sure
that chimera VIII’s resistance was not due to a flaw in the experimental
design. In addition, the authors’ claims would be strengthened if the figure
contained the results of the degree of ouabain resistance in cells only
expressing an endogenous basolateral protein or an apical protein. These
controls could be used to compare the results of the ouabain resistance
experiment on the chimeras.
Figure 7B shows that cells expressing
one of the apical chimeras, chimera III, is enzymatically active even though
it is not ouabain resistant. The first table indicates the pH level of
the apical medium when either the untransfected or transfected cells are
grown on porous filters in the absence of ouabain. The first table shows
that there is a drop in the pH level of the apical medium when chimera
III is expressed, thus indicating that the chimera is enzymatically active
and pumping protons. The second table is the same as the first, except
ouabain was added. Again, the results show that there is a slight drop
in the pH of the apical medium in cells expressing chimera III. This further
shows that chimera III is actively pumping protons and thus is still active
even though it is not ouabain resistant. I feel that the results in figure
7B are adequate to support the author’s claims that chimera III is enzymatically
active.
To conclude, the authors of
this paper performed a series of functional tests on eight different chimeric
proteins in order to determine the region of the gastric H, K-ATPase alpha
subunit that is sufficient to target the pump to the apical membrane surface
of gastric epithelial cells. The authors stated that their findings indicated
that the TM4 region of the H, K-ATPase alpha subunit was sufficient but
not necessary to direct the pump to the apical surface of gastric epithelial
cells. Overall, I felt that the immunofluorescence figures did support
these claims. However, as I have mentioned several times, I do not think
that the authors completely convinced me that the chimeras actually assembled
with the endogenous Na, K-ATPase beta subunit or that the steady-state
localization of the chimeras was not correlated to its cation specificity.
I thought that the authors use of functional tests was a good approach
to test their experimental question. However, I think they needed to use
more controls throughout the paper to help strengthen their conclusions.
I believed the authors’ claims that the apical chimeras did not associate
with GSL regions in the membrane. However, I would perform an additional
test to determine whether or not the gastric H, K-ATPase is targeted to
the apical surface of the membrane because the TM4 region interacts with
one or several other transmembrane proteins.
In a future experiment, I would
use the two hybrid system (Chien et al., 1991) to determine whether or
not the amino acids in the TM4 region of the gastric H, K-ATPase alpha
subunit interact with any additional transmembrane proteins to target the
pump to the apical surface. First, the cDNA encoding the TM4 region of
the rat gastric H, K-ATPase alpha subunit could be fused to the DNA binding
domain located on the GAL4 promoter-Lac-Z gene. I would then fuse
the cDNA from a rat genomic library to the GAL4 activation domain and place
these two domains into plasmids that are have the AmpR gene and an origin
of replication. Next, I would electroporate the plasmids containing
the TM4 gene of the H, K-ATPase alpha subunit into yeast cells and plate
the cells on x-gal plates. The presence of blue cells would indicate that
the lac-z gene was turned on and thus another protein interacted with the
TM4 region of the H, K-ATPase alpha subunit. I would isolate the
cells that turned blue and the plasmid that encodes the protein that interacted
with the TM4 region of the gastric H, K-ATPase. Once I isolated the plasmid
that encodes the protein that associated with the TM4 region, I would deduce
the cDNA encoding the interacting protein and make a monoclonal antibody
against the interacting protein.
Next, I would isolate
cells expressing the new protein and the H, K-ATPase and make cells that
express the H, K-ATPase but lacks the gene that encodes the newly found
protein using homologous recombination. I would then perform immunofluorescence
on both cell lines using two antibodies: one antibody against the H, K-ATPase
alpha subunit and the antibody that was made against the new protein. If
the new protein and the H, K-ATPase both localized in the apical membrane
in the cell line that contains both proteins, but the H, K-ATPase localized
in the basolateral in the cell line that only expresses the H, K-ATPase,
I could conclude that this new protein interacts with the TM4 region to
direct the pump to the apical surface. However, if the H, K-ATPase in the
cell line lacking the new protein was localized in the apical membrane,
I could conclude that this protein does not collaborate with the TM4 region
to target the H, K-ATPase pump to the apical membrane. If that was the
case, I would have to think of different mechanisms by which the H, K-ATPase
was directed to the apical surface. For example, since the paper showed
that the sequences flanking the TM4 region of the H, K-ATPase caused the
pump to localize at the apical surface, I could use the two hybrid system
once again but this time using the flanking sequences on the DNA binding
domain of the GAL4 promoter to see if any proteins associated with these
regions.
REFERENCES:
Chien, C.T., P.L. Bartel, R. Sternglanz, and S. Fields. 1991. The two-hybrid system: A method to identify and clone genes for proteins that interact with a protein of interest. Prot. Natl. Acad. Sci. USA. 88: 9578-9582.