Identification of a gain-of-function mutation
in a Golgi P-type ATPase that enhances
Mn2+efflux and protects against toxicity
Somshuvra Mukhopadhyay and Adam D. Linstedt1
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213
Edited by Jennifer Lippincott-Schwartz, National Institutes of Health, Bethesda, MD, and approved December 7, 2010 (received for review September
P-type ATPases transport a wide array of ions, regulate diverse
cellular processes, and are implicated in a number of human
diseases. However, mechanisms that increase ion transport by
these ubiquitous proteins are not known. SPCA1 is a P-type pump
thattransportsMn2+fromthe cytosolintothe Golgi.Wedeveloped
an intra-Golgi Mn2+sensor and used it to screen for mutations in-
troduced in SPCA1, on the basis of its predicted structure, which
could increase its Mn2+pumping activity. Remarkably, a point mu-
tation (Q747A) predicted to increase the size of its ion permeation
cavityenhanced thesensorresponseand a compensatory mutation
restoring the cavity to its original size abolished this effect. In
vivo and in vitro Mn2+transport assays confirmed the hyper-
activity of SPCA1-Q747A. Furthermore, increasing Golgi Mn2+
transport by expression of SPCA1-Q747A increased cell viability
upon Mn2+exposure, supporting the therapeutic potential of in-
creased Mn2+uptake by the Golgi in the management of Mn2+-
and thereby regulate diverse cellular processes (1). The Na+/K+
ATPase generates membrane potential in neuronal cells, the
sarco/endoplasmic reticulum Ca2+ATPase (SERCA) regulates
skeletal muscle contraction, the H+/K+ATPase mediates gastric
acidification, and lipid flippases transport phospholipids to the
outer leaflet of the plasma membrane. P-type pumps share a high
degree of sequence, structural and functional similarity, and their
invariant aspartic acid residue in the cytoplasmic domain induces
acharacteristic conformation changefrom the E1state,which has
high affinity for the ion, to the E2 state, which has low affinity for
of this conformational change (2). Devising molecular techniques
that can increase ion transport through P-type ATPases will sig-
nificantly improve our understanding of cellular processes regu-
diseases related to these processes.
Secretorypathway Ca2+ATPase1(SPCA1)is aGolgi-localized
and plays an important role in regulating Ca2+homeostasis in
mammalian cells (5). Haploinsufficiency of SPCA1 results in the
development of Hailey-Hailey disease, a blistering dermatosis
signaling in keratinocytes (5–7). Whereas much attention has
beenfocused on its Ca2+transport activity, SPCA1 can also trans-
Mn2+transport activity of SPCA1 is particularly important be-
cause mechanisms regulating Mn2+homeostasis in mammalian
cells are unknown. Furthermore, whereas Mn2+is an essential
element required for the catalytic activity of a wide array of
enzymes (5), at elevated levels, Mn2+competes with magnesium-
binding sites in proteins, enhances oxidative stress, compromises
-type ATPases are ubiquitously expressed proteins that me-
diate transport of a wide array of cations and phospholipids
The cytotoxic effects of Mn2+result in the development of man-
ganism, a Parkinson-like neurodegeneration syndrome, in adults
and cognitive and behavioral defects in children (12–14). Thus,
identifying mechanisms that reduce cytosolic Mn2+levels during
elevated exposure is of high clinical significance.
Blocking uptake of extracellular Mn2+is unlikely to succeed in
protecting against Mn2+toxicity because, in mammalian cells,
extracellular Mn2+is transported into the cytosol by multiple ion
pumps and channels that belong to different protein families and
are not specific for Mn2+. However, increasing Mn2+pumping
into the Golgi could potentially reduce cytosolic Mn2+levels and
thereby protect against toxicity. Unfortunately, the role of the
mammalian Golgi in Mn2+detoxification has thus far not been
tested for three principal reasons. First, compartment-specific
subcellular Mn2+sensors have not yet been identified, making it
impossible to rapidly screen for conditions increasing Mn2+
transport into the Golgi. Second, the Golgi localization of SPCA1
is saturable and overexpression of the wild-type (WT) protein
results in its accumulation in endosomes. Third, as there are no
known mechanisms to increase the ion transport through P-type
ATPases, it has not been possible to increase the Mn2+transport
activity of SPCA1.
In the current study we used the predicted structure of SPCA1
to generate mutations that could potentially increase its Mn2+
pumping activity. Using a unique in vivo Golgi-specific Mn2+sen-
sor to rapidly screen for conditions that increase Golgi Mn2+, we
identified Q747A as a point mutation that increased the Mn2+
transport activity of SPCA1. Analysis indicated that the structural
of its ion permeation cavity revealing, an unexpected contribution
of ion permeation to the overall rate of transport through a P-type
ATPase. Furthermore, expression of SPCA1-Q747A protected
cells from the cytotoxic effects of Mn2+, indicating that conditions
increasing Mn2+transport into the Golgi may be therapeutically
useful in the management of manganism.
Golgi Phosphoprotein of 130 kDa (GPP130) is an Intra-Golgi Mn2+
Sensor. To rapidly screen for conditions that increased Golgi
Mn2+uptake in vivo, we needed a sensor that specifically
responded to increased Mn2+within the Golgi lumen. Although
compartment-specific Mn2+sensors have not been described, we
recently reported that exposure of cells to as low as 100 μM of
Mn2+induces the cis-Golgi glycoprotein, GPP130, to traffic from
the Golgi to multivesicular bodies (MVBs) and then to lysosomes,
where it is degraded (15). Fig. 1A shows the Mn2+-induced re-
distribution and degradation response of GPP130 relative to
Author contributions: S.M. and A.D.L. designed research; S.M. performed research; S.M.
analyzed data; and S.M. and A.D.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| January 11, 2011
| vol. 108
| no. 2www.pnas.org/cgi/doi/10.1073/pnas.1013642108
the Golgi marker giantin. To test whether this response was trig-
gered by an increase in Mn2+in the Golgi lumen, we depleted
SPCA1 to block transport of Mn2+from the cytoplasm into the
Golgi. Knockdown of SPCA1 abolished the Mn2+-induced re-
distribution and degradation of GPP130 (Fig. 1 B–D). Gene re-
placement with full-length SPCA1 restored the Mn2+response of
GPP130, whereas replacement with a mutated version defective
in autophosphorylation (D350A) and thus unable to transport
either Mn2+or Ca2+(2, 16) did not (Fig. 1 E and F). Further-
more, replacement with SPCA1 containing a mutation (G309C)
that blocks Mn2+but not Ca2+pumping activity (4) also failed to
rescue the Mn2+response of GPP130 (Fig. 1 E and F). Thus, the
Mn2+-induced degradation of GPP130 required a specific in-
crease in intra-Golgi Mn2+levels, indicating that GPP130 could
be used to screen for conditions that increase Golgi Mn2+.
Identification of SPCA1-Q747A as a Potential Hyperactive Mn2+
Transporter. We first tested whether overexpression of SPCA1-
WT increased Golgi Mn2+uptake. However, the Golgi localiza-
tion of SPCA1 was saturable and the level of pump activity in-
creased by WT overexpression did not detectably enhance
GPP130 degradation when compared with untransfected cells
(Fig. 2A). Another possibility was to increase the Mn2+pumping
side chain size of a residue predicted to line the Mn2+permeation
ion-binding site in SPCA1. Remarkably, expression of SPCA1-
Q747A induced GPP130 degradation even in the absence of ex-
ternally added Mn2+(Fig.2 A andB),suggesting a hyperactivated
the loss induced by SPCA1-Q747A corresponded to the transient
transfection frequency (Fig. 2C). To test whether SPCA1 trig-
gered the GPP130 response due to its Mn2+pumping activity, we
introduced into SPCA1-Q747A mutations that block pump ac-
tivity (D350A) or block Mn2+but not Ca2+transport (G309C)
and observed that these mutations blocked its ability to induce
GPP130 degradation (Fig. 2 A and B). Although endogenous
SPCA1 was present in these experiments, it was not required as
loss of GPP130 induced by SPCA1-Q747A was also observed in
cells lacking endogenous SPCA1 due to knockdown (Fig. S1).
SPCA1-Q747A expression did not affect Golgi morphology or
alter the localization and level of other tested Golgi proteins
(Fig. S2 A–D). We also confirmed that SPCA1-Q747A did not
alter protein trafficking between the Golgi and the endocytic or
secretory pathways. Treatment of cells with monensin to block
endosome-to-Golgi protein trafficking causes rapid endosomal
accumulation of Golgi protein of 73 kDa (GP73), which consti-
tutively cycles between the Golgi and endosomes (17). The level
of the monensin-induced endosomal accumulation of GP73 did
not differ between cells that expressed SPCA1-Q747A and those
that did not (Fig. S2 E–G). Additionally, expression of SPCA1-
Q747A did not alter the distribution or localization of LAMP-2
(Fig. S2 H–J), a protein that traffics to lysosomes from the Golgi
(18). Finally, we analyzed endoplasmic reticulum-to-surface trans-
cells were treated with 500 μM of Mn2+for the indicated times and imaged to
detect GPP130 and giantin. (Scale bar, 10 μm.) (B) Cells treated with control or
anti-SPCA1 siRNAs for 48 h were transfected with HA-tagged SPCA1-WT and,
after 24 h, were exposed to 500 μM of Mn2+for 4 h and stained using anti-HA
(to detect SPCA1) and anti-GPP130. After Mn2+, GPP130 was degraded in cells
transfected with the control siRNA as well as in cells in which SPCA1 was not
depleted after knockdown (single asterisk). Cells with no detectable SPCA1
after knockdown did not exhibit Mn2+-induced loss of GPP130 (double as-
terisk). (Scale bar, 10 μm.) (C) Levels of GPP130, myc-SPCA1-WT, and tubulin
in cells treated exactly as described in B were determined by immunoblot. (D)
Quantitation after immunoblotting to determine the levels of GPP130
remaining after Mn2+, normalized to tubulin, in cells transfected with control
or anti-SPCA1 siRNAs (mean ± SE; n = 3; P < 0.05). (E) Cells treated with anti-
SPCA1 siRNA for 48 h were transfected with RNA-resistant HA-tagged SPCA1-
WT or mutated constructs. After 24 h they were exposed to 500 μM of Mn2+
for 4 h and imaged to detect SPCA1 using an anti-HA antibody and GPP130.
After Mn2+, GPP130 was lost in cells expressing SPCA1-WT but not D350A or
G309C (single asterisk). Knockdown cells not transfected with SPCA1-WT did
not exhibit a GPP130 Mn2+response (double asterisk). (Scale bar, 10 μm.) (F)
Quantitation of percentage of transfected cells that did not have detectable
GPP130 after Mn2+from E (mean ± SE; n = 100 cells per experiment from
three independent experiments; P < 0.05).
Increased intra-Golgi Mn2+induces degradation of GPP130. (A) HeLa
were transfected with HA-tagged SPCA1-WT or mutated constructs and 24 h
later imaged to detect HA and GPP130. GPP130 was degraded in cells
transfected with SPCA1-Q747A but not WT or the double mutants (single
asterisk). In the SPCA1-Q747A transfected culture, GPP130 was clearly
detected in untransfected cells (double asterisk). (Scale bar, 10 μm.) (B)
Quantitation of percentage of transfected cells without detectable GPP130
(mean ± SE; n = 100 cells per experiment from six independent experiments;
P < 0.05). (C) HeLa cells were transfected with HA-tagged SPCA1-WT or
Q747A and harvested 24 h posttransfection as described previously (15).
Immunoblotting was performed to detect GPP130, HA, and tubulin.
SPCA1-Q747A enhances the GPP130 Mn2+response. (A) HeLa cells
Mukhopadhyay and LinstedtPNAS
| January 11, 2011
| vol. 108
| no. 2
port of the temperature-sensitive mutant of vesicular stomatitis
virus G protein (VSVG) tagged with GFP (19, 20). VSVG-GFP
trafficking in cells expressing SPCA1-Q747A was indistinguishable
from controls, in each case reaching the Golgi in 20 min and the
cell surface in 60 min (Fig. S3).
We also determined that SPCA1-Q747A induced GPP130
degradation by the pathway we previously described for Mn2+-
induced GPP130 degradation (15). Upon SPCA1-Q747A ex-
with Rab7, a marker for MVBs, and LAMP-2 before degradation
(Fig. S4 A and B). GPP130 was also detected within the lumen of
giant MVBs induced by coexpression of Rab5-Q79L (Fig. S4C).
Coexpression of dominant negative Rab7 (Rab7-T22D), which
inhibits MVB-to-lysosome trafficking, blocked the GPP130 deg-
radation, whereas dominant negative Rab5 (Rab5-S34N), which
blocks early endosome-to-MVB trafficking, did not (Fig. S4 D–F).
In cells coexpressing SPCA1-Q747A and Rab7-T22D, GPP130
accumulated in large peripheral punctae (Fig. S4 D–F), consistent
with our previous description of the retention of GPP130 in an
enlarged MVB compartment in Mn2+-treated cells expressing
Rab7-T22D (15). Thus, using GPP130 as an intra-Golgi Mn2+
sensor, we identified SPCA1-Q747A as a potentially hyperactive
Substitution of Q747 with Alanine Increases Activity of SPCA1 by
Increasing the Size of the Ion Permeation Cavity. SPCA1 is >70%
similar to SERCA in its transmembrane domains (21). Muta-
genesis and computational modeling of SPCA1 on the basis of
the known SERCA structure (22–25) indicate that SPCA1 has
a single ion-binding site located between the fourth and sixth
transmembrane domains (M4 and M6) and formed by E-308, N-
738, and D-742 (2, 4, 26) (Fig. 3A). The most likely route for ions
to reach this site is through the cavity between M4 and M6. In-
deed, this route is taken by hydrated Ca2+to reach a similarly
positioned ion-binding site in SERCA (22) and mutations in
residues of M4 and M6 block access of ions to the homologous
ion-binding site in Pmr1p (21, 27, 28). Residue Q747 of SPCA1
is predicted to be at the cytoplasmic/membrane interface of M6
with an orientation that projects its side chain into the solvent
accessible cavity between M4 and M6 (Fig. 3 A–C). On the basis
of our modeling of SPCA1, the minimum distance or gap be-
tween M4 and M6 at this ion entrance point, occurring between
V313 on M4 and Q747 on M6, was 3.89 Å (Fig. 3D). Analysis of
a recomputed structure of SPCA1 containing the Q747A sub-
stitution confirmed that the side chain volume of residue 747
projecting into the cavity was reduced (Fig. 3E). As a conse-
quence, the corresponding distance between M4 and M6 in-
creased to 7.0 Å and the apparent minimum distance in the ion
permeation cavity, which now occurred between V313 and G743,
was 4.86 Å (Fig. 3E). The increase in gap between M4 and
M6 induced by the Q747A substitution is significant in com-
parison to the ∼1.6Å diameter of a hydrated Mn2+ion (29) and
could significantly increase the rate at which Mn2+reaches the
ion-binding site and thus enhance overall pumping activity of
SPCA1. Indeed, rate increases are observed in potassium chan-
nels containing mutations that increase the size of their transport
cavities (30, 31).
As a test of whether the increase in distance between M4 and
M6 caused the gain-of-function observed in SPCA1-Q747A, we
attempted to restore the WT distance in SPCA1-Q747A by
substituting isoleucine for V314 on M4. According to our mod-
eling of SPCA1, the V314I substitution rotated the side chain of
V313 bringing it to within 3.6 Å of G743 on M6 and created
a minimum gap similar to WT (Fig. 3F). In contrast to the strong
response of GPP130 to SPCA1-Q747A, the construct containing
the compensatory mutation, SPCA1-Q747A+V314I, failed to
induce GPP130 degradation (Fig. 3 G and H). This could not be
attributed to a loss of activity because SPCA1-Q747A+V314I
rescued the Mn2+response of GPP130 in cells lacking endoge-
nous SPCA1 due to knockdown (Fig. S5 A and B). As the V314I
substitution suppressed the Q747A phenotype without reintro-
ducing a polar side chain, it suggests that it is increased gap
SPCA1 ion permeation cavity. (A–C) SPCA1-WT was
modeled using the MODBASE server as described in
Materials and Methods. The M4 and M6 trans-
membrane domains are depicted in cartoon (A), surface
(B), and ribbon (C) forms using the open source PyMol
software. Ion-binding residues, E308, N738, and D742
are depicted in red; residue Q747 is in green; and V313
is in blue. Arrowheads indicate the path used by ions to
reach the ion-binding site. (D–F). Distances across the
ion permeation cavity are shown for the computed
structures of SPCA1-WT, SPCA1-Q747A, and SPCA1-
Q747A+V314I. Distances between M4 and M6 were
measured using the “Measurement Wizard” in PyMol
and the structures of the SPCA1 mutants were obtained
using the “Mutagenesis Wizard” of PyMol. Blue ar-
rowhead in F shows the rotation of the side chain of
V313 after the V314I substitution. Black arrowheads
indicate the likely path traversed by ions en route to the
ion-binding site. (G) HeLa cells were transfected with
HA-SPCA1-Q747A or HA-SPCA1-Q747A+V314I and im-
aged 24 h later to detect GPP130 and HA. GPP130 was
degraded in cells transfected with SPCA1-Q747A but
not in those expressing the V314I mutation (single as-
terisk). Untransfected cells in the SPCA1-Q747A trans-
fected culture exhibited Golgi-localized GPP130 (double
asterisk). (Scale bar, 10 μm.) (H) Quantitation of per-
centage of transfected cells lacking detectable GPP130
from G above (mean ± SE, n = 50 cells from four in-
dependent experiments, P < 0.05).
The Q747A substitution increases the size of the
| www.pnas.org/cgi/doi/10.1073/pnas.1013642108Mukhopadhyay and Linstedt
distance and not absence of partial charge that determines the
ability of SPCA1-Q747A to increase Golgi Mn2+.
Q747A Mutation Increases Mn2+Pumping Activity of SPCA1. To di-
rectly test whether the Q747A mutation hyperactivated SPCA1,
we performed an in vitro54Mn2+uptake assay on Golgi mem-
branes isolated from cells expressing SPCA1-WT and SPCA1-
Q747A. Immunoblotting confirmed recovery of comparable lev-
els of SPCA1-WT and SPCA1-Q747A in the respective pooled
Golgi membranes (Fig. 4A) and uptake of54Mn2+was specific as
it was effectively competed by excess nonradioactive Mn2+(Fig.
4B). Significantly, uptake of
Golgi vesicles containing SPCA1-Q747A (Fig. 4B), indicating
increasedpump activity. These assayslacked Ca2+,thus increased
Mn2+transport was not due to blocked Ca2+access to the ion-
binding site. Indeed, uptake of54Mn2+in SPCA1-Q747A con-
taining Golgi vesicles was effectively competed by excess non-
radioactive Ca2+(Fig. S6A). Uptake was also carried out in
permeabilized cells and again54Mn2+uptake was specific (Fig.
4C) and the presence of SPCA-Q747A conferred a twofold in-
crease (Fig. 4D). Finally, we used a pulse-chase assay to test
whether SPCA1-Q747A increased Mn2+transport into the Golgi
in intact cells. Initial controls revealed that intracellular uptake of
54Mn2+progressively increased between 5 and 30 min and was
effectively competed by addition of excess nonradioactive Mn2+
(Fig. 4E). During a subsequent 30-min chase incubation, in-
tracellular54Mn2+decreased by ∼60% (Fig. 4F) as54Mn2+was
released into the medium (Fig. 4G). The release of54Mn2+was
blocked by pretreatment of the cells with brefeldin A, a fungal
metabolite that blocks secretion (Fig. 4 F and G), indicating
that the released
was ∼3%/min as ∼30% of releasable54Mn2+was secreted every
10min(Fig.S6B),which isinaccordwithpreviousobservations of
blocking transport of Mn2+into the Golgi via knockdown of
SPCA1 (Fig. S6C) and by transferring cells to 20 °C (Fig. 4H),
a temperature that blocks Golgi-to-plasma membrane trafficking
(33). These observations indicate that efflux of intracellular Mn2+
was primarily mediated via its transport into the Golgi and se-
cretion. In support of the Q747A mutation hyperactivating Mn2+
pumping into the Golgi thereby promoting its release by secre-
tion, cells expressing SPCA1-Q747A retained 20% less54Mn2+
and secreted 20% more than WT (Fig. 4 I and J).
54Mn2+was >80% higher in the
54Mn2+was secreted. The efflux of
54Mn2+efflux (32). Efflux of
54Mn2+was inhibited by
SPCA1-Q747A Increases Cell Viability During Mn2+Toxicity. On the
basis of the finding that SPCA1-Q747A sequestered and then
secreted more Mn2+, we tested whether expression of this con-
struct protected cells exposed to toxic levels of Mn2+. A dose–
response was carried out on control cells, indicating that exposure
to 1 mM of Mn2+for 16 h reduced viability by ∼70% (Fig. 5A).
Using this condition, the viability of cells expressing SPCA1-WT
or Q747A was compared with control transfected cells. Cells
expressing SPCA1-Q747A exhibited a 70% greater survival rate
increased viability was comparable to that of increased Mn2+
transport by SPCA1-Q747A. Furthermore, SPCA1-WT, which
failed to increase Mn2+transport (Fig. 4), also did not increase
viability (Fig. 5B). On the other hand, knockdown of SPCA1,
which reduced Mn2+efflux, reduced cell viability by ∼70%,
highlighting the crucial role of Golgi Mn2+uptake in mediating
Mn2+detoxification (Fig. 5C). To confirm the protective effect of
Q747A on a cell-by-cell basis, control and Mn2+-treated cells
were imaged to identify transfected cells and score their viability.
Untreated cells expressing SPCA1-WT or Q747A, as indicated by
a transfection marker, were healthy in appearance and negative
for staining with propidium iodide, which is excluded by viable
cells (Fig. 5D). In contrast, most Mn2+-treated cells expressing
SPCA1-WT exhibited perturbed morphologies and were inviable,
on the basis of propidium iodide staining (Fig. 5 D and E). Sig-
nificantly, a large protective effect was again evident for SPCA1-
Q747A as ∼60% of cells expressing this construct remained
healthy looking and viable (Fig. 5 D and E). The protective effect
of SPCA1-Q747A was also confirmed in the neuronal PC-12 cell
line (Fig. S7). Finally, to determine whether, after uptake into the
Golgi, secretion of Mn2+was essential for detoxification, the
toxicity assay was performed at 20 °C to block secretion and, as
shownabove,Mn2+efflux.Cellviability wasunaffected at20°Cin
branes were isolated from cells transfected with HA-SPCA1-WT or Q747A as
described in Materials and Methods and 2 μg of total protein was immu-
noblotted to detect giantin, GPP130, and HA. (B) Uptake of54Mn2+by iso-
lated Golgi vesicles was performed as described in Materials and Methods.
Uptake was normalized to the uptake by untransfected cells (set to 100)
and the experiment was replicated three times (mean ± SE; P < 0.05 for the
difference in uptake in untransfected cells with and without excess cold
Mn2+and for the difference in uptake between SPCA1-WT and Q747A). (C)
Uptake of54Mn2+in permeabilized cells was done as described in Materials
and Methods. Uptake in absence of cold Mn2+was normalized to 100 for
each experiment (mean ± SE, n = 3, P < 0.05). (D) Uptake of54Mn2+on FACS
sorted cells expressing either GFP alone or GFP and SPCA1-WT or GFP and
Q747A was performed as in C above. Uptake in cells expressing GFP alone
(set to 100) was used for normalization (mean ± SE, n = 3, P < 0.05 for the
difference between SPCA1-WT and Q747A). (E) Cells were loaded with
54Mn2+and intracellular radioactivity was measured at various time points.
Uptake at 30 min (set to 100) was used for normalization (mean ± SE, n = 3,
P < 0.05). (F) Control or BFA-treated cells were loaded with54Mn2+for 30
min, washed, and chased for 30 min. Intracellular radioactivity after a 30-min
loading was normalized to 100 for control and BFA (mean ± SE, n = 3, P <
0.05 for the difference in intracellular radioactivity retained after the chase
in control cultures). (G) Distribution of intracellular and extracellular radio-
activity after a 30-min chase in the presence or absence of BFA. (H) Cells were
loaded with54Mn2+for 30 min at 37 °C, washed, and chased for 30 min at
37 °C or 20 °C. Intracellular radioactivity after a 30-min loading was nor-
malized to 100 (mean ± SE, n = 3, P < 0.05 for the difference in intracellular
radioactivity retained between 37 °C and 20 °C groups). (I and J) FACS sorted
cells expressing SPCA1-WT or Q747A were loaded with54Mn2+for 30 min
and subsequently chased for an additional 30 min. Intracellular radioactivity
retained and secreted after the 30-min chase was normalized to 50 for
WT-expressing cells (mean ± SE, n = 3, P < 0.05 for the difference in radio-
activity retained and radioactivity secreted between WT and Q747A).
SPCA1-Q747A is a hyperactive Mn2+transporter. (A) Golgi mem-
Mukhopadhyay and Linstedt PNAS
| January 11, 2011
| vol. 108
| no. 2
the absence of Mn2+but, after exposure to Mn2+, the protective
effect of SPCA1-Q747A was no longer evident (Fig. 5F). Thus,
Mn2+efflux via uptake by the Golgi and secretion is essential for
Mn2+detoxification and increasing Mn2+transport into the
Golgi protects against Mn2+-induced cytotoxicity (Fig. 5G).
Mutations inhibiting transport through P-type pumps are often
described but gain-of-function mutations are not. After con-
firming that the previously described Mn2+-induced trafficking
and degradation of GPP130 (15) requires a specific increase in
intra-Golgi Mn, we used GPP130 as a sensor of intra-Golgi Mn2+
and identified Q747A as a hyperactivating mutation of SPCA1.
Structural modeling indicated that the Q747A substitution in-
creased ion permeation to the binding site, which is significant
because the rate of conformational change between the E1 and
E2 states is considered rate limiting for P-type ATPases (2). It is
unlikely that the Q747A substitution directly influenced the rate
of this conformational change because Q747 does not appear to
interact with neighboring residues in any of the SPCA1 con-
formations computationally determined on the basis of SERCA.
A more likely possibility, based on the compensatory mutation, is
that access to the ion-binding site is partially impeded by Q747
and reducing the size of Q747 allows quicker reloading of the
pump when it reverts back to the E1 conformation.
There are a few known mutations of P-type ATPases that in-
crease activity but these mutations relieve negative regulation to
restore constitutive activity (34–36). The best-characterized ex-
ample is for plasma membrane calcium ATPase (PMCA), which
has an autoinhibitory domain that binds calmodulin (35, 36).
Deletion of this domain increases Ca2+transport to the level
normally seen when calmodulin binds and counteracts the effect
of the inhibitory domain (36).
Enlargement of the ion access cavity may be a generally ap-
plicable strategy to increase the intrinsic rates of P-type ATPa-
ses. Sequence alignments indicate that residues with bulky side
chains are present at the entrance face of ion permeation cavities
of other P-type pumps. Residues corresponding to Q747 of
SPCA1 include T804 in SERCA and L888 in PMCA4. Con-
ceivably, alanine substitution at these sites could hyperactivate
the pumps. However, success in increasing pump activity would
also depend on the nature of the other residues lining the cavity.
For example, a mutation homologous to Q747A in the yeast
homolog of SPCA1, Pmr1p (the mutation in Pmr1p is Q783A),
blocks rather than increases Mn2+transport (21, 27). This dif-
ference in activity likely occurs because, in contrast to the valine
at 313 opposite Q747 in SPCA1, Pmr1p has an isoleucine in this
position. The bulkier side chain of isoleucine may alter helix
packing when present opposite the alanine residue in Pmr1p-
Q783A and thereby occlude access of Mn2+to the ion-binding
site (21, 27). Thus, although using the SERCA structure to target
the ion access gateway of other P-type ATPases is now an ex-
citing possibility, careful attention will need to be paid to dif-
ferences in neighboring residues.
Increased Mn2+transport into the Golgi by SPCA1-Q747A
protected cells against Mn2+-induced cytotoxicity, whereas
blocking Mn2+transport into the Golgi or out of the Golgi to the
cell surface had the opposite effect, showing the importance of
the Golgi apparatus in Mn2+homeostasis and detoxification in
mammalian cells. Consistent with our results, HEK293 cells,
which express low levels of endogenous SPCA1 (2), wererecently
shown to be sensitive to Mn2+and protected from toxicity by
SPCA1 expression (37). The identification of the Golgi as the
major route of Mn2+detoxification is significant, given that Mn2+
efflux via bile is the sole mechanism for Mn2+excretion in hu-
mans, and patients with compromised liver function due to dis-
eases like cirrhosis develop Mn-induced neurotoxicity without
exposure to elevated Mn2+(38). These results are also consistent
with the Mn2+hypersensitivity of ΔPMR1 yeast strains (39) but,
without a hyperactive Mn2+pump, earlier investigations were
unable to address whether increasing Mn2+uptake into the Golgi
offered any protective effect. Our findings reveal that increased
Golgi Mn2+uptake actually leads to less Mn2+retention by cells
due to secretion. Thus, increased pumping into the Golgi likely
alters ion flux such that, at least within a particular concentra-
tion range of extracellular Mn2+, efflux of Mn2+becomes greater
Our results highlight the therapeutic potential of increasing
cytosol-to-Golgi Mn2+transport in the management of man-
ganism, a disease that remains incurable. Clearly, gene therapy
with SPCA1-Q747A is not easily feasible, but a promising avenue
is a drug targeting the machinery responsible for SPCA1 locali-
HeLa cells were exposed to 1, 2, and 5 mM of Mn2+for 16 h and viability was
determined using the MTT assay. Controls were untreated (0 mM of Mn2+).
Samples were normalized using absorption at 570 nm of the controls (set to
100) for each experiment (mean ± SE, n = 3, P < 0.05). (B) FACS sorted cells
expressing GFP alone, GFP andSPCA1-WT, orGFP andSPCA1-Q747A were left
untreated or exposed to 1 mM of Mn2+for 16 h and viability was determined
using the MTT assay. Viability in cells expressing GFP alone (set to 100) was
used for normalization (mean ± SE, n = 3, P < 0.05 for the difference in via-
bility between SPCA1-WT and Q747A). (C) Cells were treated with control or
anti-SPCA1 siRNAs for 48 h and then exposed to 0 or 1 mM of Mn2+for 16 h.
Cell viability was assessed using the MTT assay and viability in cells treated
with thecontrolsiRNA (set to 100) was used for normalization (mean ±SE, n =
3, P < 0.05 for the difference in viability between control and anti-SPCA1
siRNA treated groups). (D) Cells were transfected with SPCA1-WT or Q747A
and mCherry for 24 h and then exposed to 1 mM of Mn2+for 16 h. Propidium
iodide staining was performed as described in Materials and Methods. (Scale
bar, 10 μm.) (E) Quantitation of percentage of transfected cells positive for
propidium iodide from D above (mean ± SE, n = 25 cells from three in-
dependent experiments per group, P < 0.05 for the difference between
SPCA1-WT and Q747A after Mn2+). (F) Cells were transfected with a GFP
marker aloneorcotransfected with GFPandSPCA1-WTorQ747A for24h and
then exposed to 1 mM of Mn2+for 16 h at 20 °C. Control cultures did not
receive Mn2+. Cell viability was then assayed using propidium iodide staining
(mean ± SE, n = 25 cells from three independent experiments per group, P >
0.05 for the difference in cell viability between GFP, SPCA1-WT, and Q747A
after Mn2+and P < 0.05 for the difference in cell viability with and without
Mn2+in the GFP group). (G) Schematic showing that Mn2+detoxification is
mediated via transport into the Golgi in mammalian cells. Increasing Golgi
Mn2+uptake reduces cytosolic Mn2+and protects against Mn2+toxicity. De-
creasing transport of Mn2+into or out of the Golgi has the opposite effect.
Expression of SPCA1-Q747A protects cells from Mn2+toxicity. (A)
| www.pnas.org/cgi/doi/10.1073/pnas.1013642108Mukhopadhyay and Linstedt
zation in the Golgi. We observed that Golgi targeting of SPCA1 is
readily saturated leading to endosome localization. Interestingly,
a pool of endosomal SPCA1 has recently been detected in bile
canalicular WIF-B cells (37), suggesting that SPCA1 in a physio-
logically relevant cell type may already be expressed in excess of
that required for Golgi saturation. Thus, preventing SPCA1 exit
from the Golgi in these cells should protect against Mn2+toxicity.
In conclusion, we have elucidated an unexpected mode of
hyperactivating the pumping activity of a P-type ATPase and
demonstrated that increased Mn2+pumping into the Golgi
protects mammalian cells from the cytotoxic effects of Mn2+.
Materials and Methods
Cell culture, transfections, immunofluorescence microscopy, image analysis,
and immunoblot analyses were done as described by us previously (15, 19, 20,
40, 41). Tertiary structure of SPCA1 was derived using the algorithms of
the MODBASE server (http://modbase.compbio.ucsf.edu/modbase-cgi/index.
cgi; ref. 42). Purification of Golgi membranes on discontinuous sucrose gra-
dients was done essentially as described (43, 44). The in vitro uptake of
54Mn2+in pooled Golgi membranes was done as described (16). To ensure
100% transfection frequency of SPCA1-WT or Q747A in a transient trans-
fection system, cells were cotransfected with SPCA1-WT or Q747A and Rab5-
GFP-WT and sorted using fluorescence-activated cell sorting (Vantage SF;
Becton Dickinson) in the GFP channel. FACS sorted cells were used for up-
take assays using permeabilized cells and for the viability assays using
methylthiazolylphenyl-tetrazolium bromide (MTT). Uptake of
permeabilized cells was done as described (4, 45). MTT assay was performed
as described (15). Propidium iodide was from Sigma-Aldrich and was used as
described by the manufacturer. See SI Materials and Methods for details.
ACKNOWLEDGMENTS. We thank Yehuda Creeger for help with the FACS
assays; Claudia Almaguer for performing transfections in the PC-12 cells;
Collin Bachert, Tina Lee, and Manojkumar Puthenveedu for critical reading
of the manuscript; Donald R. Smith (University of California, Santa Cruz, CA)
for advice; and Rajini Rao (The Johns Hopkins Medical School, Baltimore,
MD) for sharing of unpublished data. This work was funded by National
Institutes of Health Grant R01 GM-084111 (to A.L.).
1. Kühlbrandt W (2004) Biology, structure and mechanism of P-type ATPases. Nat Rev
Mol Cell Biol 5:282–295.
2. Dode L, et al. (2005) Functional comparison between secretory pathway Ca2+/
Mn2+-ATPase (SPCA) 1 and sarcoplasmic reticulum Ca2+-ATPase (SERCA) 1 isoforms
by steady-state and transient kinetic analyses. J Biol Chem 280:39124–39134.
3. Dode L, et al. (2006) Dissection of the functional differences between human
secretory pathway Ca2+/Mn2+-ATPase (SPCA) 1 and 2 isoenzymes by steady-state and
transient kinetic analyses. J Biol Chem 281:3182–3189.
4. Fairclough RJ, et al. (2003) Effect of Hailey-Hailey disease mutations on the function
of a new variant of human secretory pathway Ca2+/Mn2+-ATPase (hSPCA1). J Biol
5. Missiaen L, et al. (2004) SPCA1 pumps and Hailey-Hailey disease. Biochem Biophys Res
6. Sudbrak R, et al. (2000) Hailey-Hailey disease is caused by mutations in ATP2C1
encoding a novel Ca(2+) pump. Hum Mol Genet 9:1131–1140.
7. Missiaen L, Dode L, Vanoevelen J, Raeymaekers L, Wuytack F (2007) Calcium in the
Golgi apparatus. Cell Calcium 41:405–416.
8. Ton VK, Mandal D, Vahadji C, Rao R (2002) Functional expression in yeast of the
human secretory pathway Ca(2+), Mn(2+)-ATPase defective in Hailey-Hailey disease.
J Biol Chem 277:6422–6427.
9. Milatovic D, Zaja-Milatovic S, Gupta RC, Yu Y, Aschner M (2009) Oxidative damage
and neurodegeneration in manganese-induced neurotoxicity. Toxicol Appl Pharmacol
10. Roth JA, Horbinski C, Higgins D, Lein P, Garrick MD (2002) Mechanisms of manganese-
11. Zhao P, et al. (2008) Manganese chloride-induced G0/G1 and S phase arrest in A549
cells. Toxicology 250:39–46.
12. Olanow CW (2004) Manganese-induced parkinsonism and Parkinson’s disease. Ann N
Y Acad Sci 1012:209–223.
13. Wright RO, Amarasiriwardena C, Woolf AD, Jim R, Bellinger DC (2006) Neuro-
psychological correlates of hair arsenic, manganese, and cadmium levels in school-age
children residing near a hazardous waste site. Neurotoxicology 27:210–216.
14. Bouchard M, Laforest F, Vandelac L, Bellinger D, Mergler D (2007) Hair manganese
and hyperactive behaviors: Pilot study of school-age children exposed through tap
water. Environ Health Perspect 115:122–127.
15. Mukhopadhyay S, Bachert C, Smith DR, Linstedt AD (2010) Manganese-induced
trafficking and turnover of the cis-Golgi glycoprotein GPP130. Mol Biol Cell 21:
16. Sorin A, Rosas G, Rao R (1997) PMR1, a Ca2+-ATPase in yeast Golgi, has properties
distinct from sarco/endoplasmic reticulum and plasma membrane calcium pumps.
J Biol Chem 272:9895–9901.
17. Puri S, Bachert C, Fimmel CJ, Linstedt AD (2002) Cycling of early Golgi proteins via the
cell surface and endosomes upon lumenal pH disruption. Traffic 3:641–653.
18. Shestakova A, Zolov S, Lupashin V (2006) COG complex-mediated recycling of Golgi
glycosyltransferases is essential for normal protein glycosylation. Traffic 7:191–204.
19. Puthenveedu MA, Linstedt AD (2004) Gene replacement reveals that p115/SNARE
interactions are essential for Golgi biogenesis. Proc Natl Acad Sci USA 101:1253–1256.
20. Yadav S, Puri S, Linstedt AD (2009) A primary role for Golgi positioning in directed
secretion, cell polarity, and wound healing. Mol Biol Cell 20:1728–1736.
21. Mandal D, Rulli SJ, Rao R (2003) Packing interactions between transmembrane helices
alter ion selectivity of the yeast Golgi Ca2+/Mn2+-ATPase PMR1. J Biol Chem 278:
22. Toyoshima C, Nakasako M, Nomura H, Ogawa H (2000) Crystal structure of the
calcium pump of sarcoplasmic reticulum at 2.6 A resolution. Nature 405:647–655.
23. Toyoshima C, Nomura H (2002) Structural changes in the calcium pump accompanying
the dissociation of calcium. Nature 418:605–611.
celldeath and celldifferentiation.
24. Toyoshima C, Nomura H, Tsuda T (2004) Lumenal gating mechanism revealed in
calcium pump crystal structures with phosphate analogues. Nature 432:361–368.
25. Toyoshima C, Mizutani T (2004) Crystal structure of the calcium pump with a bound
ATP analogue. Nature 430:529–535.
26. Ton VK, Rao R (2004) Functional expression of heterologous proteins in yeast: Insights
into Ca2+ signaling and Ca2+-transporting ATPases. Am J Physiol Cell Physiol 287:
27. Mandal D, Woolf TB, Rao R (2000) Manganese selectivity of pmr1, the yeast secretory
pathway ion pump, is defined by residue gln783 in transmembrane segment 6.
Residue Asp778 is essential for cation transport. J Biol Chem 275:23933–23938.
28. Wei Y, et al. (2000) Phenotypic screening of mutations in Pmr1, the yeast secretory
pathway Ca2+/Mn2+-ATPase, reveals residues critical for ion selectivity and transport.
J Biol Chem 275:23927–23932.
29. Marcus Y (1983) Ionic-radii in aqueous-solutions. J Solution Chem 12:271–275.
30. Hänelt I, et al. (2010) Gain of function mutations in membrane region M2C2 of KtrB
open a gate controlling K+ transport by the KtrAB system from Vibrio alginolyticus.
J Biol Chem 285:10318–10327.
31. Xia M, et al. (2005) A Kir2.1 gain-of-function mutation underlies familial atrial
fibrillation. Biochem Biophys Res Commun 332:1012–1019.
32. Aschner M, Gannon M, Kimelberg HK (1992) Manganese uptake and efflux in
cultured rat astrocytes. J Neurochem 58:730–735.
33. Ladinsky MS, Wu CC, McIntosh S, McIntosh JR, Howell KE (2002) Structure of the Golgi
and distribution of reporter molecules at 20 degrees C reveals the complexity of the
exit compartments. Mol Biol Cell 13:2810–2825.
34. Curran AC, et al. (2000) Autoinhibition of a calmodulin-dependent calcium pump
involves a structure in the stalk that connects the transmembrane domain to the
ATPase catalytic domain. J Biol Chem 275:30301–30308.
35. Enyedi A, et al. (1989) The calmodulin binding domain of the plasma membrane Ca2+
pump interacts both with calmodulin and with another part of the pump. J Biol Chem
36. Enyedi A, Verma AK, Filoteo AG, Penniston JT (1993) A highly active 120-kDa
truncated mutant of the plasma membrane Ca2+ pump. J Biol Chem 268:
37. Leitch S, et al. (2010) Vesicular distribution of secretory pathway Ca(2+)-ATPase
isoform 1 and a role in manganese detoxification in liver-derived polarized cells.
Biometals, in press.
38. Aschner M, Erikson KM, Herrero Hernández E, Hernández EH, Tjalkens R (2009)
Manganese and its role in Parkinson’s disease: From transport to neuropathology.
Neuromolecular Med 11:252–266.
39. Culotta VC, Yang M, Hall MD (2005) Manganese transport and trafficking: Lessons
learned from Saccharomyces cerevisiae. Eukaryot Cell 4:1159–1165.
40. Sengupta D, Truschel S, Bachert C, Linstedt AD (2009) Organelle tethering by
a homotypic PDZ interaction underlies formation of the Golgi membrane network.
J Cell Biol 186:41–55.
41. Linstedt AD, Mehta A, Suhan J, Reggio H, Hauri HP (1997) Sequence and
overexpression of GPP130/GIMPc: Evidence for saturable pH-sensitive targeting of
a type II early Golgi membrane protein. Mol Biol Cell 8:1073–1087.
42. Pieper U, et al. (2006) MODBASE: A database of annotated comparative protein
structure models and associated resources. Nucleic Acids Res 34(Database issue):
43. Xu H, Shields D (1993) Prohormone processing in the trans-Golgi network:
Endoproteolytic cleavage of prosomatostatin and formation of nascent secretory
vesicles in permeabilized cells. J Cell Biol 122:1169–1184.
44. Jesch SA, Linstedt AD (1998) The Golgi and endoplasmic reticulum remain inde-
pendent during mitosis in HeLa cells. Mol Biol Cell 9:623–635.
45. Van Baelen K, Vanoevelen J, Missiaen L, Raeymaekers L, Wuytack F (2001) The Golgi
PMR1 P-type ATPase of Caenorhabditis elegans. Identification of the gene and
demonstration of calcium and manganese transport. J Biol Chem 276:10683–10691.
Mukhopadhyay and Linstedt PNAS
| January 11, 2011
| vol. 108
| no. 2