Plant Physiol. (1999) 119: 1165-1176
Identification of a Calmodulin-Regulated Ca2+-ATPase
in the Endoplasmic Reticulum1
Bimei Hong,
Audrey Ichida,
Yuwen Wang,
J. Scott Gens,
Barbara G. Pickard, and
Jeffrey F. Harper*
Department of Cell Biology, The Scripps Research Institute, BCC283,
10550 North Torrey Pines Road, La Jolla, California 92037 (B.H., Y.W.,
J.F.H.); and Department of Biology, Washington University, St.
Louis, Missouri 63130-4899 (A.I., J.S.G., B.G.P.)
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ABSTRACT |
A unique subfamily of
calmodulin-dependent Ca2+-ATPases was recently identified
in plants. In contrast to the most closely related pumps in animals,
plasma membrane-type Ca2+-ATPases, members of this new
subfamily are distinguished by a calmodulin-regulated autoinhibitor
located at the N-terminal instead of a C-terminal end. In addition, at
least some isoforms appear to reside in non-plasma membrane locations.
To begin delineating their functions, we investigated the subcellular
localization of isoform ACA2p (Arabidopsis
Ca2+-ATPase, isoform 2
protein) in Arabidopsis. Here we provide evidence that ACA2p
resides in the endoplasmic reticulum (ER). In buoyant density sucrose
gradients performed with and without Mg2+, ACA2p
cofractionated with an ER membrane marker and a typical "ER-type"
Ca2+-ATPase, ACA3p/ECA1p. To visualize its subcellular
localization, ACA2p was tagged with a green fluorescence protein at its
C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We
collected fluorescence images from live root cells using confocal and
computational optical-sectioning microscopy. ACA2-GFPp appeared as a
fluorescent reticulum, consistent with an ER location. In addition, we
observed strong fluorescence around the nuclei of mature epidermal
cells, which is consistent with the hypothesis that ACA2p may also
function in the nuclear envelope. An ER location makes ACA2p distinct
from all other calmodulin-regulated pumps identified in plants or
animals.
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INTRODUCTION |
Ca2+ is thought to function as an important
second messenger in all eukaryotes (Bootman and Berridge, 1995
;
Clapham, 1995). In addition, Ca2+ is required for
the stability and activity of many proteins and appears to play a
critical role in protein processing in the secretory pathway (Rudolph
et al., 1989; Gill et al., 1996). To control Ca2+
concentrations in different compartments, cells commonly use two active
transport systems, Ca2+-ATPases (pumps) and
H+- or Na+-coupled antiporters. In plants there
is evidence for multiple Ca2+ pumps (Evans and
Williams, 1998) and low-affinity
Ca2+/H+ antiporters (Hirschi et al.,
1996).
Ca2+ pumps belong to a large superfamily of
P-type ATPases that include the
Na+/K+-ATPase of animals and the
H+-ATPase of plants and fungi. Axelsen and
Palmgren (1998) have proposed two distinct families of
Ca2+ pumps, type IIA and IIB, based on protein
sequence homologies. Type IIA and IIB pumps include the "ER-type"
and the "PM-type" Ca2+ pumps, respectively,
first characterized in animal cells. Previously, homologs of ER- or
PM-type pumps were distinguished by three criteria: (a)
localization to either the ER or PM, respectively, (b)
differential sensitivity to inhibitors (e.g. ER-type inhibition by
cyclopiazonic acid and thapsigargin), and (c) direct activation of
PM-type pumps by calmodulin. However, not all plant homologs conform to
these criteria (Bush, 1995; Evans and Williams, 1998).
In plants several genes encoding type IIA pumps (ER-type homologs) have
been cloned, including LCA1 from tomato (Wimmers et al., 1992), OsCA
from rice (Chen et al., 1997), and ACA3/ECA1 (Arabidopsis
Ca2+-ATPase, isoform
3/ER-Ca2+-ATPase
isoform 1) from Arabidopsis (Liang et al., 1997).
Consistent with the criteria for a typical ER-type pump, ACA3p
(ACA isoform 3 protein) appears to
reside in the ER (Liang et al., 1997). However, non-ER locations have
been suggested for other isoforms. For example, Ferrol and Bennett
(1996) obtained evidence for tonoplast and PM isoforms from membrane
fractionation and immunodetection of pumps cross-reacting with an
anti-LCA1 antibody.
Three plant genes encoding type IIB pumps (PM-type homologs) have also
been identified: ACA1 and ACA2 from Arabidopsis (Huang et al., 1993;
Harper et al., 1998) and BCA1 from Brassica oleracea pimprivate (Malmstrom et al., 1997). These plant homologs are distinguished from animal PM-type pumps by a unique structural arrangement with putative autoinhibitory sequences located in the
N-terminal instead of C-terminal domain and proposed non-PM locations
(Harper et al., 1998). Nevertheless, as expected for type IIB pumps, at
least some plant homologs have calmodulin-dependent Ca2+-ATPase activity. For isoform ACA2p, this
contention is supported by three lines of evidence (Harper et al.,
1998). First, ACA2p was expressed in yeast and shown to have a
Ca2+ and calmodulin-stimulated ATPase activity.
Second, a calmodulin-binding sequence was mapped within 36 residues of
the N terminus, indicating that the pump could interact directly with
calmodulin. Third, a partial deletion of the N-terminal domain resulted
in a constitutively active Ca2+-ATPase (i.e.
calmodulin independent). Together, these results support the hypothesis
that the N-terminal domain functions as a calmodulin-regulated
autoinhibitor.
Harper et al. (1998) previously obtained evidence that ACA2p is
targeted to a non-PM location from aqueous two-phase partitioning of
microsomal membranes. However, these studies did not determine a
specific endomembrane location for ACA2p. Non-PM locations have also
been proposed for ACA1p and BCA1p. ACA1p is thought to target to a
plastid inner membrane, based on membrane fractionation and immunodetection with an anti-ACA1 polyclonal antibody (Huang et al.,
1993). BCA1p is thought to target to the tonoplast, based on
correspondence to the peptide sequence obtained from a purified tonoplast ATPase (Askerlund, 1996; Malmstrom et al., 1997).
However, none of these studies used an isoform-specific probe, nor were they confirmed by cytological evidence.
Here we show that ACA2p is most abundant in the ER, as indicated by
membrane fractionation and corroborated by fluorescence imaging in live
cells of ACA2p tagged with a GFP. An ER location establishes ACA2p as
the first calmodulin-regulated Ca2+-ATPase to be
identified in the ER of any organism. The ER in Arabidopsis also
contains a typical ER-type Ca2+ pump. Thus, to
our knowledge, our results provide the first example of an organism in
which the ER has been found to function with two different types of
Ca2+ pumps.
| |
MATERIALS AND METHODS |
Plant experiments were conducted with Arabidopsis cv Columbia.
Yeast experiments were conducted with the Saccharomyces
cerevisiae strain K616 (MATa pmr1::HIS3
pmc1::TRP1 cnb1::LEU2, ura3 [Cunningham and
Fink, 1994]). DNA cloning was done in Escherichia coli
strain XL1-Blue (Stratagene) or DH10
(a derivative of DH5;
Stratagene). Unless noted otherwise, we used standard molecular
techniques according to the method of Sambrook et al. (1989).
Antibodies
Anti-ACA2 (no. 1371) rabbit polyclonal antiserum against a
GST fusion protein with ACA2 residues Val-119 to Pro-161 was
described by Harper et al. (1998). Anti-CTF2 was made against a GST
fusion protein with the C-terminal domain of a PM
H+-ATPase (AHA2) (DeWitt et al., 1996). Anti-ACA3
(no. 1374) was generated against a GST fusion protein containing the
last 27 residues of ECA1p/ACA3p, an ER-type
Ca2+-ATPase from Arabidopsis (Liang et al.,
1997). Dr. M. Chrispeels (University of California, San Diego) kindly
provided the anti-BiP and anti-
-tonoplast intrinsic protein. We
purchased anti-GFP from Clontech (Palo Alto, CA).
For immunocytochemistry, we used affinity-purified anti-ACA2
antibodies. Serum was first precipitated with 50% ammonium sulfate and
resuspended in PBS. Anti-GST antibodies were removed using a column
coupled with GST protein. Anti-ACA2 antibodies were then allowed to
bind to a column containing the fusion protein encoded by pACA2-Ns
(Harper et al., 1998). Columns were made using cyanogen bromide-activated Sepharose 4B according to the manufacturer's instructions (Pharmacia). Anti-ACA2 antibodies were eluted with 0.1 M Gly, pH 2.7, immediately neutralized with 0.1 volume of 1 M Tris-HCl, pH 8.0, and dialyzed against PBS. Control
preimmune serum was purified over a protein-A Sepharose CL-4B column
(Sigma).
Plasmid Constructs
We used standard PCR reactions and subcloning procedures to
engineer ACA2 sequences into the clones described below. We used Ampli-Taq (Perkin-Elmer) or Taka Ra Ex Taq (PanVera, Madison, WI) to
perform PCR. All PCR-derived sequences were sequenced to ensure the
absence of PCR mistakes. DNA sequencing was done at The Scripps
Biochemistry Core Facility using an automated sequencer (Prism 373XL,
ABI, Foster City, CA).
pYX-ACA2-1 and pYX-ACA2-2 encode full-length and N-terminally
truncated versions, respectively, of ACA2p for expression in yeast
(Harper et al., 1998). The parent vector used for all yeast constructs
in this study was pYX-112-TEV, which contains a URA3 gene for selection
in yeast.
pYX-ACA2-7 (mp11) encodes an ACA2-GFPp identical to that diagrammed in
Figure 3 but engineered for expression in yeast. The construct was
engineered as an insertion between the SalI and NheI sites of pYX-112-TEV. The sequence at the 5
end is
GTCGACATG (start codon is underlined). The coding sequence
contains a BstB1 and SacII site as shown for
pACA2-wt (Harper et al., 1998).
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| Figure 3.
Diagram of p35S-ACA2-GFP used for expression of a
GFP-tagged pump in transgenic plants. ACA2-GFP was placed under the
control of a CaMV-35S promoter. A translational enhancer sequence
derived from the tobacco etch virus was used as a 5-untranslated
leader sequence (Harper et al., 1998). For the junction between ACA2
and the C-terminal GFP, the nucleotide and amino acid sequences are
shown. Pro-1013 is the last amino acid contributed by ACA2p. The
downstream M1 in bold corresponds to the "start" Met
for GFP. A six-His motif present at the C-terminal end of the GFP
domain, encoded by the following sequence, starts with the
XbaI site: T CTA GAC CCG GGA ATG CAT CAC CAT CAC CAT CAC
GGA TCC TGA). Some useful enzyme sites are shown for diagnostic and
subcloning purposes.
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pYX-ACA2-8 (mp20) encodes an N-terminally truncated ACA2p (Harper et
al., 1998) fused to a C-terminal GFP, as described for pYX-ACA2-7. The
sequence at the modified 5 end is GTCGACATGAGT (start
codon for M1 is underlined, followed by the codon for "S81").
p35S-ACA2-GFP1 encodes a full-length ACA2-GFPp fusion as shown in
Figure 3. The 35S-GFP vector used was p35S-GFP-JFH1 (mp16). This vector
was derived from pBIN19 (accession no. U09365; Frisch et al., 1995),
modified to include a CaMV-35S promoter from a pRT-derived
vector (Topfer et al., 1987), a translational enhancer derived from a
tobacco etch virus (sequence shown from 20 to 155 [Harper et al.,
1998]), a synthetic GFP sequence with an S65-T mutation (derived from
a clone provided by J. Sheen; Sheen, 1996), and a six-His affinity tag.
The coding sequence for ACA2p was cut from clone pACA2-BSX-3 (mp 62) as
an SalI-Spe1 insert and subcloned into the
XhoI-Spe1 site of p35S-GFP-JFH1. The 3 end of
pACA2-BSX-3 had been modified by site-specific mutagenesis to include
three Gly residues followed by an Spe1.
Yeast Transformations
For transformations, K616 was grown in standard yeast extract,
peptone, and dextrose medium supplemented with 10 mM
CaCl2. Yeast were transformed by lithium
acetate/PEG methods (Elble, 1992) and selected for uracil prototrophy
by plating on synthetic medium minus uracil supplemented with 2% Suc
as a carbon source and 1.5% agar (Rose et al., 1990). For
complementation studies, Ura+ colonies were
streaked on plates containing 10 mM EGTA (pH 6.0) and
incubated for 2 to 3 d at 30°C as described by Liang et al. (1997).
Plant Transformation
Transgenic plants were generated by a vacuum-infiltration protocol
(Bechtold et al., 1993) using Agrobacterium tumefaciens strain GV3101. Infiltration was performed a few days after clipping primary bolts. Dry seeds were harvested, sterilized in 20% bleach for
20 min, and plated on Gamborg's B5 medium containing kanamycin (50 mg
L
1), and carbenicillin (300 mg
L1). Kanamycin-resistant plants
(T0) were identified and grown from seed. We
conducted experiments with T1 or
T2 plants and selected two independent transgenic
plant lines for detailed analysis of ACA2-GFPp expression and
localization (identification nos. 1284 and 1289).
Membrane Fractionation
We prepared microsomal membranes by modifying a procedure
previously described by Serrano (1984). All manipulations were
conducted on ice or in a cold room with prechilled buffers. Membranes
came from plants grown in liquid Gamborg's B5 medium with 0.05% (w/v) Mes (pH 5.7) on a shaker at about 125 rpm. Fresh tissues were cut into
small pieces under an extraction buffer (290 mM Suc, 2 mM EDTA, 250 mM Tris-HCl, pH 8.5, 2 mM PMSF, and 76 mM 
-mercaptoethanol) and
homogenized with a mortar and pestle. For some preparations, the
homogenization buffer contained 5 mM
MgCl2. Homogenates were filtered through
cheesecloth to remove large debris and then centrifuged at
5,000g to remove intact organelles and cell walls.
Supernatants were spun at 100,000g for 1 h to pellet
microsomal membranes. Membrane pellets were resuspended in
homogenization buffer (1 mL/10 g starting material) using a glass
homogenizer. Microsomes (0.5 mL) were then layered onto a gradient of
15% to 45% (w/w) Suc in a centrifugation buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 1 mM DTT, 2 mM benzamidine, and 0.1 mM
PMSF). For all the plus Mg2+ Suc gradients, 5 mM MgCl2 was added to the
homogenization and centrifugation buffers. Gradients were centrifuged
at 110,000g for 16 h and 1-mL fractions were collected,
frozen in liquid nitrogen, and stored at 
80°C. A refractometer was
used to measure the Suc concentration of each fraction.
Western Blots
For SDS-PAGE, samples were mixed with 3× loading buffer (100 mM Tris, pH 6.8, 3.7% [w/v] SDS, 5% [w/v] DTT, 20%
[w/v] Suc or glycerol, and 0.3% [w/v] bromphenol blue) and
incubated for 15 min at 37°C. After the sample was electrophoresed, a
transfer apparatus (Bio-Rad) transferred proteins to nitrocellulose.
The transfer buffer consisted of 192 mM Gly, 25 mM Tris-HCl (pH 8.3), 20% (v/v) methanol, and 0.02% (w/v)
SDS.
Blots were incubated for 2 h in a blocking buffer (20 mM Tris, [pH 7.6], 137 mM NaCl, and 0.5%
[w/v] Tween 20 [TBS-T] with 5% [w/v] nonfat dry milk). We
diluted the primary antisera accordingly in the blocking buffer. The
secondary antibody used for immunodetection on western blots was a
donkey anti-rabbit IgG conjugated with horseradish peroxidase
(Amersham), and it was diluted at 1:5000 in a blocking buffer. Two-hour
primary and secondary antibody incubations at room
temperature were followed by four 15-min washes in TBS-T. Secondary
antibodies were detected using ECL (Amersham).
Marker Enzyme Assays
Chlorophyll a and b concentrations were
determined spectrophotometrically by mixing 10-µL samples with 750 µL of 95% ethanol, and the A648.6 and
A664.2 were measured. Chlorophyll a
and b content was determined using the equation
Ca+b = 5.24A664.2 + 22.24A648.6 (Lichtenthaler, 1987).
Triton-stimulated UDPase activity was assayed by adding 10-µL samples
of membrane fractions to 100 µL of assay buffer consisting of 3 mM UDP, 3 mM MnSO4, and
30 mM Mes-Tris, pH 6.5 (Nagahashi and Kane, 1982). A
parallel reaction was conducted with the addition of 0.03% (w/v)
Triton X-100. Reactions were incubated at 37°C for 20 min, and we
used the Malachite Green method (Lanzetta et al., 1979) to determine
the released phosphate. We calculated latent activity as the difference
in activity between the presence and absence of detergent.
Immunocytochemistry
Roots were dissected from 1-week-old seedlings grown vertically on
Gamborg's B5 medium with 1% agar and fixed for 1 h at 25°C in
freshly prepared 4% paraformaldehyde with 5% Suc in 100 mM phosphate, pH 7.3. Fixed tissues were washed three times
at 10-min intervals in 100 mM phosphate and dehydrated in a
graded ethanol series. Dehydrated tissues were infiltrated and embedded
in London Resin White at 50°C for 16 h. Sections of 1-µm
thickness were cut with a glass knife on a microtome (JB-4, Sorvall)
and placed on glass slides.
For cyrosectioning, roots were fixed for 2 h in freshly prepared
periodate-Lys-paraformaldehyde (McClean and Nakane, 1974). Fixed
tissues were washed three times at 10-min intervals in 100 mM phosphate and embedded with frozen tissue medium
(Electron Microscopy Sciences, Fort Washington, PA) at 70°C.
Sections were cut 8 µm thick at 20°C on a cryostat (Cryocut 1800, Reichert-Jung, Heidelberg, Germany). Frozen sections were affixed to
Poly-L-Lys-coated glass slides (Sigma) and allowed to soak
for 15 min in PBS-T (PBS with 0.1% Tween 20).
Tissue sections were treated with blocking solution (PBS-T containing
10% normal goat serum and 1% BSA) for 1 h at room temperature and then incubated for 2 h with 10 µg
mL1 affinity-purified anti-ACA2 antibodies or
protein A-purified preimmune IgG in the blocking solution. After the
sections were washed in three changes of PBS-T (10 min each), they were
incubated for 2 h at room temperature with goat anti-rabbit IgG
coupled to fluorescein isothiocyanate (Molecular Probes, Eugene, OR)
diluted 1:100 in a blocking solution. The sections were washed with
three changes of PBS-T (10 min each) and mounted with SlowFade
(Molecular Probes).
Fluorescence Confocal Microscopy and Computational
Optical-Sectioning Microscopy
For imaging GFP fluorescence, root tips were excised from young
seedlings and mounted in Gamborg's B5 medium under glass coverslips. Confocal images were collected on a confocal laser-scanning microscope (MRC-600, Bio-Rad) attached to an inverted microscope (Nikon) equipped
with a fluorescein filter. For computational optical sectioning,
specimens were sometimes irrigated with 0.1% azide to slow or stop
cytoplasmic streaming to generate three-dimensional stacks. The
wide-field computational optical-sectioning microscope system was
described by Gens et al. (1996). The objective lens used was an X60,
1.4 NA Plan apo lens (Nikon). CCD (charge-coupled device) wells of 6.8 µm width were binned 2 × 2 or 4 × 4, with the cubic voxel
size set at either 0.23 × 0.23 × 0.23 or 0.45 × 0.45 × 0.45 µm. Restoration of Figure 7, E through G, was by the maximum-likelihood algorithm with 1000 iterations and with an
intensity penalty of 5 × 106 for F and G,
and these were also subjected to light-Gaussian filtering. The
algorithm and user-friendly interfaces are available at
http://ibc.wustl.edu:80/bcl/.
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| Figure 7.
Imaging of ACA2-GFPp fluorescence reveals ER-like
structures in root cells. A, Longitudinal section from a primary root
shows undifferentiated cells at the root tip. Cells are stained with
toluidine blue to emphasize densely stained nuclei. Toluidine
blue-stained sections were photographed on an inverted microscope
(model IMT2, Olympus) using Kodak Ektachrome P1600. B, Two
undifferentiated cells from A are shown at higher magnification to
emphasize the position of the large central nuclei. These cells are
representative of live cells imaged for GFP fluorescence in C and D. C
and D, Confocal images of green fluorescence from two undifferentiated
cells expressing a GFPp-only control (C) or ACA2-GFPp (D). E and F,
Green fluorescence from mature root epidermal cells expressing a
GFPp-only control (E) or ACA2-GFPp (F), as imaged by computational
optical-sectioning microscopy. G, The nuclear region from two
neighboring epidermal cells expressing ACA2-GFPp. Imaging of the
nuclear region required shorter exposure times, which left the image of
the surrounding network only faintly fluorescent. The thickness and
angle of this section does not show the cell wall, which lies at an
oblique angle between two nuclei in two separate epidermal cells. Scale
bars = 50 µm in A; 5 µm in B, C, and D; and 10 µm in E, F,
and G. Images were arranged using Adobe PhotoShop (Adobe Systems,
Mountain View, CA).
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RESULTS |
Immunocytology Indicates That ACA2p Is an Endomembrane Pump
Harper et al. (1998) previously showed ACA2p to be most abundant
in the roots, as determined by a western-blot analysis. As a first
approach to determine the subcellular localization of ACA2p, root
sections were examined by immunofluorescence microscopy (Fig.
1). Cryosections were immunodecorated
with an affinity-purified anti-ACA2 polyclonal antibody. The antiserum
was generated against residues Val-119 to Pro-161 within the N-terminal
domain of ACA2p (Harper et al., 1998). Affinity-purified antibodies
detected a single protein band of 110 kD in a western blot of total
cellular protein (Harper et al., 1998). These antibodies appeared to be highly specific for ACA2p, as indicated by their failure to detect cross-reacting proteins in tobacco or onion (data not shown). Nevertheless, the possibility that these antibodies cross-react with a
second, closely related isoform in Arabidopsis has not been excluded.
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| Figure 1.
Immunofluorescence microscopy of root cells
showing ACA2p in an endomembrane system. A, Longitudinal section of a
root tip labeled with preimmune IgG and fluorescein
isothiocyanate-conjugated secondary antibody. B, A similar section
labeled with affinity-purified anti-ACA2p and fluorescein
isothiocyanate-conjugated secondary antibody. Figure 7A shows a similar
section stained with toluidine to show structural preservation of root
tip cells and the presence of large, densely stained nuclei in the
center of most cells. Bar = 50 µm.
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Confocal microscopy revealed anti-ACA2p staining of all cell types
within roots. Immunodecorated proteins were observed inside the cell
boundary, with an apparent exclusion from the nucleoplasm. These
results are consistent with a non-PM location, as previously suggested,
based on membrane-fractionation studies using aqueous two-phase
partitioning (Harper et al., 1998). However, our immunocytology did not
provide sufficient resolution to identify specific subcellular structures.
ACA2p Cofractionates with ER
To further define the endomembrane location of ACA2p, microsomal
membranes were fractionated on Suc gradients and characterized by
western blots and marker enzyme assays (Fig.
2). ACA2p cofractionated with ER
membranes, as indicated by sedimentation profiles overlapping with two
other ER resident proteins, ACA3p and BiP. BiP is a commonly used ER
marker (Haas, 1994) and ACA3p (ECA1p) is a typical ER-type Ca2+ pump in Arabidopsis (Liang et al., 1997). We
analyzed two independent sets of gradients with equivalent results. In
gradients prepared with 5 mM Mg2+,
ACA2p was most abundant at a Suc density of 34% to 40% (w/v), as
detected in western blots using anti-ACA2p antibodies. In gradients prepared without Mg2+ (i.e. + 5 mM
EDTA), the ACA2p peak shifted to a lighter Suc density between 28% and
32%. A large Mg2+-dependent density shift is
characteristic of ER membranes and it occurs when
Mg2+ is chelated and polyribosomes are
dissociated from the ER. Controls also identified peak fractions for
marker enzymes, detecting the PM (H+-ATPase
marker at 40%-44% Suc), tonoplast (-tonoplast intrinsic protein
marker at 25%-32% Suc), chloroplast thylakoid membranes (chlorophyll
marker at 40% Suc), and Golgi (latent UDPase marker at 32%-34%
Suc). None of these markers revealed a fractionation profile comparable
to ACA2p. Thus, our fractionation effectively separated the ER from
other major membrane systems and supported an ER localization for
ACA2p.
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| Figure 2.
Suc-gradient membrane fractionation showing
cofractionation of ACA2p with ER markers. Microsomal membranes were
fractionated over 15% to 45% Suc gradients, and 1-mL fractions were
collected. Mg2+ + or indicate parallel gradients run
with 5 mM MgCl2 (+) to stabilize
membrane-associated proteins or with 5 mM EDTA () to
dissociate membrane-associated proteins. A, Western-blot analysis of
membrane fractions probed with anti-ACA2, ACA3 (ER marker), BiP (ER
marker; Denecke et al., 1991; Hofte and Chrispeels, 1992), -TIP
(tonoplast marker; Johnson et al., 1990), and H+-ATPase
antibodies (PM marker; DeWitt et al., 1996). Black bars highlight peak
fractions. Samples (10 µL) from each fraction were separated by
SDS-PAGE, blotted, and probed with anti-ACA2 (1:2000), anti-ACA3
(1:2000), anti-BiP (1 2000), anti--TIP (1:500), or anti-CTF2
(H+-ATPase) (1:7500). B, Spectrophotometric analysis of
membrane fractions (10-µL samples) for chlorophyll a
and b (chloroplast marker). C, Marker enzyme analysis of
membrane fractions (10-µL samples) for Triton-stimulated UDPase
activity (Golgi marker; Nagahashi and Kane, 1982). The maximal activity
in the Mg2+ gradient was 0.2 µmol min1
mL1, and in the +Mg2+ gradient it was 0.3 µmol min1 mL1.
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Engineering Plants with ACA2-GFPp
To provide cytological corroboration for an ER localization, we
tagged ACA2p with a GFP from jellyfish (Aequorea victoria) and visualized its subcellular location in transgenic plants. A GFP was
fused to the C-terminal end of ACA2p immediately following the
penultimate Pro (Fig. 3). Three Gly
residues were included in the linker sequence to provide a flexible
attachment. The GFP sequence contained an S65-T mutation that provided
an optimal excitation of approximately 490 nm and an emission at 510 nm
(Chiu et al., 1996). In addition, a six-His motif was included at the C
terminus to permit nickel-resin affinity purification. Figure 3
diagrams the engineering of ACA2-GFP into a plant-expression vector,
with transcription under the control of a CaMV-35S promoter.
There were two rationales for choosing a C-terminal location for the
GFP tag. First, DeWitt et al. (1996) previously showed that adding
epitopes to the C-terminal end of H+-ATPase
isoforms AHA2p and AHA3p did not alter membrane insertion or
PM-targeting properties. Second, the predicted C-terminal domain of
ACA2p had no proposed function and contained only five residues (KTIPV). We considered an N-terminal location to be a risky alternative because the N-terminal domain functions as an autoinhibitor and a tag
in this location could easily disrupt the autoinhibitor and generate a
hyperactive pump. This was a serious concern because a
"deregulated" pump might result in pleiotropic cellular
dysfunctions and thereby raise uncertainties about whether normal
targeting pathways were being observed.
To confirm that our engineered ACA2-GFPp retained the expected
functional properties, we genetically tested its activity in a yeast
host, K616. This yeast strain harbors a disruption of both endogenous
Ca2+ pumps and does not grow on
Ca2+-depleted media (e.g. with 10 mM
EGTA). Previous studies of an untagged ACA2p demonstrated that only a
deregulated mutant (ACA2-2p) provided complementation and allowed K616
to grow on Ca2+-depleted media (Harper et al.,
1998). Complementation is thought to require the activity of a
high-affinity Ca2+ pump to transport
Ca2+ into the ER/Golgi. Enzyme assays established
that, in contrast to the
Ca2+/calmodulin-regulated activity of the
full-length pump (ACA2-1p), the mutant ACA2-2p was constitutively
active. This calmodulin-independent activity is proposed to allow
ACA2-2p to restore growth to the yeast K616 on
Ca2+-depleted media. Under these same growth
conditions, the full-length pump probably remains inactive, since the
yeast calmodulin would likely be in an apo state because of the
expected low levels of cytosolic Ca2+. Consistent
with these earlier studies, the complementation properties of ACA2p
were not altered by the addition of a GFP tag to either the full-length
or the deregulated versions (Fig. 4 shows
complementation by the "deregulated ACA2-2 + GFP" = ACA2-8).
These results indicated that a C-terminal GFP did not disrupt the
potential of ACA2p to function as a Ca2+ pump or
for the pump to be regulated by its autoinhibitor. Therefore, the C
terminus of ACA2p appeared to be a suitable location for a GFP
tag.
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| Figure 4.
Genetic evidence that ACA2-GFPp retains normal
functional properties when expressed in yeast. The yeast mutant K616
was transformed with different constructs and tested for its ability to
grow on synthetic medium supplemented with 10 mM
CaCl2 (control) or on synthetic medium supplemented with 10 mM EGTA. Growth of K616 harboring different constructs is
shown. Both tagged and untagged versions of a deregulated pump (ACA2-8
and ACA2-2, respectively) were able to restore growth of the K616
mutant on EGTA. The constructs shown are: Vector, the parent vector
pYX-112; GFP, pYX-GFP-JFH1, which encodes a GFP(S65/T) (J.F. Harper,
unpublished data); ACA2-2, pYX-ACA2-2, which encodes a deregulated
ACA2p (Harper et al., 1998); and ACA2-8, pYX-ACA2-8, which encodes
the same deregulated ACA2-2p with an additional C-terminal GFP tag.
The tagged and untagged full-length versions of ACA2p (respectively
encoded by pYX-ACA2-7 and pYX-ACA2-1) did not complement and are not
shown.
|
|
In working with plant cells, our initial evaluation of ACA2-GFPp
expression was conducted by electroporating the 35S-ACA2-GFP plasmid
into tobacco cv BY2 protoplasts (not shown). Transient expression was
easily detected by fluorescence microscopy. The fluorescence was
concentrated in subcellular structures similar to those observed in
stable, transformed Arabidopsis cells (see below).
We pursued subcellular localization further in transgenic Arabidopsis.
The rationale was to examine ACA2-GFPp localization in cell types in
which the endogenous ACA2p was known to be expressed. Of 20 independent
transgenic plant lines showing ACA2-GFPp expression, all grew into
normal, healthy plants, indicating that ACA2-GFPp expression did not
adversely alter cellular functions. Expression levels and subcellular
localization were examined in detail for two transgenic plant lines,
and both expressed ACA2GFPp at approximately 5-fold higher
levels than observed for the endogenous ACA2p in wild-type plants (see
below). Thus, in the following subcellular localization study healthy
cells with only modest levels of transgene overexpression were used.
ACA2-GFPp Cofractionates with ER
To examine whether the targeting of tagged ACA2-GFPp was
comparable to the endogenous ACA2p, we performed parallel
membrane-fractionation analyses. We obtained equivalent cofractionation
results for the two independent transgenic plants lines that we
analyzed. As observed for the endogenous ACA2p (Fig. 2) the tagged
ACA2-GFPp cofractionated with two ER markers, BiP and ACA3p, in Suc
gradients performed with and without Mg2+ (Fig.
5). Not shown are additional marker
assays for the PM, tonoplast, chloroplast, and Golgi, which also showed
fractionation profiles equivalent to those shown in Figure 2.
ACA2-GFPp was detected in these gradients using two different
assays: a western-blot analysis using anti-GFP to immunodetect the
chimeric protein at 136 kD (Fig. 5A) and fluorescence spectroscopy to
detect the GFP fluorescence (Fig. 5B).
View larger version (39K):
[in this window]
[in a new window]
| Figure 5.
Suc-gradient fractionation of membranes from
35S-ACA2-GFP transgenic plants showing cofractionation of ACA2-GFPp
with ER markers. Microsomal membranes were fractionated through a 15%
to 45% (w/w) Suc gradient and analyzed as described in Figure 2. A,
Western-blot analysis of membrane fractions probed for ACA2-GFPp,
ACA3p, and BIP. Western blots were probed with anti-GFP (1:2000) to
specifically detect the ACA2-GFP fusion. ACA3p and BIP were detected as
described in Figure 2. B, Fractionation profile of ACA2-GFP as detected
by GFP fluorescence. Fluorescence was measured at 510 nm (±5 nm) with
excitation at 480 nm. The values shown were derived by subtracting the
background fluorescence detected in parallel gradients from a wild-type
control (normalized per microgram of protein). Each point is the mean
from the analysis of two gradients.
|
|
ACA2p Expression Is Silenced in ACA2-GFPp Plants
To more directly assess whether ACA2-GFPp targets the same
membrane as ACA2p, we attempted to simultaneously detect the endogenous ACA2p in the same Suc gradients. Figure 6
shows that both pumps can be separated by SDS-PAGE and detected on a
western blot by anti-ACA2 antibodies as bands of 136 and 110 kD,
respectively. However, in both transgenic plant lines analyzed, the
endogenous ACA2p was not detectable in any fraction, even after long
overexposures (Fig. 6 shows a representative comparison). We estimated
that expression of the endogenous ACA2p was suppressed more than
10-fold, based on western-blot analyses conducted with wild-type and
two transgenic plant lines. We also examined, as a control, the
expression levels of two other resident ER proteins, ACA3p and BiP. In
contrast to ACA2p, normal expression levels were observed for ACA3p
(Fig. 6) and BiP (not shown). These results indicate that ACA2p
suppression was specific and not caused by a general suppression of ER
resident proteins.
View larger version (36K):
[in this window]
[in a new window]
| Figure 6.
Western-blot analysis showing suppression of
endogenous ACA2p in transgenic plants expressing ACA2-GFPp. Suc
gradients from two independent 35S-ACA2-GFP plants were simultaneously
analyzed for endogenous and GFP-tagged ACA2p using anti-ACA2 or
anti-GFP antibodies, respectively. Shown here is a representative
comparison of two 10-µg protein samples taken from fraction 9 of the
+Mg2+ gradients derived from wild-type and 35S-ACA2-GFP
transgenic plants (see Figs. 2 and 5). Parallel samples were analyzed
for the expression levels of ACA3p using anti-ACA3 antibodies. Proteins
were separated by SDS-PAGE, blotted, and probed with anti-ACA2 (1:2000)
or anti-ACA3 (1:2000).
|
|
ACA2-GFPp Images as a Fluorescent Reticulum
To visualize the subcellular structures containing ACA2-GFPp, we
imaged GFP fluorescence in living cells using confocal microscopy and
computational optical-sectioning microscopy. We used both approaches to
observe similar fluorescent images. No detectable signals were observed
from nontransgenic plants (data not shown). Two separate transgenic
plant lines were analyzed with equivalent results. Fluorescent images
were evaluated from hundreds of cells showing both high and low levels
of expression. Even within a single plant root tip there was
considerable cell-to-cell variation in expression levels. The source of
this variability is not known but may be related to the cosuppression
phenomena discussed above (Fig. 6). This variability allowed us to make
a direct comparison of strong and weak fluorescent patterns in
neighboring cells, and the comparison revealed similar reticulate
structures. Therefore, the images shown appear to be representative of
a normal subcellular localization pattern and not an artifact produced
by the overexpression and mislocalization of a GFP-tagged fusion
protein.
Confocal images (Fig. 7, C and D) are
shown for cells near the root tip from plants expressing ACA2-GFPp or a
control GFP only. The root tip is shown as a toluidine blue-stained
section in Figure 7A for reference. We chose the root tip for detailed analysis because the endogenous ACA2p was highly expressed in this
tissue, as indicated by immunocytology with an anti-ACA2 antibody (Fig.
1). Toluidine blue-stained root tip cells appear at higher
magnification in Figure 7B to illustrate the central location of a
large, densely stained nucleus. Comparable cell types in Figure 7, C
and D, show the fluorescent images of a GFP-only control and ACA2-GFPp.
In the GFP control, the strongest fluorescent signal corresponded to
the central nuclear region. This apparent nuclear accumulation is
consistent with previous reports of imaging GFP expression in plants
(Haseloff et al., 1997). In contrast, the signal from ACA2-GFPp was
excluded from the nuclear region and showed a network of connecting
strands throughout the cell, most similar to patterns seen for an
actin/ER network (Boevink et al., 1998).
We also examined images from cells in more mature regions of the root.
Figure 7, E and F, shows representative fluorescence images from mature
root epidermal cells expressing a GFP-only control or the ACA2-GFP
fusion. As with the subcellular localization in undifferentiated cells,
the GFP-only control appeared to accumulate in the nucleus, whereas
ACA2-GFPp was excluded from the nucleoplasm and formed a network of
connecting strands. This network differed from that in undifferentiated
cells, showing more evidence of membrane sheets in addition to
interconnecting membrane strands. In addition, the perinuclear region
of many epidermal cells showed strong fluorescence, as illustrated by
an image of two nuclei in Figure 7G.
| |
DISCUSSION |
A PM-Type Ca2+ Pump in the ER
Our results indicate that ACA2p is localized to the ER, which
makes ACA2p distinct from all other calmodulin-regulated
Ca2+ pumps identified in any organism. Two lines
of evidence support this novel, subcellular localization. First,
membrane fractionation by Suc gradients showed that ACA2p and the
tagged isoform ACA2-GFPp cofractionated with two ER markers, BiP and a
second, more typical ER-type Ca2+ pump,
ACA3p/ECA1p (Figs. 2 and 5). Second, fluorescence microscopy of cells
expressing ACA2-GFPp revealed an interconnecting network of sheets and
strands, forming a reticular pattern characteristic of plant ER (Fig.
7). Although both lines of evidence corroborated an ER localization, we
cannot exclude the possibility that lesser amounts of ACA2p were
targeted to other locations, such as the Golgi. These results
identified ACA2-like pumps as a source of calmodulin-regulated
Ca2+-ATPase activity previously reported in plant
ER membrane fractions (Gavin et al., 1993; Hwang et al., 1997; Evans
and Williams, 1998).
It is not clear whether the appearance of ACA2-GFPp around some nuclei
reflects a preferential targeting of the pump to the nuclear envelope
or to multiple layers of ER that can sometimes wrap around the nucleus.
Ca2+ pumps are expected to be associated with the
perinuclear region to control Ca2+ levels in this
subcellular location. Nuclear-localized Ca2+
fluxes have been observed in plant and animal cells. For example, Ehrhardt et al. (1996) observed an oscillating
Ca2+ signal around nuclei in alfalfa root
epidermal cells exposed to a nodulation factor. In both plant and
animal cells there is evidence for a typical ER-type
Ca2+ pump in the nuclear envelope (Lanini et al.,
1992; Santella and Carafoli, 1997; Downie et al., 1998). However,
results here prompt speculation that plant cells differ from animal
cells and use an additional calmodulin-regulated
Ca2+ pump in the perinuclear region.
Use of a GFP-Tagged ACA2p
Tagging proteins with a GFP provides a powerful approach for
imaging subcellular locations for proteins. In this study, imaging a
GFP-tagged ACA2p provided important corroboration for our
membrane-fractionation analysis for two reasons. First, the GFP tag
provided isoform-specific information. This was important because
immunocytology and membrane-fractionation studies both relied on
detecting the endogenous pump with a polyclonal antibody. Although this
antibody appeared to be highly specific, we could not exclude the
possibility that it also detected a second, more abundant
cross-reacting isoform. Second, Suc gradient fractionation protocols do
not cleanly separate all membrane systems and the diversity of membrane
systems in plants is still undefined. Thus, without cytological
verification, membrane-fractionation studies may provide misleading
information.
On the other hand, an investigation based solely on GFP tagging also
requires a cautious interpretation. A protein tagged with GFP may not
target the correct location for many reasons; e.g. the GFP tag may
disrupt the normal targeting signals of the subject protein or
overexpression of a tagged protein may overload a targeting pathway and
cause significant missorting of membranes; and/or ectopic expression of
a tagged protein may show an artifactual localization in a cell type
where the organelles, interacting proteins, or modifying enzymes
required for proper targeting are not present. With these concerns in
mind, in this study we imaged ACA2-GFPp under conditions in which the
transgene was expressed at low to moderate levels; we focused on cell
types that showed expression of the endogenous gene. We further
demonstrated, in a membrane-fractionation analysis (Fig. 5), that the
behavior of the tagged pump was like that of the endogenous pump.
Therefore, we believe that our imaging of ACA2-GFPp accurately reflects
the subcellular distribution of the endogenous ACA2p.
Regulation of Ca2+-Pump Expression
We observed that ACA2p expression was suppressed in both of the
transgenic plant lines analyzed in the present localization study (Fig.
6). This silencing may have two nonexclusive explanations. First, the
endogenous ACA2p may be silenced by "transgene-cosuppression" (Baulcombe and English, 1996; Meyer and Saedler, 1996), or second and
alternatively, it may be down-regulated by a feedback system that
maintains an appropriate level of Ca2+-ATPase
activity.
Although we cannot distinguish between these two mechanisms of
down-regulation, there is precedence for an integrated feedback system
that coordinates the expression of both PM- and ER-type Ca2+ pumps in animal cells (Guerini et al., 1995;
Kuo et al., 1997). For example, overexpression of a PM-type
Ca2+-ATPase in mammalian tissue-cultured cells
correlated with the down-regulation of the ER-type pump (Kuo et al.,
1997). In contrast to that example, we failed to observe any
interdependence between the overexpression of ACA2-GFPp and the
expression levels for the ER-type pump ACA3p (e.g. ACA3p levels were
not down-regulated in response to ACA2-GFP overexpression). This result
supports the hypothesis that, even though both the ACA2p and the ACA3p are located in the ER, they provide functionally separate
Ca2+-efflux pathways that are not coordinately
regulated at the protein expression level.
ER Targeting Signals
Exactly how P-type ATPases are targeted to different membrane
systems in plants or animals is not known. A working hypothesis for ER
localization of ACA3p (i.e. the more typical ER-type pump) is that a
Lys-rich sequence (KQKEE) at the C-terminal end provides an
ER-retention motif (Jackson et al., 1990; Townsley and Pelham, 1994).
In animal cells many resident ER membrane proteins contain a C-terminal
retention motif with the consensus (K/X)(K/X)KXX-stop (Jackson et al.,
1993). Because the sequence KKXX can function as an ER retention signal
in yeast (Townsley and Pelham, 1994), this retention motif appears to
be conserved in distantly related phyla.
However, not all ER resident membrane proteins are localized by a
common mechanism. A notable exception is an ER-type
Ca2+ pump from chicken (SERCa1p), which does not
contain a consensus C-terminal retention motif (its C-terminal sequence
is DERRK; Karin and Settle, 1992). Instead, a targeting analysis of
chimeras, made between animal ER-type- and PM-type
Ca2+-pumps, suggests that the structure or sequence of the
first transmembrane domain provides an ER-retention signal (Foletti et
al., 1995). It is even less clear how ACA2p could be localized to the
ER and how it could accumulate to high levels around the nucleus. A
consensus ER-retention motif was not present in the C-terminal domain
of ACA2p, and the sequence of the first transmembrane domain was not
well conserved with the animal ER-type
Ca2+-ATPase pumps.
In animals, calmodulin-regulated pumps are thought to be exclusively
localized to the PM. By contrast, the identification of ACA2p in the
plant ER and the proposed plastid and tonoplast locations of ACA1p and
BCA1p, respectively, suggest a fundamental difference between the ways
that plant and animal cells use calmodulin-regulated Ca2+ pumps.
Why Are There Two Ca2+ Pumps in the ER?
Our results indicate that both ACA3p and ACA2p cofractionate with
ER isolated from Arabidopsis roots. It is likely that at least some
cells in the root contain both pumps, because both ACA2p and ACA3p were
predominantly expressed in the root, and immunocytology of the root tip
suggested that ACA2p was expressed in every cell type. However, we have
not investigated the issue of whether the colocalization of both types
of Ca2+ pumps in the ER is a common feature of
all plant cells.
We offer three hypotheses for considering the potential significance of
two different Ca2+ pumps in the ER. First, the
two pumps may be spatially separated into different functional domains.
Separate domains of the ER have been reported in animal cells, each
maintaining a distinct pool of Ca2+ (Golovina and
Blaustein, 1997). Second, each pump may provide a unique enzymatic
activity, perhaps the ability to pump a secondary cation such as
Mn2+ (Liang et al., 1997). Third, the two pumps
may act differently to control
cytoplasmic/ER-Ca2+ levels in response to
different stimuli. "Differential activation" is an attractive
hypothesis because calmodulin directly activates ACA2p but not ACA3p.
In any event, the observation that a plant ER contains both type IIA
and IIB pumps highlights the complexity of Ca2+
transport into this organelle.
| |
FOOTNOTES |
1
This research was supported by a U.S. Department
of Energy grant (no. DE-FG03-94ER20152) to J.F.H. and a joint grant
(no. IBN-9416038) from the National Aeronautics and Space
Administration and the National Science Foundation for the Plant
Sensory Systems Collaborative Research Network. Support for
computational microscopy was provided by a National Institutes of
Health grant (no. RR01380) to the Institute for Biomedical Computing at
Washington University and to J.S.G. through a Monsanto predoctoral
fellowship in plant biology.
*
Corresponding author; e-mail Harper{at}Scripps.edu; fax
1- 619-784-9840.
Received September 21, 1998;
accepted December 21, 1998.
| |
ABBREVIATIONS |
Abbreviations:
BiP, a homolog of the ER-resident immunoglobulin
heavy chain-binding protein.
CaMV, cauliflower mosaic virus.
GFP, green
fluorescence protein.
GST, glutathione S-transferase.
PM, plasma membrane.
| |
ACKNOWLEDGMENTS |
The authors thank Catharine Conley for helpful discussions, Woo
Sik Chung for discussions and assistance with subclonings, Maarten
Chrispeels for providing anti-BiP and anti--TIP antibodies, and
Malcom Wood for assistance with confocal imaging. We thank Mrs.
Frederick Garry and Mr. and Mrs. Kenneth E. Hill for general support to
The Scripps Research Institute for operation of the laboratory for
J.F.H.
| |
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Abstract
Introduction