§
* Division of Cellular and Molecular Medicine, Department of Pathology,
Department of Pharmacology, and § Howard Hughes
Medical Institute, University of California San Diego, La Jolla, California 92093-0651
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We previously demonstrated that CALNUC,
a Ca2+-binding protein with two EF-hands, is the major
Ca2+-binding protein in the Golgi by 45Ca2+ overlay
(Lin, P., H. Le-Niculescu, R. Hofmeister, J.M. McCaffery, M. Jin, H. Henneman, T. McQuistan, L. De Vries,
and M. Farquhar. 1998. J. Cell Biol. 141:1515-1527). In
this study we investigated CALNUC's properties and
the Golgi Ca2+ storage pool in vivo. CALNUC was
found to be a highly abundant Golgi protein (3.8 µg
CALNUC/mg Golgi protein, 2.5 × 105 CALNUC molecules/NRK cell) and to have a single high affinity, low
capacity Ca2+-binding site (Kd = 6.6 µM, binding capacity = 1.1 µmol Ca2+/µmol CALNUC). 45Ca2+ storage was increased by 2.5- and 3-fold, respectively, in
HeLa cells transiently overexpressing CALNUC-GFP
and in EcR-CHO cells stably overexpressing CALNUC. Deletion of the first EF-hand helix from CALNUC completely abolished its Ca2+-binding capability.
CALNUC was correctly targeted to the Golgi in transfected cells as it colocalized and cosedimented with the Golgi marker,
-mannosidase II (Man II). Approximately 70% of the 45Ca2+ taken up by HeLa and CHO
cells overexpressing CALNUC was released by treatment with thapsigargin, a sarcoplasmic/endoplasmic
reticulum calcium ATPase (SERCA) (Ca2+ pump)
blocker. Stimulation of transfected cells with the agonist ATP or IP3 alone (permeabilized cells) also resulted in a significant increase in Ca2+ release from
Golgi stores. By immunofluorescence, the IP3 receptor type 1 (IP3R-1) was distributed over the endoplasmic
reticulum and codistributed with CALNUC in the
Golgi. These results provide direct evidence that
CALNUC binds Ca2+ in vivo and together with
SERCA and IP3R is involved in establishment of the
agonist-mobilizable Golgi Ca2+ store.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE Golgi complex is involved in posttranslational
modification of newly synthesized proteins and
serves as the main sorting station for protein and
vesicular traffic (Farquhar and Hauri, 1997; Farquhar
and Palade, 1998
). Calcium is well known to be essential
for cell signaling (Tsien and Tsien, 1990
; Meldolesi and
Pozzan, 1998
) but also for cell processes such as protein processing and membrane traffic to and through the Golgi
(Davidson et al., 1988
; Ivessa et al., 1995
; Duncan and Burgoyne, 1996
). Recently the Golgi has been identified as a
Ca2+-enriched compartment using ion microscopy and
electron energy loss spectroscopy-electron spectroscopic
imaging (EELS-ESI) (Chandra et al., 1991
; Grohovaz et al.,
1996
; Pezzati et al., 1997
). Ca2+ can be released from the
Golgi by the Ca2+ ionophore A23187 (Chandra et al.,
1991
), the Ca2+ channel blocker La3+ (Zha and Morrison,
1995
), and histamine, an agonist known to be coupled to
IP3 generation (Pinton et al., 1998
). How the high level of
Ca2+ in the Golgi is maintained is unknown at present.
The ER Ca2+ pool (or Ca2+ store) has been studied
more extensively and is known to be maintained by organelle-associated Ca2+ ATPase (Ca2+ pumps) and lumenal Ca2+-binding proteins of which there are many (Bastianutto et al., 1995; Meldolesi and Pozzan, 1998
). There is
also evidence for the existence of Ca2+ pumps on the
Golgi based on ATP-dependent Ca2+ uptake into mammalian (Baumrucker and Keenan, 1975
; Hodson, 1978
; Neville et al., 1981
; Virk et al., 1985
) and yeast (Sorin et al., 1997
) Golgi fractions. Both sarcoplasmic/ER calcium ATPase (SERCA)1 and plasma membrane calcium ATPase
en route to the plasma membrane are essential for Ca2+
uptake into isolated Golgi fractions (Taylor et al., 1997
).
However, information on Golgi calcium-binding proteins
is still limited and the detailed mechanisms of Ca2+ uptake, storage, and release from the Golgi apparatus remain to be elucidated. Previously, we demonstrated that
CALNUC (nucleobindin) (Miura et al., 1992
; Wendel et al.,
1995
), a Golgi resident protein that faces the Golgi lumen,
is the major Ca2+-binding protein in the Golgi based on
45Ca2+ overlay (Lin et al., 1998
).
In this study we have investigated the role of CALNUC in establishing the Golgi Ca2+ pool in vivo by examining the effects of overexpression of CALNUC on Ca2+ uptake. We provide direct evidence that CALNUC possesses high affinity/low capacity Ca2+ binding properties and binds Ca2+ in the Golgi in vivo. The finding that the majority of the Ca2+ sequestered by overexpressed CALNUC is released by thapsigargin (Tg), ATP, and IP3 provides additional in vivo evidence for the existence of SERCA and inositol 1, 4, 5 trisphosphate receptor (IP3R) on the Golgi. CALNUC together with SERCA and IP3R on Golgi membranes constitute a cellular Ca2+ pool in the Golgi which may have distinct functions.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials
Polyclonal antibody (F-5059) against full-length, recombinant CALNUC
was generated and affinity purified as previously described (Lin et al.,
1998). Polyclonal anti-
-mannosidase II (Man II) was prepared as described (Velasco et al., 1993
). Monoclonal anti-Man II (53FC3) and polyclonal antibody against denatured Man II were gifts from Drs. B. Burke
(University of Alberta, Alberta, Canada) and K. Moremen (University of
Georgia, Athens, GA), respectively. Monoclonal anti-mouse IP3R-1
(18A10) was kindly provided by Drs. A. Miyawaki and K. Mikoshiba
(University of Tokyo, Tokyo, Japan) (Furuichi et al., 1989
). Polyclonal antibody against calnexin was a gift from Dr. J.J.M. Bergeron (McGill University, Montreal, Canada). Cross-absorbed Texas red-conjugated donkey anti-rabbit F(ab')2 was obtained from Jackson ImmunoResearch
Laboratories, and affinity-purified goat anti-rabbit IgG (H+L) conjugated to HRP was from Bio-Rad. 45CaCl2 was obtained from NEN Life
Science Products. Supersignal chemiluminescent reagent was purchased
from Pierce. All chemical reagents were from Sigma Chemical Co. except
as indicated.
Preparation and Purification of His6-CALNUC
Full-length CALNUC cDNA was amplified by PCR using 5'-CGCGCGGCAGCCATATGCCTACCTCTGTG-3' and 5'-CGGAATTCGGATCCTTATAAATGCTGAGAATC-3' as primers. PCR was carried out using 100 pmol of each primer, 2 ng CALNUC cDNA, 200 µM dNTP, 2.5 U
PFU polymerase (Stratagene), and PCR reaction buffer in a total volume
of 50 µl. PCR products were purified using a QIAquick PCR Purification
kit (Qiagen) and subcloned into the pET-28a(+) vector (Novagen) at
BamHI/NdeI restriction sites, followed by transformation into Escherichia coli BL21(DE3). Expression of CALNUC protein was induced with
1 mM isopropyl -D-thiogalactoside (IPTG) (Pharmacia Biotech) at 18°C
for 4 h at a bacterial density of OD600
1.0.
To purify His6-CALNUC protein, transformed E. coli were suspended in binding buffer containing 20 mM sodium phosphate and 500 mM NaCl (pH 7.5), and sonicated using a Microson Ultrasonic Cell Disrupter (Heat Systems). Lysates were incubated with Ni-NTA agarose (Qiagen), washed with 20 mM sodium phosphate, 500 mM NaCl at decreasing pH (8.0, 6.0, and 5.3), and bound proteins were sequentially eluted with an imidazole step gradient (10 mM to 1 M). Fractions containing a single band of purified CALNUC detected by silver staining were pooled and dialyzed against TBS containing 150 mM NaCl and 10 mM Tris-HCl (pH 7.4) at 4°C, and subsequently concentrated using an Ultrafree-15 (Biomax-50K) filter (Millipore). Highly purified His6-CALNUC [0.6 mg/liter of transformed BL21(DE3)] was obtained.
Ca2+-binding Analysis
Equilibrium dialysis was performed essentially as previously published
(MacLennan and Wong, 1971; Baksh and Michalak, 1991
). Ca2+-free solution was prepared by treatment of deionized water with a UniPure I Water Purification System (Solution Consultants) and Chelex 100 ion exchange resin (Bio-Rad) (Thielens et al., 1990
). Equilibrium dialysis was
performed using a Dialysis System (GIBCO BRL). 0.25 mg Ca2+-depleted
CALNUC (Thielens et al., 1990
) was incubated with 0.35 µCi/ml 45CaCl2
and different concentrations of cold Ca2+ at 4°C for 16 h, followed by assessment of radioactivity using a LS 6000IC Liquid Scintillation System
(Beckman Instruments) in EcoLume liquid scintillation cocktail (ICN).
Scatchard analysis was performed using CA Cricket Graph III software
(Computer Associates International).
Primary Structure Comparison
Amino acid sequences of CALNUC, Cab45 (Scherer et al., 1996), and
calmodulin (CaM) were obtained through Entrez on the National Center
for Biotechnology Information's (NCBI) World Wide Web home page.
Alignment of EF-hand motifs was performed using MacVector 6.0 software (Oxford Molecular Groups-IBI).
Cell Culture
HeLa cells were maintained in DME high glucose medium (Irvine Scientific) supplemented with 10% FCS (Life Technologies Inc.). Cells were
used as 80% confluent monolayers for transfection. Transfected EcR-CHO cells were cultured in Ham's F12 medium (CORE Cell Culture Facility, University of California, San Diego, CA) with 10% FCS (Life Technologies), 250 µg/ml Zeocin (Invitrogen), and 750 µg/ml G418 sulfate
(Calbiochem). All media contained 100 U/ml of penicillin G and 100 µg/
ml of streptomycin sulfate. NRK cells were cultured as previously described (Lin et al., 1998).
Transient Overexpression of CALNUC-Green Fluorescent Protein (GFP) or Truncated CALNUC-GFP in HeLa Cells
CALNUC cDNA was amplified by PCR with the primers 5'-CGCGGATCCATGCCTACCTCTGTG-3' and 5'-CCATGCCATGGCTAAATGCTGAGAATCC-3'. GFP cDNA was also amplified by PCR with the primers 5'-TCATGCCATGGTGAGCAAGGG-3' and 5'-ATAGTTTAGCGGCCGCTTACTTGTACAGCTC-3'. PCR products were purified and digested, respectively, with BamHI, NcoI, and NotI (New England Biolabs). CALNUC and GFP cDNA were subcloned into the pcDNA3 vector (Invitrogen) by three-fragment ligation to obtain a CALNUC-GFP/pcDNA3 construct with GFP ligated to the 3' (COOH terminus) of CALNUC.
CALNUC(EF-1), in which the
helix (Asp227-Leu239) of the first EF-hand (EF-1) domain (see Fig. 1) was deleted, was obtained by PCR with
the primers 5'-CGCGGATCCATGCCTACCTCTGTG-3'/5'-CCCAAGCTTATGCAGTATGAAGAA-3', and 5'-CCCAAGCTTGAAGCTCTGTTTACC-3' / 5 ' -CCATGCCATGGCTAAATGCTGAGAATCC-3 ' . CALNUC(
EFs-1,2), in which both EF-1 and EF-2 domains (Asp227- Phe291) (see Fig. 1) were deleted, was prepared with primers of 5'-CGCGGATCCATGCC TACC TCTGTG- 3 ' / 5 ' -CCCAAGCT TATGCAGTATGAAGAA-3', and 5'-CCCAAGCTTCTGGCATCCACACAG-3'/5'-CCA-
TGCCATGGCTAAATGCTGAGAATCC-3'. CALNUC mutants and
the GFP tag were subcloned into the pcDNA3 vector by four-fragment ligation with HindIII and NcoI as internal restriction linker sites. Fidelity of
the constructs was verified by automated DNA sequencing (CFAR, University of California, San Diego, CA). cDNA constructs were transformed
into E. coli DH5
, followed by extraction and purification using
QIAGEN Plasmid Midi/Mega Kits (Qiagen) and UltraPure CsCl (optical
grade) (GIBCO BRL).
|
To express wild-type or truncated CALNUC-GFP in HeLa cells, 1 µg purified DNA was transfected into HeLa cells (33-mm dish, 80% confluence) using 6 µg lipofectamine (GIBCO BRL). Transfected cells were grown in serum and antibiotic-free high glucose DME medium for 5 h followed by replacement with regular culture medium.
Establishment of a Stable HeLa Cell Line Overexpressing GFP Using Flow Cytometry
GFP cDNA amplified by PCR with the primers 5'-TCGCGGATCCATGGTGAGCAAGGG-3' and 5'-ATAGTTTAGCGGCCGCTTACTTGTACAGCTC-3' was subcloned into the pcDNA3 vector at BamHI/ NotI restriction sites, followed by transfection into HeLa cells as described above and G418 selection (0.75 mg/ml) for 4 d. Cells expressing GFP were sorted by flow cytometry (Ex/Em: 488/530 ± 15) (FACStar Plus®; Becton Dickinson) in the UCSD Flow Cytometry Core Facility. The top 0.12% of the positive cells was collected and maintained in media containing 0.75 mg/ml G418 until confluent. Selection by sorting was repeated three times until 100% of the cells (HeLa-GFP, GPH-1216) expressed GFP (data not shown).
Establishment of Stable Cell Lines Overexpressing CALNUC in the Ecdysone-inducible Mammalian Expression System
CALNUC cDNA was amplified by PCR and subcloned into the pIND vector (Invitrogen) at BamHI/NotI restriction sites. EcR-CHO cells (Invitrogen) stably expressing the ecdysone receptor (RxR and VgEcR) were transfected with CALNUC/pIND plasmid DNA using lipofectamine as described above followed by selection for G418 resistance (0.4 mg/ml) for 18 d. Cells were split into 96-well plates by serial dilution, 0.5 cells/well, and subsequently reselected with G418 (0.75 mg/ml). Four clones overexpressing CALNUC after induction with muristerone A (Invitrogen) were obtained; one of these, EcR-CHO-CALNUC-1 (CPC-22A), was used for these experiments.
Immunocytochemistry
CALNUC-GFP was directly visualized using a Zeiss Axiophot microscope and an FITC-filter (Ex/Em: 485/510). For immunofluorescence, cells on coverslips were fixed with 2% paraformaldehyde (50 min), permeabilized with 0.1% Triton X-100 (10 min), and incubated with affinity-purified anti-CALNUC IgG (6 µg/ml), anti-Man II serum (1:300), or
anticalnexin serum (1:100) as previously described (Lin et al., 1998).
Detection was with Texas red- or FITC-conjugated donkey anti-rabbit
F(ab')2. In some cases cells were doubly stained for CALNUC and either
a mouse mAb against Man II (40 µg/ml) or the IP3R-1 (1.25 µg/ml) and
appropriate secondary antibodies. Specimens were examined with either a
Zeiss Axiophot equipped for epifluorescence or a Bio-Rad confocal microscopy (MRC 1024) equipped with Lasersharp 3.1 software (Bio-Rad)
and a krypton-argon laser. Images were processed with Scion Image and
Adobe Photoshop (Adobe Systems) software.
Subcellular Fractionation
Sucrose gradient flotation of Golgi fractions was carried out using a protocol similar to those previously published (Fries and Rothman, 1980;
Brown and Farquhar, 1987
) with minor modifications. In brief, microsomal membranes were resuspended in 1.5 ml 55% sucrose (wt/wt), loaded
at the bottom of a sucrose step gradient consisting of 40, 35, 30, 25, and
20% (wt/wt in 1 mM Tris-HCl, pH 7.5), and centrifuged at 85,500 g for 16 h
at 4°C using a SW-40Ti rotor (Beckman). 20 fractions were collected from
the bottom, followed by SDS-PAGE and immunoblotting for calnexin (an
ER marker), Man II (a Golgi marker), and CALNUC.
Rat liver Golgi fractions, membrane (100,000 g pellet) and cytosolic
(100,000 g supernatant) fractions were prepared from postnuclear supernatants of transfected HeLa or EcR-CHO-CALNUC cells as previously
described (Saucan and Palade, 1994; Jin et al., 1996
; Lin et al., 1998
).
Immunoblotting and SDS-PAGE
Proteins were separated by 5 or 10% SDS-PAGE, transferred to PVDF
membranes, and immunoblotted with affinity-purified anti-CALNUC IgG, anticalnexin, and anti-Man II serum followed by HRP-conjugated anti-rabbit IgG and detection by ECL (Lin et al., 1998).
45Ca2+ Equilibrium Uptake and Release
The procedures followed were those reported previously (Bastianutto et al.,
1995). Cells (2 × 106) transfected with CALNUC-GFP or GFP alone were
incubated with 45Ca2+ (2 µCi/ml) for 48 h to reach 45Ca2+ equilibrium after
which they were washed three times in Krebs-Ringer-Hepes (KRH)
buffer (125 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2 mM
CaCl2, 6 mM glucose, and 25 mM Hepes, pH 7.4) and five times with PBS.
45Ca2+ was extracted with 0.1 N HCl (30 min at room temperature) and radioactivity assessed as described above. To examine 45Ca2+ release after
stimulation, washed cells were resuspended in KRH supplemented with
3 mM EGTA and stimulated at room temperature with 100 µM ATP or
sequentially stimulated with 0.1 µM Tg, 2 µM ionomycin, and 2 µM monensin, 5 min each. Equal aliquots (106 cells) were collected after each
stimulation, followed by centrifugation at 14,000 rpm (30 s) and quantification of 45Ca2+ in the supernatant.
Ca2+ Imaging
Noninduced or induced EcR-CHO-CALNUC cells were loaded with 1 mM Fura-2 AM (Molecular Probes Inc.) in Ham's F12 medium/0.5% FCS at 22°C for 1 h, washed with Ca2+-free HBSS (Irvine Scientific) followed by addition of 100 µM ATP. Ca2+ release was monitored by Ca2+ imaging performed on a Zeiss Axiovert microscope equipped with a cooled charge-coupled CCD camera (Photometrics) and MetaFluor software (Universal Imaging). Dual-excitation ratio imaging was obtained using two excitation filters (340DF20 and 380DF20) (Omega Optical and Chroma Technology) mounted on a filter wheel (Lambda 10-2; Sutter Instruments), a 420DRLP dichroic mirror, and a 510DF80 emission filter.
Assessment and Mobilization of Stored Ca2+ by IP3 in Permeabilized Cells
The procedures used were basically similar to those published (Berridge
et al., 1984; De Smedt et al., 1997
) with minor modifications. To examine
equilibrium 45Ca2+ uptake, EcR-CHO-CALNUC cells induced with 5 µM
ponasterone A for 24 h in a 6-well culture plate (106 cells/well) were permeabilized at 20°C for 4 min with saponin (50 µg/ml) in loading buffer
(140 mM KCl, 20 mM NaCl, 2 mM MgCl2, 2 mM ATP, 0.1 mM EGTA,
20 mM Pipes, pH 6.80), and 0.13 µM free Ca2+ calculated for conditions of
pH 6.80, at 20°C (Tsien and Pozzan, 1989
). Cells were washed four times
with loading buffer and subsequently loaded with 45Ca2+ (10 µCi/ml) for
various times (10-60 min). They were then rinsed five times with loading
buffer (30 s), stored 45Ca2+ was extracted with 1 ml 0.1 N HCl for 30 min,
and 0.5-ml aliquots were counted.
To investigate 45Ca2+ mobilization by IP3, induced and permeabilized EcR-CHO-CALNUC cells were loaded with 10 µCi/ml of 45Ca2+ as above for 45 min. After washing (five times over 1-1.5 min), cells were challenged with 10 µM IP3 (D-myo-inositol 1,4,5-trisphosphate potassium salt) in loading buffer, 1 ml/well. Solutions were collected at 2-min intervals, replaced with loading buffer containing IP3, and counted.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Quantification of Endogenous CALNUC in Rat Liver Golgi Fractions and NRK Cells
To quantify endogenous CALNUC in rat liver Golgi fractions, a linear standard curve was obtained for purified
His6-CALNUC (1.3-40 ng) by immunoblotting and densitometric analysis (data not shown). Endogenous CALNUC was found to be present in pooled Golgi light and
heavy fractions from rat liver (Saucan and Palade, 1994;
Jin et al., 1996
) at a concentration of 3.8 µg/mg Golgi protein, i.e., ~0.4% of the total Golgi protein (includes both Golgi resident proteins and cargo in transit through the
Golgi). NRK cells were found to have 0.02 µg CALNUC/
106 cells, or 2.5 × 105 CALNUC molecules/NRK cell.
CALNUC EF-1 Is an Ideal EF-Hand Ca2+-binding Motif and Constitutes a High Affinity, Low Capacity Ca2+-binding Site
An ideal EF-hand Ca2+-binding motif has an helix-
loop-
helix structure in which oxygen ligands (O) provided by carboxy side chains of Asp (D)/Glu (E), carbonyl
groups (C'O) of the peptide main chain and H2O constitute the Ca2+-binding site, and a hydrophobic amino acid
(
) and a Gly (G) are essential for Ca2+ binding (Kretsinger, 1987
; Branden and Tooze, 1991
). CALNUC and Cab45 (Scherer et al., 1996
) are the only EF-hand, Ca2+-binding proteins identified so far in the Golgi. Since their Ca2+-binding constants are not yet known, in order to predict and compare the Ca2+-binding properties of these two
proteins, we compared the EF-hand primary structures of
CALNUC (two EF-hands), Cab45 (six EF-hands), and
CaM (four EF-hands). Cab45's and CaM's EF-hand motifs
are similar (Scherer et al., 1996
), and CaM's Ca2+-binding
properties have been well characterized.
As shown in Fig. 1 A, CALNUC EF-1, Cab45 EF-2 and -5, and all four CaM EF-hand structures constitute ideal EF-hand motifs. CALNUC EF-1 and EF-2 are strikingly similar to CaM EF-4 and Cab45 EF-5, but CALNUC EF-2 has an Arg (R) instead of Gly at residue 6 (Fig. 1 B). This suggests that CALNUC has only a single ideal EF-hand motif, EF-1.
To investigate the binding affinity of CALNUC for
Ca2+, we performed equilibrium dialysis. Purified recombinant His6-CALNUC was used based on a report that recombinant calreticulin (CRT) was comparable to native
CRT in its Ca2+-binding capability (Baksh and Michalak,
1991). Scatchard analysis of the binding curve (Fig. 1 C)
indicates that CALNUC binds Ca2+ with a high affinity
binding constant (Kd = 6.6 µM) and a low capacity, ~1.1
µmol Ca2+/µmol protein, suggesting only one high affinity, low capacity Ca2+-binding site on CALNUC.
Overexpressed CALNUC Colocalizes with the Golgi Marker Man II
To further investigate Ca2+ binding to CALNUC in vivo, we expressed CALNUC-GFP by transient transfection in HeLa cells and generated an inducible cell line, EcR-CHO-CALNUC, stably expressing CALNUC. By immunoblotting, CALNUC-GFP (90 kD) was detected in transiently transfected HeLa cells but not in nontransfected cells (Fig. 2 A). The majority of the CALNUC (~85%) was associated with membranes (100,000 g pellet) and the remainder (15%) was present in the cytosolic fraction (100,000 g supernatant). Three additional bands (Fig. 2 A), also visualized after in vitro translation (data not shown), were also seen. They could be products of protein degradation or mistranslated CALNUC retained in the cytosol. By immunofluorescence the distribution of CALNUC-GFP overlapped with that of the Golgi marker Man II (Fig. 3 A), indicating that the majority of the CALNUC-GFP is correctly targeted to the Golgi.
|
|
In EcR-CHO-CALNUC cells induced with muristerone A or ponasterone A (0.1-10 µM) for 24 h, we found a linear increase in the expression of CALNUC with increasing amounts of added hormone (Fig. 2 B). The ratio of CALNUC in membrane versus cytosolic fractions was similar to that of CALNUC-GFP in HeLa cells (data not shown). By immunofluorescence the distribution of CALNUC again overlapped with that of Man II in the Golgi region (Fig. 3 C) in EcR-CHO-CALNUC cells induced with 10 µM muristerone A for 24 h, and was distinct from that of the ER marker, calnexin (Fig. 3 B).
Cosedimentation of Overexpressed CALNUC and Man II in Sucrose Gradients
Next we analyzed the distribution of CALNUC in induced
EcR-CHO-CALNUC cells using an established procedure
for flotation of Golgi membranes and their separation
from ER membranes. As shown in Fig. 4, we found that
CALNUC and Man II cosedimented and peaked in fractions 12-15 with sucrose densities similar to those previously reported (1.10-1.14 g/ml) (Dunphy and Rothman,
1983; Brown and Farquhar, 1987
) for CHO cells. By contrast, the ER marker, calnexin, peaked in denser fractions
7-11 (1.16-1.19 g/ml). These results together with the
immunofluorescence findings demonstrate that overexpressed CALNUC is found in the Golgi and is consistent
with our previous conclusion (Lin et al., 1998
) that overexpression does not lead to mistargeting of CALNUC.
|
Overexpression of CALNUC-GFP or CALNUC in the Golgi Increases 45Ca2+ Uptake
To assess whether overexpressed CALNUC-GFP binds
Ca2+ in the Golgi, we carried out in vivo equilibrium Ca2+
uptake. The 45Ca2+ loading time was ~48 h, the time
shown previously to be long enough to reach 45Ca2+ equilibrium in cultured cells (Mery et al., 1996). 45Ca2+ uptake
by HeLa cells transiently overexpressing CALNUC-GFP
was 2.5-fold that of nontransfected HeLa cells or those stably expressing GFP alone (Fig. 5 A). Similarly, there was a
threefold increase in 45Ca2+ taken up by induced (5 µM
muristerone A for 48 h) versus noninduced EcR-CHO-CALNUC cells (Fig. 5 B). These results demonstrate that Golgi-associated CALNUC binds Ca2+ in vivo and most
likely is responsible for sequestering Ca2+ in the Golgi
lumen.
|
To investigate whether EF-1 is indeed the sole Ca2+-binding motif in CALNUC, we examined Ca2+ binding in
HeLa cells transiently transfected with truncated CALNUC-GFP mutants. When the helix of EF-1 (Asp227-
Leu239) or both EF-1 and EF-2 (Asp227-Phe291) were deleted from CALNUC, its Ca2+-binding capability was
completely abolished (Fig. 5 A). Mistargeting could be
ruled out since the majority of the mutant CALNUC-GFP was detected in the Golgi region by fluorescence. The results obtained from this in vivo Ca2+-binding analysis provide direct evidence that CALNUC binds Ca2+ in the
Golgi, and EF-1 constitutes the sole Ca2+-binding site on
CALNUC. The latter is in agreement with the data shown
in Fig. 1.
Release of Sequestered 45Ca2+ by the SERCA Inhibitor, Tg
To further investigate the characteristics of the Golgi Ca2+
pool, we performed experiments similar to those done previously to characterize the ER Ca2+ pool in cells overexpressing CRT (Bastianutto et al., 1995; Mery et al., 1996
).
When HeLa cells transiently overexpressing CALNUC-GFP or EcR-CHO-CALNUC cells stably expressing
CALNUC were treated with the SERCA inhibitor Tg
(Thastrup et al., 1990
), ~73% and 70%, respectively, of
the 45Ca2+ was released (Fig. 6), suggesting the existence
of SERCA on Golgi membranes. Since some Tg-insensitive organelles are capable of retaining Ca2+ after Tg
treatment, we subsequently treated cells with the Ca2+
ionophore ionomycin to release the remaining stored
45Ca2+. Nearly all the remaining 45Ca2+ (~20-25%) was
released by ionomycin (Fig. 6). In view of the fact that ionomycin is inactivated in acidic compartments such as
secretory granules and endosomes, we further treated cells with monensin, a carboxylic sodium proton ionophore
which releases Ca2+ from acidic compartments (Bastianutto et al., 1995
; Mery et al., 1996
) and found <5% of
the 45Ca2+ was released. Cells overexpressing CALNUC-GFP or induced EcR-CHO-CALNUC cells released twice
as much 45Ca2+ as nontransfected HeLa cells, HeLa cells
stably expressing GFP alone, or noninduced EcR-CHO-CALNUC cells. The fact that the majority of the 45Ca2+
taken up by CALNUC was released by Tg suggests that
both CALNUC and SERCA play a key role in sequestering 45Ca2+ in the Golgi, a conclusion in agreement with the
recent description of SERCA associated with isolated
Golgi fractions (Taylor et al., 1997
).
|
Release of Sequestered Ca2+ from the Golgi by Extracellular ATP
We next examined whether or not Ca2+ sequestered in the
Golgi is released after agonist challenge. Extracellular
ATP is known to activate phospholipase C (PLC) (Brown
et al., 1991) via binding to G protein-coupled nucleotide
receptors on the cell surface (O'Connor, 1992
). Activated
PLC promotes production of IP3 which binds to IP3R on
the ER and triggers Ca2+ mobilization. To investigate
whether Ca2+ sequestered by overexpressed CALNUC in
the Golgi could be released by agonist, we examined Ca2+
release in EcR-CHO-CALNUC cells by Ca2+ imaging after ATP challenge. The results (Fig. 7 A) demonstrated that the ratio, 340:380, was doubled in cells induced with
2.5 µM ponasterone A for 24 h compared with noninduced
cells, suggesting that more Ca2+ was released from induced cells. Similar results were also obtained when induced EcR-CHO-CALNUC cells were loaded with 45Ca2+
(Fig. 7 B). These results obtained by two different methods suggest that the Golgi Ca2+ store is sensitive to IP3
generated after ATP binding.
|
Release of 45Ca2+ Sequestered in the Golgi by IP3
To obtain direct evidence that IP3 is able to release Ca2+
from the Golgi, 45Ca2+ uptake and release studies were
performed on permeabilized EcR-CHO-CALNUC cells.
Fig. 8 A reveals that 45Ca2+ is rapidly taken up by both induced and noninduced permeabilized cells, but approximately twice the amount of 45Ca2+ was sequestered by
cells overexpressing CALNUC. Steady state was achieved
45 min after loading, which was slower than reported for
Swiss 3T3 cells (20 min) (Berridge et al., 1984). 45Ca2+ release was then stimulated by addition of IP3 (Fig. 8 B). The ratio of 45Ca2+ released from induced versus noninduced
cells was ~2:1. These results support the previous report
of Pinton and colleagues (1998) suggesting that both Golgi
membranes and ER membranes bear IP3R.
|
Localization of the IP3 Receptor on the Golgi and ER by Immunofluorescence
In view of the functional evidence for the existence of IP3R on the Golgi, we carried out immunofluorescence studies on NRK cells and induced EcR-CHO-CALNUC cells using a mAb that recognizes IP3R-1. IP3R-1 was found throughout the cytoplasm and concentrated in the Golgi region (Fig. 3 D) which is compatible with both an ER and Golgi localization. Confocal analysis (Fig. 3 E) showed that the distribution of IP3R-1 overlaps with that of CALNUC in the juxtanuclear region, suggesting that IP3R-1 and CALNUC colocalize on Golgi membranes. As mentioned by Pinton and co-workers (1998), it was not possible to carry out reproducible immunogold localization by immunoelectron microscopy with the antibody available.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Golgi complex has been recently identified as a Ca2+-enriched compartment whose total Ca2+ concentration is
>0.1 mM (Chandra et al., 1991; Pezzati et al., 1997
; Pinton
et al., 1998
), but the question of how Ca2+ is sequestered
in the Golgi has remained unanswered. Previously we
showed that CALNUC is the major Ca2+-binding protein
in Golgi fractions from rat liver detected by 45Ca2+ overlay
(Lin et al., 1998
). In this study we provide evidence that
CALNUC binds Ca2+ in the Golgi in vivo, because overexpression of CALNUC in the Golgi led to a two- to
threefold increase in Ca2+ storage based on Ca2+ equilibrium loading. This suggests that CALNUC is directly involved in maintenance of Ca2+ storage and thereby in
Ca2+ homeostasis in the Golgi. Equilibrium dialysis demonstrated the existence of only a single high affinity (Kd =
6.6 µM)/low capacity (~1 mol Ca2+/mol protein) binding
site on recombinant CALNUC. CALNUC's low Ca2+-binding capacity in the Golgi might be compensated for by
its abundance (3.8 µg/mg Golgi protein).
The demonstration of a single, high affinity Ca2+-binding site is in keeping with the fact that CALNUC possesses
two EF-hand motifs but only one, EF-1, has the structure
expected for high affinity calcium binding. EF-2 has an
Arg (R) instead of a Gly (G) at residue 6 of the EF-hand
loop region. Arg is supposed to disrupt the EF-hand motif
and abolish its Ca2+-binding capacity (Branden and
Tooze, 1991). CALNUC's EF-1 has the highest homology
to the COOH-terminal EF-4 of CaM which constitutes the
high affinity Ca2+-binding site of CaM (Crouch and Klee,
1980
). Moreover, the Ca2+-binding capability of CALNUC EF-1 was demonstrated previously by 45Ca2+ overlay
on truncated CALNUC. When EF-2 was deleted, Ca2+
binding was maintained, but when both EF-1 and EF-2
were deleted, Ca2+-binding capability was lost (Miura et
al., 1994
). In this study, we further demonstrated that truncated CALNUC with either the EF-1
helix (Asp227-
Leu239) or both EF-1 and EF-2 domains (Asp227-Phe291)
deleted lost Ca2+-binding capability completely. The majority of each of the CALNUC mutant proteins was still
targeted to the Golgi region as monitored via the GFP tag.
Collectively, these data suggest that EF-1 may constitute
the sole high affinity Ca2+-binding site on CALNUC.
Characterization of the Ca2+ pool in HeLa and CHO
cells overexpressing CALNUC provides several important
new pieces of information. 45Ca2+ sequestered in the Golgi
in cells overexpressing CALNUC was largely released by
Tg, an irreversible inhibitor of the SERCA Ca2+ pump,
providing in vivo evidence for the existence of SERCAs on Golgi membranes. SERCAs were also assumed to be
localized on Golgi membranes because it was shown previously that the p-type, Tg-sensitive SERCA Ca2+ pump was
essential for Ca2+ uptake into isolated Golgi fractions in
vitro (Taylor et al., 1997). Our results also suggest that the
increase in 45Ca2+ uptake in cells overexpressing CALNUC is not likely to be due to the presence of CALNUC
in the cytosol or another recently reported Tg- and IP3-insensitive Ca2+ pool (Pizzo et al., 1997
) since the majority of
the Ca2+ was released only after SERCA was inhibited.
Our finding that only a small amount of the Ca2+ remaining after Tg treatment was released by subsequent
ionomycin treatment might be due to incomplete depletion of Ca2+ from the Golgi by Tg, since the existence of a
Tg-insensitive/ionomycin-sensitive-plasma membrane calcium ATPase Ca2+ pump on Golgi membranes has also
been reported recently (Taylor et al., 1997). The fact that
monensin treatment which depletes Ca2+ from acidic compartments (secretory vesicles, granules, trans-Golgi network) (Fasolato et al., 1991
) did not release a significant amount of Ca2+ demonstrates that Ca2+ was not sequestered in an acidic compartment. Thus, our current results
from in vivo studies suggest that the Ca2+-binding protein
CALNUC together with SERCA Ca2+ pumps are responsible for the maintenance of the Golgi Ca2+ storage pool.
We also investigated the agonist sensitivity of the Golgi
Ca2+ pool. It was shown recently that the Golgi Ca2+ store
is sensitive to histamine, an agonist known to be coupled to IP3 generation (Pinton et al., 1998), suggesting that
there may be IP3R on Golgi membranes. Here we used extracellular ATP, another agonist known to generate IP3 after binding to plasma membrane nucleotide receptors
(P2y-purinoceptors) (O'Connor, 1992
), to investigate the sensitivity of the Golgi Ca2+ store to IP3. ATP challenge is
coupled to IP3 production via activation of PLC (Brown et
al., 1991
), and binding of IP3 to IP3R on the surface of
Ca2+ pool releases intracellular Ca2+ (Iredale and Hill,
1993
). When ATP was added to induced EcR-CHO-CALNUC cells, there was a rapid release of sequestered Ca2+
revealed by both Ca2+ imaging and 45Ca2+ which far exceeded that released from noninduced cells. Moreover, IP3
directly triggered 45Ca2+ mobilization from the Golgi in
permeabilized EcR-CHO-CALNUC cells. Thus, our biochemical results and those of Pinton et al. (1998)
using histamine as agonist suggest that the Golgi apparatus bears IP3R. The assumption that IP3R are expressed on the
Golgi is supported by our immunofluorescence observations suggesting a dual localization of IP3R-1 on both
ER and Golgi membranes. CHO cells were found previously to express ample IP3R-1 by immunoprecipitation
(Monkawa et al., 1995
) using mAb 18A10 (Furuichi et al.,
1989
) which specifically recognizes the COOH terminus of
IP3R-1.
A major controversy in the physiology of intracellular
Ca2+ stores concerns the mechanism by which their depletion triggers influx of Ca2+ through the plasma membrane.
In vertebrate cells, it has been assumed generally that the
relevant store is the ER (Randriamampita and Tsien,
1993; Parekh and Penner, 1997
). However, because both ER and Golgi accumulate Ca2+ via SERCAs and release
Ca2+ via IP3 receptors, both should undergo depletion
roughly in parallel, so one cannot yet exclude a role for the
Golgi in controlling plasma membrane Ca2+ influx. In
yeast, store-operated Ca2+ influx appears to be controlled
mainly at the Golgi, because genetic deletion of the Golgi
Ca2+ pump encoded by PMR1 increases the influx of extracellular Ca2+ (Halachmi and Eilam, 1996
). Therefore,
we tried to distinguish between ER and Golgi contributions by testing whether overexpression of CALNUC in
Xenopus oocytes affected the store-operated Ca2+ current,
Isoc (Yao and Tsien, 1997
). If the Golgi were important, increasing the quantity of Ca2+ buffer in its lumen should
diminish or delay Isoc (Mery et al., 1996
; Fasolato et al.,
1998
). Overexpression of CALNUC (via microinjection of
its mRNA) increased the 45Ca2+ content of oocytes analogously with Fig. 5 and appeared by fluorescence microscopy to be colocalized with the Golgi marker galactosyltransferase fused to GFP (Llopis et al., 1998
). However,
CALNUC overexpression did not significantly affect Isoc,
either partially activated by the membrane-permeant Ca2+
buffer TPEN (Arslan et al., 1985
; Hofer et al., 1998
) or
maximally activated by the ionophore ionomycin. This
negative result might seem to argue against a major role
for the Golgi in controlling Ca2+ influx into oocytes, but a
firm conclusion would require additional controls such as
immunoelectron microscopic localization of CALNUC and evidence that comparable increases in ER buffering
do affect Isoc.
Previously, we demonstrated significant homology between CALNUC and CRT and two conserved motifs,
AY(I/A)EE and QRLX(Q/E)E(I/E)E, located in the
C-domain of CRT (aa337-341 and 365-372) (Lin et al., 1998).
However, the homologous regions do not involve Ca2+-binding domains. CRT lacks EF-hand motifs but possesses
a high affinity/low capacity and a low affinity (Kd = 2 mM)/
high capacity (21 µmol Ca2+/µmol protein) Ca2+-binding
site (Baksh and Michalak, 1991
) constituted by clusters of
~35 Asp (D)/Glu (E) located in CRT's C-domain. In the
future it will be of interest to examine whether CALNUC
can function like CRT, its ER-resident counterpart (Lin et
al., 1998
), to maintain a high Ca2+ concentration required
for Golgi functions, e.g., sorting, lectin binding, budding,
and concentration of cargo into regulated secretory granules.
In summary, this study demonstrates that CALNUC, an abundant Golgi resident protein and the major Golgi Ca2+-binding protein, together with SERCA Ca2+ pumps and IP3R are involved in the maintenance of the Ca2+ storage pool in the Golgi. Further investigation of several remaining intriguing questions including whether the binding of Ca2+ to CALNUC regulates membrane traffic or posttranslational processing events in the Golgi, should shed light on the biological functions of CALNUC and on the Golgi Ca2+ pool.
![]() |
Footnotes |
---|
Address correspondence to Marilyn G. Farquhar, Ph.D., Division of Cellular and Molecular Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0651. Tel.: (619) 534-7711. Fax: (619) 534-8549. E-mail: mfarquhar{at}ucsd.edu
Received for publication 15 October 1998 and in revised form 17 March 1999.
We thank Dr. Larry Goldstein (Howard Hughes Medical Institute, UCSD) for use of his confocal microscope, and Dr. Ralf-Peter Czekay for assistance in the confocal analysis. We also thank Michele Wilhite and Tammie McQuistan (Immunoelectron Microscopy Core Facility) for their valuable assistance in the immunocytochemical studies, and Dennis Young (Flow Cytometry Core Facility, UCSD) for assistance in FACS® sorting.
This work was supported by National Institutes of Health grants DK17780 and CA58689 to M.G. Farquhar, and National Institutes of Health grant NS27177 and a Human Frontiers Science Program Grant to R.Y. Tsien.
![]() |
Abbreviations used in this paper |
---|
CaM, calmodulin;
CRT, calreticulin;
GFP, green fluorescent protein;
IP3R, inositol 1, 4, 5 trisphosphate receptor;
Man II, -mannosidase II;
PLC, phospholipase C;
SERCA, sarcoplasmic/ER calcium ATPase;
Tg, thapsigargin.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Arslan, P., F. Di Virgilio, M. Beltrame, R.Y. Tsien, and T. Pozzan. 1985. Cytosolic Ca2+ homeostasis in Ehrlich and Yoshida carcinomas. A new, membrane-permeant chelator of heavy metals reveals that these ascites tumor cell lines have normal cytosolic free Ca2+. J. Biol. Chem. 260: 2719-2727 [Abstract]. |
2. |
Baksh, S., and
M. Michalak.
1991.
Expression of calreticulin in Escherichia coli
and identification of its Ca2+ binding domains.
J. Biol. Chem.
266:
21458-21465
|
3. | Bastianutto, C., E. Clementi, F. Codazzi, P. Podini, F. De Giorgi, R. Rizzuto, J. Meldolesi, and T. Pozzan. 1995. Overexpression of calreticulin increases the Ca2+ capacity of rapidly exchanging Ca2+ stores and reveals aspects of their lumenal microenvironment and function. J. Cell Biol. 130: 847-855 [Abstract]. |
4. | Baumrucker, C.R., and T.W. Keenan. 1975. Membranes of mammary gland. X. Adenosine triphosphate dependent calcium accumulation by Golgi apparatus rich fractions from bovine mammary gland. Exp. Cell Res. 90: 253-260 |
5. | Berridge, M.J., J.P. Heslop, R.F. Irvine, and K.D. Brown. 1984. Inositol trisphosphate formation and calcium mobilization in Swiss 3T3 cells in response to platelet-derived growth factor. Biochem. J. 222: 195-201 |
6. | Branden, C., and J. Tooze. 1991. Motifs of protein structure. In Introduction to Protein Structure. C. Branden and J. Tooze, editors. Garland Publishing, New York. |
7. | Brown, H.A., E.R. Lazarowski, R.C. Boucher, and T.K. Harden. 1991. Evidence that UTP and ATP regulate phospholipase C through a common extracellular 5'-nucleotide receptor in human airway epithelial cells. Mol. Pharmacol. 40: 648-655 [Abstract]. |
8. | Brown, W.J., and M.G. Farquhar. 1987. The distribution of 215-kilodalton mannose 6-phosphate receptors within cis (heavy) and trans (light) Golgi subfractions varies in different cell types. Proc. Natl. Acad. Sci. USA. 84: 9001-9005 [Abstract]. |
9. | Chandra, S., E.P. Kable, G.H. Morrison, and W.W. Webb. 1991. Calcium sequestration in the Golgi apparatus of cultured mammalian cells revealed by laser scanning confocal microscopy and ion microscopy. J. Cell Sci. 100: 747-752 [Abstract]. |
10. | Crouch, T.H., and C.B. Klee. 1980. Positive cooperative binding of calcium to bovine brain calmodulin. Biochemistry. 19: 3692-3698 |
11. | Davidson, H.W., C.J. Rhodes, and J.C. Hutton. 1988. Intraorganellar calcium and pH control proinsulin cleavage in the pancreatic beta cell via two distinct site-specific endopeptidases. Nature. 333: 93-96 |
12. |
De Smedt, F.,
L. Missiaen,
J.B. Parys,
V. Vanweyenberg,
H. De Smedt, and
C. Erneux.
1997.
Isoprenylated human brain type I inositol 1,4,5-trisphosphate
5-phosphatase controls Ca2+ oscillations induced by ATP in Chinese hamster ovary cells.
J. Biol. Chem.
272:
17367-17375
|
13. | Duncan, J.S., and R.D. Burgoyne. 1996. Characterization of the effects of Ca2+ depletion on the synthesis, phosphorylation and secretion of caseins in lactating mammary epithelial cells. Biochem. J. 317: 487-493 |
14. | Dunphy, W.G., and J.E. Rothman. 1983. Compartmentation of asparagine-linked oligosaccharide processing in the Golgi apparatus. J. Cell Biol. 97: 270-275 [Abstract]. |
15. | Farquhar, M.G., and H.-P. Hauri. 1997. Protein sorting and vesicular traffic in the Golgi apparatus. In The Golgi Apparatus. E.G. Berger and J. Roth, editors. Birkhauser Verlag, Basel. 63-129. |
16. | Farquhar, M.G., and G.E. Palade. 1998. The Golgi apparatus: 100 years of progress and controversy. Trends Cell Biol. 8: 2-11 . |
17. |
Fasolato, C.,
M. Zottini,
E. Clementi,
D. Zacchetti,
J. Meldolesi, and
T. Pozzan.
1991.
Intracellular Ca2+ pools in PC12 cells. Three intracellular pools are
distinguished by their turnover and mechanisms of Ca2+ accumulation, storage, and release.
J. Biol. Chem.
266:
20159-20167
|
18. |
Fasolato, C.,
P. Pizzo, and
T. Pozzan.
1998.
Delayed activation of the store-operated calcium current induced by calreticulin overexpression in RBL-1
cells.
Mol. Biol. Cell.
9:
1513-1522
|
19. | Fries, E., and J.E. Rothman. 1980. Transport of vesicular stomatitis virus glycoprotein in a cell-free extract. Proc. Natl. Acad. Sci. USA. 77: 3870-3874 [Abstract]. |
20. | Furuichi, T., S. Yoshikawa, A. Miyawaki, K. Wada, N. Maeda, and K. Mikoshiba. 1989. Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400. Nature. 342: 32-38 |
21. |
Grohovaz, F.,
M. Bossi,
R. Pezzati,
J. Meldolesi, and
F.T. Tarelli.
1996.
High
resolution ultrastructural mapping of total calcium: electron spectroscopic
imaging/electron energy loss spectroscopy analysis of a physically/chemically
processed nerve-muscle preparation.
Proc. Natl. Acad. Sci. USA.
93:
4799-4803
|
22. | Halachmi, D., and Y. Eilam. 1996. Elevated cytosolic free Ca2+ concentrations and massive Ca2+ accumulation within vacuoles, in yeast mutant lacking PMR1, a homolog of Ca2+-ATPase. FEBS Lett. 392: 194-200 |
23. | Hodson, S.. 1978. The ATP-dependent concentration of calcium by a Golgi apparatus-rich fraction isolated from rat liver. J. Cell Sci. 30: 117-128 [Abstract]. |
24. |
Hofer, A.M.,
C. Fasolato, and
T. Pozzan.
1998.
Capacitative Ca2+ entry is
closely linked to the filling state of internal Ca2+ stores: a study using simultaneous measurements of ICRAC and intraluminal [Ca2+].
J. Cell Biol.
140:
325-334
|
25. | Iredale, P.A., and S.J. Hill. 1993. Increases in intracellular calcium via activation of an endogenous P2-purinoceptor in cultured CHO-K1 cells. Br. J. Pharmacol. 110: 1305-1310 [Abstract]. |
26. |
Ivessa, N.E.,
C. De Lemos-Chiarandini,
D. Gravotta,
D.D. Sabatini, and
G. Kreibich.
1995.
The Brefeldin A-induced retrograde transport from the
Golgi apparatus to the endoplasmic reticulum depends on calcium sequestered to intracellular stores.
J. Biol. Chem.
270:
25960-25967
|
27. |
Jin, M.,
L. Saucan,
M.G. Farquhar, and
G.E. Palade.
1996.
Rab1a and multiple
other Rab proteins are associated with the transcytotic pathway in rat liver.
J. Biol. Chem.
271:
30105-30113
|
28. | Kretsinger, R.H.. 1987. Calcium coordination and the calmodulin fold: divergent versus convergent evolution. Cold Spring Harbor Symp. Quant. Biol. 52: 499-510 |
29. |
Lin, P.,
H. Le-Niculescu,
R. Hofmeister,
J.M. McCaffery,
M. Jin,
H. Henneman,
T. McQuistan,
L. De Vries, and
M. Farquhar.
1998.
The mammalian
calcium-binding protein, nucleobindin (CALNUC), is a Golgi resident protein.
J. Cell Biol.
141:
1515-1527
|
30. |
Llopis, J.,
J.M. McCaffery,
A. Miyawaki,
M.G. Farquhar, and
R.Y. Tsien.
1998.
Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells
with green fluorescent proteins.
Proc. Natl. Acad. Sci. USA.
95:
6803-6808
|
31. | MacLennan, D.H., and P.T. Wong. 1971. Isolation of a calcium-sequestering protein from sarcoplasmic reticulum. Proc. Natl. Acad. Sci. USA. 68: 1231-1235 [Abstract]. |
32. | Meldolesi, J., and T. Pozzan. 1998. The endoplasmic reticulum Ca2+ store: a view from the lumen. Trends Biochem. Sci. 23: 10-14 |
33. |
Mery, L.,
N. Mesaeli,
M. Michalak,
M. Opas,
D.P. Lew, and
K.H. Krause.
1996.
Overexpression of calreticulin increases intracellular Ca2+ storage and decreases store-operated Ca2+ influx.
J. Biol. Chem.
271:
9332-9339
|
34. | Miura, K., K. Titani, Y. Kurosawa, and Y. Kanai. 1992. Molecular cloning of nucleobindin, a novel DNA-binding protein that contains both a signal peptide and a leucine zipper structure. Biochem. Biophys. Res. Commun. 187: 375-380 |
35. | Miura, K., Y. Kurosawa, and Y. Kanai. 1994. Calcium-binding activity of nucleobindin mediated by an EF hand moiety. Biochem. Biophys. Res. Commun. 199: 1388-1393 |
36. |
Monkawa, T.,
A. Miyawaki,
T. Sugiyama,
H. Yoneshima,
M. Yamamoto-Hino,
T. Furuichi,
T. Saruta,
M. Hasegawa, and
K. Mikoshiba.
1995.
Heterotetrameric complex formation of inositol 1,4,5-trisphosphate receptor subunits.
J. Biol. Chem.
270:
14700-14704
|
37. | Neville, M.C., F. Selker, K. Semple, and C. Watters. 1981. ATP-dependent calcium transport by a Golgi-enriched membrane fraction from mouse mammary gland. J. Membr. Biol. 61: 97-105 |
38. | O'Connor, S.E.. 1992. Recent developments in the classification and functional significance of receptors for ATP and UTP, evidence for nucleotide receptors. Life Sci. 50: 1657-1664 |
39. |
Parekh, A.B., and
R. Penner.
1997.
Store depletion and calcium influx.
Physiol.
Rev.
77:
901-930
|
40. | Pezzati, R., M. Bossi, P. Podini, J. Meldolesi, and F. Grohovaz. 1997. High-resolution calcium mapping of the endoplasmic reticulum-Golgi-exocytic membrane system. Electron energy loss imaging analysis of quick frozen-freeze dried PC12 cells. Mol. Biol. Cell. 8: 1501-1512 [Abstract]. |
41. |
Pinton, P.,
T. Pozzan, and
R. Rizzuto.
1998.
The Golgi apparatus is an inositol
1,4,5-trisphosphate-sensitive Ca2+ store, with functional properties distinct
from those of the endoplasmic reticulum.
EMBO (Eur. Mol. Biol. Organ.) J.
17:
5298-5308
|
42. |
Pizzo, P.,
C. Fasolato, and
T. Pozzan.
1997.
Dynamic properties of an inositol
1,4,5-trisphosphate- and thapsigargin-insensitive calcium pool in mammalian cell lines.
J. Cell Biol.
136:
355-366
|
43. | Randriamampita, C., and R.Y. Tsien. 1993. Emptying of intracellular Ca2+ stores releases a novel small messenger that stimulates Ca2+ influx. Nature. 364: 809-814 |
44. | Saucan, L., and G.E. Palade. 1994. Membrane and secretory proteins are transported from the Golgi complex to the sinusoidal plasmalemma of hepatocytes by distinct vesicular carriers. J. Cell Biol. 125: 733-741 [Abstract]. |
45. | Scherer, P.E., G.Z. Lederkremer, S. Williams, M. Fogliano, G. Baldini, and H.F. Lodish. 1996. Cab45, a novel Ca2+-binding protein localized to the Golgi lumen. J. Cell Biol. 133: 257-268 [Abstract]. |
46. |
Sorin, A.,
G. Rosas, and
R. Rao.
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
|
47. |
Taylor, R.S.,
S.M. Jones,
R.H. Dahl,
M.H. Nordeen, and
K.E. Howell.
1997.
Characterization of the Golgi complex cleared of proteins in transit and examination of calcium uptake activities.
Mol. Biol. Cell.
8:
1911-1931
|
48. | Thastrup, O., P.J. Cullen, B.K. Drobak, M.R. Hanley, and A.P. Dawson. 1990. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc. Natl. Acad. Sci. USA. 87: 2466-2470 [Abstract]. |
49. | Thielens, N.M., A. Van Dorsselaer, J. Gagnon, and G.J. Arlaud. 1990. Chemical and functional characterization of a fragment of C1-s containing the epidermal growth factor homology region. Biochemistry. 29: 3570-3578 |
50. | Tsien, R.W., and R.Y. Tsien. 1990. Calcium channels, stores, and oscillations. Annu. Rev. Cell Biol. 6: 715-760 . |
51. | Tsien, R.Y., and T. Pozzan. 1989. Measurement of cytosolic free Ca2+ with Quin2. Methods Enzymol. 172: 230-262 |
52. |
Velasco, A.,
L. Hendricks,
K.W. Moremen,
D.R. Tulsiani,
O. Touster, and
M.G. Farquhar.
1993.
Cell type-dependent variations in the subcellular distribution of ![]() |
53. | Virk, S.S., C.J. Kirk, and S.B. Shears. 1985. Ca2+ transport and Ca2+-dependent ATP hydrolysis by Golgi vesicles from lactating rat mammary glands. Biochem. J. 226: 741-748 |
54. |
Wendel, M.,
Y. Sommarin,
T. Bergman, and
D. Heinegard.
1995.
Isolation,
characterization, and primary structure of a calcium-binding 63-kD bone
protein.
J. Biol. Chem.
270:
6125-6133
|
55. |
Yao, Y., and
R.Y. Tsien.
1997.
Calcium current activated by depletion of calcium stores in Xenopus oocytes.
J. Gen. Physiol.
109:
703-715
|
56. |
Zha, X., and
G.H. Morrison.
1995.
Ion microscopy evidence that La3+ releases
Ca2+ from Golgi complex in LLC-PK1 cells.
Am. J. Physiol.
269:
C923-C928
|