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PubMed1994

Characterisation of a novel Ca2+ pump inhibitor (bis-phenol) and its effects on intracellular Ca2+ mobilization.

Bis-phenol, a phenolic antioxidant, is an inhibitor of sarcoplasmic reticulum (SR), endoplasmic reticulum (ER) and plasma membrane Ca2+ ATPases. The concentration of bis-phenol giving half-maximal inhibition of the SR Ca(2+)-ATPase is 2 microM. On binding to the SR Ca(2+)-ATPase it shifts the E2 to E1 transition towards the E2 state and slows the transition between E2 to E1. Bis-phenol completely inhibits Ca(2+)-dependent ATP hydrolysis and Ca2+ uptake by rat cerebellar microsomes at a concentration of 30 microM. The plasma membrane Ca(2+)-ATPase is also completely inhibited at similar concentrations, however, the Na+/K(+)-ATPase is only marginally affected. Other inhibitors of the ER Ca(2+)-ATPases, thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ), inhibit Ca2+ uptake by approximately 75%. Bis-phenol therefore inhibits all types of ER Ca(2+)-ATPases present in cerebellum. This inhibitor is also able to mobilize Ca2+ from intracellular Ca2+ stores, including those sensitive to InsP3, in intact HL-60 cells.

Adenosine Triphosphate↗
PubMed1995

Functional significance of the beta-subunit for heterodimeric P-type ATPases.

We have reviewed the structural and functional role of the beta-subunit in a subfamily of the P-ATPases known as the alpha/beta-heterodimeric, cation-exchange ATPases. The subfamily consists of the various isoforms of Na+/K(+)-ATPase and H+/K(+)-ATPase, both of which pump a cation out of the cell (Na+ or H+, respectively) in recycle exchange for K+. Much of the earlier work has emphasized the functional activities of the alpha-subunit, which shares many characteristics with the broader P-ATPase family. It is now clear that the glycosylated beta-subunit is an essential component of the cation-exchange ATPase subfamily. All beta-subunit isoforms have three highly conserved disulfide bonds within the extracellular domain that serve to stabilize the alpha-subunit, alpha/beta interaction and functional activity of the holoenzyme. Evidence strongly suggests that the beta-subunit is involved in the K(+)-dependent reactions of the enzymes, such as the E1-E2 transition and K+ occlusion, and that the extracellular domain of the beta-subunit plays an important role in determining the kinetics of K+ interaction. In most vertebrate cells, the unassociated alpha-subunit is restricted to the endoplasmic reticulum (ER), and assembly of the alpha/beta complex occurs within the ER. Signals for exiting the ER and directing the correct intracellular trafficking are primarily determined by the beta-subunit; Na+/K(+)-ATPase typically terminates in the plasma membrane facing the basolateral membrane, whereas all isoforms of H+/K(+)-ATPase terminate in the apical membrane. The C-terminal extracellular domain of the beta-subunit is important for proper interaction with the alpha-subunit and for correct intracellular trafficking. Oligosaccharides on the beta-subunit are not essential for enzyme function, but do serve to enhance the efficiency of alpha/beta association by increasing the lifetime of the unassociated beta-subunit and the stability of the alpha/beta complex to tryptic attack. We propose that highly specialized glycosylation on the beta-subunit of the gastric H+/K(+)-ATPase may help to protect that enzyme from the harsh extracellular environment of the stomach.

Adenosine Triphosphatases↗
PubMed2004

Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA).

The anti-apoptotic effect of Bcl-2 is well established, but the detailed mechanisms are unknown. In the present study, we show in vitro a direct interaction of Bcl-2 with the rat skeletal muscle SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), leading to destabilization and inactivation of the protein. Recombinant human Bcl-2D21, a truncated form of Bcl-2 with a deletion of 21 residues at the C-terminal membrane-anchoring region, was expressed and affinity-purified as a glutathione S-transferase fusion protein. Bcl-2D21 co-immunoprecipitated and specifically interacted with SERCA in an in vitro-binding assay. The original level of Bcl-2 in sarcoplasmic reticulum vesicles was very low, i.e. hardly detectable by immunoblotting with specific antibodies. The addition of Bcl-2D21 to the sarcoplasmic reticulum resulted in the inhibition of the Ca2+-ATPase activity dependent on the Bcl-2D21/SERCA molar ratio and incubation time. A complete inactivation of SERCA was observed after 2.5 h of incubation at approx. 2:1 molar ratio of Bcl-2D21 to SERCA. In contrast, Bcl-2D21 did not significantly change the activity of the plasma-membrane Ca2+-ATPase. The redox state of the single Cys158 residue in Bcl-2D21 and the presence of GSH did not affect SERCA inhibition. The interaction of Bcl-2D21 with SERCA resulted in a conformational transition of SERCA, assessed through a Bcl-2-dependent increase in SERCA thiols available for the labelling with a fluorescent reagent. This partial unfolding of SERCA did not lead to a higher sensitivity of SERCA towards oxidative inactivation. Our results suggest that the direct interaction of Bcl-2 with SERCA may be involved in the regulation of apoptotic processes in vivo through modulation of cytoplasmic and/or endoplasmic reticulum calcium levels required for the execution of apoptosis.

Animals↗
PubMed2002

Analysis of Ca(2+) uptake into the smooth endoplasmic reticulum of permeabilised sternal epithelial cells during the moulting cycle of the terrestrial isopod Porcellio scaber.

In terrestrial isopods, large amounts of Ca(2+) are transported across anterior sternal epithelial cells during moult-related deposition and resorption of CaCO(3) deposits. Because of its toxicity and function as a second messenger, resting cytosolic Ca(2+) levels must be maintained below critical concentrations during epithelial Ca(2+) transport, raising the possibility that organelles play a role during Ca(2+) transit. We therefore studied the uptake of Ca(2+) into Ca(2+)-sequestering organelles by monitoring the formation of birefringent calcium oxalate crystals in permeabilised anterior and posterior sternal epithelium cells of Porcellio scaber during Ca(2+)-transporting and non-transporting stages of the moulting cycle using polarised-light microscopy. The results indicate ATP-dependent uptake of Ca(2+) into organelles. Half-maximal crystal growth at a Ca(2+) activity, a(Ca), of 0.4 micromol l(-1) and blockade by cyclopiazonic acid suggest Ca(2+) uptake into the smooth endoplasmic reticulum by the smooth endoplasmic reticulum Ca(2+)-ATPase. Analytical electron microscopical techniques support this interpretation by revealing the accumulation of Ca(2+)-containing crystals in smooth membranous intracellular compartments. A comparison of different moulting stages demonstrated a virtual lack of crystal formation in the early premoult stage and a significant fivefold increase between mid premoult and the Ca(2+)-transporting stages of late premoult and intramoult. These results suggest a contribution of the smooth endoplasmic reticulum as a transient Ca(2+) store during intracellular Ca(2+) transit.

Animals↗
PubMed2002

Release of mitochondrial Ca2+ via the permeability transition activates endoplasmic reticulum Ca2+ uptake.

Regulatory interactions between the endoplasmic reticulum (ER) and the mitochondria in the control of intracellular free Ca2+ concentration ([Ca2+]I), may be of importance in the control of many cell functions, and particularly those involved in initiating cell death. We used targeted Ca2+ sensors (cameleons) to investigate the movement of Ca2+ between the ER and mitochondria of intact cells and focused on the role of the mitochondrial permeability transition (MPT) in this interaction. We hypothesized that release of Ca2+ from mitochondria in response to a known MPT agonist (atractyloside) would cause release of ER Ca2+, perpetuating cellular Ca2+ overload, and cell death. Targeted cameleons (mitochondria and ER) were imaged with confocal microscopy 2-3 days following transient transfection of human embryonic kidney 293 cells. Opening of the MPT resulted in specific loss of mitochondrial Ca2+ (blocked by cyclosporin A), which was sequestered initially by ER. The ER subsequently released this Ca2+ load, leading to a global Ca2+ elevation, a response that was not observed when ER Ca2+-ATPases were blocked with cyclopiazonic acid. Thus, ER plays an important role in moderating changes in intracellular Ca2+ following MPT and may play a key role in cell death initiated by mitochondrial mechanisms.

Atractyloside↗
PubMed1998

Evidence for mitochondrial Ca(2+)-induced Ca2+ release in permeabilised endothelial cells.

Generally most intracellular Ca2+ is stored in the endoplasmic reticulum (ER) and mitochondria. Recently a mitochondrial Ca(2+)-induced Ca2+ release (mCICR) mechanism, unconnected with ryanodine receptors (RyR's), has been shown in tumour cells. The existence of a mitochondrial Ca2+ release mechanism in BAE cells was investigated using saponin-permeabilised BAE cells. When buffered intracellular solution were 'stepped' from 10 nM to 10 microM free Ca2+, the mitochondrial inhibitors CN (2 mM), FCCP (1 microM), and RR (20 microM) significantly reduced total CICR by approximately 25%. The ER Ca(2+)-ATPase inhibitor thapsigargin (100 nM) had no effect. Furthermore, cyclosporin A (200 nM), an inhibitor of the mitochondrial permeability transition pore (PTP), abolished total CICR. Therefore, the novel ryanodine-caffeine insensitive CICR mechanism previously reported in BAE cells involves mitochondrial Ca2 release. It is proposed that in BAE cells, mCICR occurs via the mitochondrial PTP and may be physiologically important in endothelial cell Ca2+ signalling.

Animals↗
PubMed2000

Role of p97 and syntaxin 5 in the assembly of transitional endoplasmic reticulum.

Transitional endoplasmic reticulum (tER) consists of confluent rough and smooth endoplasmic reticulum (ER) domains. In a cell-free incubation system, low-density microsomes (1.17 g cc(-1)) isolated from rat liver homogenates reconstitute tER by Mg(2+)GTP- and Mg(2+)ATP-hydrolysis-dependent membrane fusion. The ATPases associated with different cellular activities protein p97 has been identified as the relevant ATPase. The ATP depletion by hexokinase or treatment with either N-ethylmaleimide or anti-p97 prevented assembly of the smooth ER domain of tER. High-salt washing of low-density microsomes inhibited assembly of the smooth ER domain of tER, whereas the readdition of purified p97 with associated p47 promoted reconstitution. The t-SNARE syntaxin 5 was observed within the smooth ER domain of tER, and antisyntaxin 5 abrogated formation of this same membrane compartment. Thus, p97 and syntaxin 5 regulate assembly of the smooth ER domain of tER and hence one of the earliest membrane differentiated components of the secretory pathway.

Adenosine Triphosphatases↗
PubMed2007

Matrix regulation of skeletal cell apoptosis III: mechanism of ion pair-induced apoptosis.

Our previous work has demonstrated that while the Ca(2+) and Pi ions acting in concert function as a potent osteoblast apoptogen, the underlying mechanisms by which it activates cell death is not known. We hypothesize that the ion pair causes release of Ca(2+) from intracellular stores ([Ca(2+)]i); the increase in intracellular calcium prompts the mitochondria to uptake more calcium. This accumulation of calcium eventually results in the loss of mitochondrial membrane potential (MMP) and, subsequently, apoptosis. To test this hypothesis, we evaluated apoptosome formation in MC3T3-E1 osteoblast-like cells treated with the ion pair. Western blot analysis indicated migration of cytochrome-c and Smac/DIABLO from mitochondria to the cytoplasm. Inhibition of either the electron transfer chain (with antimycin a and rotenone), or the activation of a MMP transition (with bongkrekic acid) inhibited apoptosis in a dose-dependent manner. Pre-treating osteoblasts with ruthenium red, a Ca(2+) uniporter inhibitor of both mitochondria and the endoplasmic reticulum (ER), also completely abolished Ca(2+.)Pi-induced apoptosis. Moreover, we showed that an increase in [Ca(2+)]i preceded the increase in MMP over the first 45 min of treatment; a mitochondrial membrane permeability transition was evident at 75 min. To determine the role of ER, Ca(2+) stores in the generation of the apoptotic signal by the ion pair, cells were treated with several inhibitors. Apoptosis was inhibited when cells were treated with dantrolene, an inhibitor of ER ryanodine receptors, and 2-aminodiphenylborate, an IP3 Ca(2+) channel inhibitor, but not cyclopiazonic acid, an ER Ca(2)-ATPase inhibitor. Together, these data demonstrate that Ca(2+) Pi-induced osteoblast apoptosis is characterized by the generation of an apoptosome and that Ca(2+) release from ER stores may promote ion pair-dependent cell death.

3T3 Cells↗
PubMed1989

Membrane protein sorting: biosynthesis, transport and processing of yeast vacuolar alkaline phosphatase.

The Saccharomyces cerevisiae PHO8 gene product, repressible alkaline phosphatase (ALP), is a glycoprotein enzyme that is localized to the yeast vacuole (lysosome). Using antibodies raised against synthetic peptides corresponding to two distinct hydrophilic sequences in ALP, we have been able to examine the biosynthesis, sorting and processing of this protein. ALP is synthesized as an inactive precursor containing a C-terminal propeptide that is cleaved from the protein in a PEP4-dependent manner. The precursor and mature protein are anchored in the membrane by an N-terminal hydrophobic domain that also appears to function as an uncleaved internal signal sequence. ALP has the topology of a type-II integral membrane protein containing a short basic N-terminal cytoplasmic tail that is accessible to exogenous protease when associated both with the endoplasmic reticulum and the vacuole. Similar to the soluble vacuolar hydrolases carboxypeptidase Y (CPY) and proteinase A (PrA), ALP transits through the early stages of the secretory pathway prior to vacuolar delivery. Two observations indicate, however, that ALP is localized to the vacuole by a mechanism which is in part different from that used by CPY and PrA: (i) maturation of proALP, which is indicative of vacuolar delivery, is less sensitive than CPY and PrA to the defects exhibited by certain of the vacuolar protein sorting (vps) mutants; and (ii) maturation of proALP proceeds normally in the presence of a potent vacuolar ATPase inhibitor, bafilomycin A1, which is known to block vacuole acidification and leads to the mis-sorting and secretion of precursor forms of CPY and PrA. These results indicate that ALP will be a useful model protein for studies of membrane protein sorting in yeast.

Alkaline Phosphatase↗
PubMed2001

Functional coupling between the caffeine/ryanodine-sensitive Ca2+ store and mitochondria in rat aortic smooth muscle cells.

We investigated the role of mitochondria in the agonist-induced and/or caffeine-induced Ca2+ transients in rat aortic smooth muscle cells. We explored the possibility that proliferation modulates the coupling between mitochondria and endoplasmic reticulum. Ca2+ transients induced by either ATP or caffeine were measured in presence or absence of drugs interfering with mitochondrial activity in freshly dissociated cells (day 1) and in subconfluent primary culture (day 12). We found that the mitochondrial inhibitors, rotenone or carbonyl cyanide m-chlorophenylhydrazone, as well as the permeability transition pore inhibitor, cyclosporin A, had no effect on the ATP-induced Ca2+ transient at either day 1 or day 12, but prevented caffeine-induced cytosolic Ca2+ increase at day 12 but not at day 1. Close connections between ryanodine receptors and mitochondria were observed at both day 1 and 12. Thapsigargin (TG) prevented ATP- and caffeine-induced Ca2+ transients at day 1. At day 12, where only 50% of the cells were sensitive to caffeine, TG did not prevent the caffeine-induced Ca2+ transient, and prevented ATP-induced Ca2+ transient in only half of the cells. Together, these data demonstrate that rat aortic smooth muscle cells at day 1 have an ATP- and caffeine-sensitive pool, which is functionally independent but physically closely linked to mitochondria and totally inhibited by TG. At day 12, we propose the existence of two cell populations: half contains IP3 receptors and TG-sensitive Ca2+ pumps only; the other half contains, in addition to the IP3-sensitive pool independent from mitochondria, a caffeine-sensitive pool. This latter pool is linked to mitochondria through the permeability transition pore and is refilled by both TG-sensitive and insensitive mechanisms.

Adenosine Triphosphate↗
PubMed1997

Immunoglobulin binding protein (BiP) function is required to protect cells from endoplasmic reticulum stress but is not required for the secretion of selective proteins.

BiP/GRP78 is a lumenal stress protein of the endoplasmic reticulum (ER) that interacts with polypeptide folding intermediates transiting the secretory compartment. We have studied the secretion and the stress response in Chinese hamster ovary (CHO) cells that overexpress either wild-type immunoglobulin binding protein (BiP) or a BiP deletion molecule (residues 175-201) that can bind peptides and ATP but is defective in ATP hydrolysis and concomitant peptide release. Overexpressed wild-type BiP was localized to the ER and unique vesicles within the nucleus, whereas overexpressed ATPase-defective BiP was localized to the ER and cytoplasmic vesicles but was absent from the nucleus. Compared with wild-type CHO cells, overexpression of ATPase-defective BiP prevented secretion of factor VIII, a coagulation factor that extensively binds BiP in the lumen of the ER. Under these conditions factor VIII was stably associated with the ATPase-defective BiP. In contrast, the secretion of monocyte/macrophage colony stimulating factor, a protein that is not detected in association with BiP, was not affected by overexpression of ATPase-defective BiP. These results show that BiP function is not required for secretion of some proteins and suggest that some proteins do not interact with BiP upon transport through the ER. The presence of unfolded protein in the ER induces transcription of BiP and also elicits a general inhibition of protein synthesis. Overexpression of wild-type BiP prevented the stress-mediated transcriptional induction of BiP in response to either calcium ionophore A23187 treatment or tunicamycin treatment. In contrast, overexpression of ATPase-defective BiP did not prevent the stress induction of BiP, showing that the ATPase activity is required to inhibit transcriptional induction. Overexpression of wild-type BiP, but not ATPase-defective BiP, increased survival of cells treated with A23187. The increased survival mediated by overexpressed wild-type BiP correlated with reduced translation inhibition in response to the stress condition. These results indicate that overexpressed BiP alleviated the stress in the ER to prevent BiP transcriptional induction and permit continued translation of cellular mRNAs.

Adenosine Triphosphatases↗
PubMed2005

Involvement of the actin cytoskeleton and homotypic membrane fusion in ER dynamics in Caenorhabditis elegans.

The endoplasmic reticulum (ER) is the major intracellular membrane system. The ER is essential for protein and lipid biosynthesis, transport of proteins along the secretory pathway, and calcium storage. Here, we describe our investigations into the dynamics and regulation of the ER in the early Caenorhabditis elegans embryo. Using a GFP fusion to the ER-resident signal peptidase SP12, we observed the morphological transitions of the ER through fertilization and the early cell-cycles in living embryos. These transitions were tightly coordinated with the division cycle: upon onset of mitosis, the ER formed structured sheets that redispersed at the initiation of cleavage. Although microtubules were not required for the transition of the ER between these different states, the actin cytoskeleton facilitated the dispersal of the ER at the end of mitosis. The ER had an asymmetric distribution in the early embryo, which was dependent on the establishment of polarity by the PAR proteins. The small GTPase ARF-1 played an essential role in the ER dynamics, although this function appeared to be unrelated to the role of ARF-1 in vesicular traffic. In addition, the ER-resident heat shock protein BiP and a homologue of the AAA ATPase Cdc48/p97 were found to be crucial for the ER transitions. Both proteins have been implicated in homotypic ER membrane fusion. We provide evidence that homotypic membrane fusion is required to form the sheet structure in the early embryo.

ADP-Ribosylation Factor 1↗
PubMed2005

Conformational changes of p97 during nucleotide hydrolysis determined by small-angle X-Ray scattering.

Valosin-containing protein (VCP)/p97 is an AAA family ATPase that has been implicated in the removal of misfolded proteins from the endoplasmic reticulum and in membrane fusion. p97 forms a homohexamer whose protomers consist of an N-terminal (N) domain responsible for binding to effector proteins, followed by two AAA ATPase domains, D1 and D2. Small-angle X-ray scattering (SAXS) measurements of p97 in the presence of AMP-PNP (ATP state), ADP-AlF(x) (hydrolysis transition state), ADP, or no nucleotide reveal major changes in the positions of the N domains with respect to the hexameric ring during the ATP hydrolysis cycle. Nucleotide binding and hydrolysis experiments indicate that D2 inhibits nucleotide exchange by D1. The data suggest that the conversion of the chemical energy of ATP hydrolysis into mechanical work on substrates involves transmission of conformational changes generated by D2 through D1 to move N.

Adenosine Triphosphatases↗
PubMed2003

Dependence of endoplasmic reticulum-associated degradation on the peptide binding domain and concentration of BiP.

ER-associated degradation (ERAD) removes defective and mis-folded proteins from the eukaryotic secretory pathway, but mutations in the ER lumenal Hsp70, BiP/Kar2p, compromise ERAD efficiency in yeast. Because attenuation of ERAD activates the UPR, we screened for kar2 mutants in which the unfolded protein response (UPR) was induced in order to better define how BiP facilitates ERAD. Among the kar2 mutants isolated we identified the ERAD-specific kar2-1 allele (Brodsky et al. J. Biol. Chem. 274, 3453-3460). The kar2-1 mutation resides in the peptide-binding domain of BiP and decreases BiP's affinity for a peptide substrate. Peptide-stimulated ATPase activity was also reduced, suggesting that the interdomain coupling in Kar2-1p is partially compromised. In contrast, Hsp40 cochaperone-activation of Kar2-1p's ATPase activity was unaffected. Consistent with UPR induction in kar2-1 yeast, an ERAD substrate aggregated in microsomes prepared from this strain but not from wild-type yeast. Overexpression of wild-type BiP increased substrate solubility in microsomes obtained from the mutant, but the ERAD defect was exacerbated, suggesting that simply retaining ERAD substrates in a soluble, retro-translocation-competent conformation is insufficient to support polypeptide transit to the cytoplasm.

Animals↗
PubMed2006

Involvement of endoplasmic reticulum in hepatitis B virus replication.

The mitochondrial calcium and downstream proline-rich tyrosine kinase-2 (PyK2) signaling pathway are critical to hepatitis B virus (HBV) replication, and the endoplasmic reticulum (ER) plays an important role in intracellular calcium regulation. To investigate the role of ER in HBV replication, the HBV genome transfected HepG2.2.15 cells were treated by cyclosporine A (CsA), cyclopiazonic acid (CPA), ryanodine and U73122, which are all specific blockers of calcium channels located in either ER or mitochondria. The HBV replication level was evaluated by two methods: slot blot hybridization analysis of intracellular HBV DNA and real-time polymerase chain reaction (PCR) analysis of secreted HBV DNA in supernatant; the activation of PyK2 kinase was detected by Western blot analysis. Results indicated that the HBV replication was inhibited when mitochondrial permeability transition pore, ER Ca2+ -ATPase and ER inositol 1,4,5-trisphosphate receptor (IP3R) were blocked by CsA, CPA and U73122, respectively; but not inhibited when ER ryanodine receptor was blocked by ryanodine. The PyK2 phosphorylation level declined after treatment of 2 microg/ml CsA, 5 microM CPA and 25 microM U73122, but not changed apparently after 50 microM ryanodine treatment. Compared with monotreatment, a more powerful inhibitory effect was achieved when the CsA, CPA and U73122 were combined used in twosome or triple manner, while the HBV replication level did not change apparently when ryanodine combined with CsA, CPA or U73122. In conclusion, besides the mitochondria, the ER also participates in the HBV replication through calcium-PyK2 signaling pathway; the calcium channels of ER Ca2+ -ATPase and ER IP3R are responsible for this role; during this complicated process, an interaction between ER and mitochondria maybe involved.

Calcium↗
PubMed1999

Depletion of divalent cations within the secretory pathway inhibits the terminal glycosylation of complex carbohydrates of thyroglobulin.

Newly synthesized thyroglobulin transiting the secretory pathway is posttranslationally modified by addition of oligosaccharides to asparagine N-linked residues. The effect of divalent cation depletion on oligosaccharide processing of Tg was studied in FRTL-5 cells. Treatment with an ionophore, A23187, or thapsigargin, an inhibitor of the sarcoplasmic/endoplasmic reticulum ATPases delayed Tg secretion. These effects were accompanied by a normal distribution of the marker of the endoplasmic reticulum protein disulfide isomerase. Analysis of the thyroglobulin oligosaccharides by Bio-gel P4 chromatography showed that in the presence of A23187 and thapsigargin the addition of peripheral sialic acid and possibly galactose is inhibited. These findings were strengthened by experiments of exoglycosidase digestion and SDS-PAGE analysis of the resulting products. These results reveal a cellular mechanism of production of thyroglobulin with incompletely processed complex chains, i.e., the ligand of the recently described GlcNAc and asialoglycoprotein receptors of the thyroid. Since A23187 and thapsigargin inhibit biosynthetically the addition of peripheral sugars on N-linked oligosaccharides chains, the thyroglobulin molecules secreted in the presence of A23187 and thapsigargin should greatly facilitate studies on the function of the GlcNAc and asialoglycoprotein receptors of the thyroid.

Animals↗
PubMed1997

Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin-ankyrin G119 skeleton in Madin Darby canine kidney cells.

Spectrin (betaISigma*) and ankyrin (AnkG119) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of betaI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of alpha- and beta-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of beta-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as AnkG119, Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular-tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin-ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins.

Animals↗
PubMed2007

Dual effect of thapsigargin on cell death in porcine aortic smooth muscle cells.

A sustained increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) can cause cell death. In this study, we found that, in cultured porcine aortic smooth muscle cells, endoplasmic reticulum (ER) stress, triggered by depletion of Ca(2+) stores by thapsigargin (TG), induced an increase in the [Ca(2+)](i) and cell death. However, the TG-induced death was not related to the [Ca(2+)](i) increase but was mediated by targeting of activated Bax to mitochondria and the opening of mitochondrial permeability transition pores (PTPs). Once the mitochondrial PTPs had opened, several events, including collapse of the mitochondrial membrane potential, cytochrome c release, and caspase-3 activation, occurred and the cells died. TG-induced cell death was completely inhibited by the pan-caspase inhibitor Z-VAD-fmk and was enhanced by the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), suggesting the existence of a Ca(2+)-dependent anti-apoptotic mechanism. After TG treatment, Ca(2+)-sensitive mitogen-activated protein kinase (MAPK) activation was induced and acted as a downstream effector of phosphatidylinositol 3-kinase (PI 3-kinase). The protective effect of Z-VAD-fmk on TG-induced cell death was reversed by BAPTA, PD-098059 (an MAPK kinase inhibitor), or LY-294002 (a PI 3-kinase inhibitor). Taken together, our data indicate that ER stress simultaneously activate two pathways, the mitochondrial caspase-dependent death cascade and the Ca(2+)-dependent PI 3-kinase/MAPK anti-apoptotic machinery. The Bax activation and translocation, but not the [Ca(2+)](i) increase, may activate mitochondrial PTPs, which, in turn, causes activation of caspases and cell death, whereas Ca(2+)-dependent MAPK activation counteracts death signaling; removal of Ca(2+) activated a second caspase-independent death pathway.

Animals↗