A homolog of voltage-gated Ca(2+) channels stimulated by depletion of secretory Ca(2+) in yeast

In animal cells, capacitative calcium entry (CCE) mechanisms become activated specifically in response to depletion of calcium ions (Ca(2+)) from secretory organelles. CCE serves to replenish those organelles and to enhance signaling pathways that respond to elevated free Ca(2+) concentrations in the cytoplasm. The mechanism of CCE regulation is not understood because few of its essential components have been identified. We show here for the first time that the budding yeast Saccharomyces cerevisiae employs a CCE-like mechanism to refill Ca(2+) stores within the secretory pathway. Mutants lacking Pmr1p, a conserved Ca(2+) pump in the secretory pathway, exhibit higher rates of Ca(2+) influx relative to wild-type cells due to the stimulation of a high-affinity Ca(2+) uptake system. Stimulation of this Ca(2+) uptake system was blocked in pmr1 mutants by expression of mammalian SERCA pumps. The high-affinity Ca(2+) uptake system was also stimulated in wild-type cells overexpressing vacuolar Ca(2+) transporters that competed with Pmr1p for substrate. A screen for yeast mutants specifically defective in the high-affinity Ca(2+) uptake system revealed two genes, CCH1 and MID1, previously implicated in Ca(2+) influx in response to mating pheromones. Cch1p and Mid1p were localized to the plasma membrane, coimmunoprecipitated from solubilized membranes, and shown to function together within a single pathway that ensures that adequate levels of Ca(2+) are supplied to Pmr1p to sustain secretion and growth. Expression of Cch1p and Mid1p was not affected in pmr1 mutants. The evidence supports the hypothesis that yeast maintains a homeostatic mechanism related to CCE in mammalian cells. The homology between Cch1p and the catalytic subunit of voltage-gated Ca(2+) channels raises the possibility that in some circumstances CCE in animal cells may involve homologs of Cch1p and a conserved regulatory mechanism.     

Mitochondria recycle Ca(2+) to the endoplasmic reticulum and prevent the depletion of neighboring endoplasmic reticulum regions

To study Ca(2+) fluxes between mitochondria and the endoplasmic reticulum (ER), we used "cameleon" indicators targeted to the cytosol, the ER lumen, and the mitochondrial matrix. High affinity mitochondrial probes saturated in approximately 20% of mitochondria during histamine stimulation of HeLa cells, whereas a low affinity probe reported averaged peak values of 106 +/- 5 microm, indicating that Ca(2+) transients reach high levels in a fraction of mitochondria. In concurrent ER measurements, [Ca(2+)](ER) averaged 371 +/- 21 microm at rest and decreased to 133 +/- 14 microm and 59 +/- 5 microm upon stimulation with histamine and thapsigargin, respectively, indicating that substantial ER refilling occur during agonist stimulation. A larger ER depletion was observed when mitochondrial Ca(2+) uptake was prevented by oligomycin and rotenone or when Ca(2+) efflux from mitochondria was blocked by CGP 37157, indicating that some of the Ca(2+) taken up by mitochondria is re-used for ER refilling. Accordingly, ER regions close to mitochondria released less Ca(2+) than ER regions lacking mitochondria. The ER heterogeneity was abolished by thapsigargin, oligomycin/rotenone, or CGP 37157, indicating that mitochondrial Ca(2+) uptake locally modulate ER refilling. These observations indicate that some mitochondria are very close to the sites of Ca(2+) release and recycle a substantial portion of the captured Ca(2+) back to vicinal ER domains. The distance between the two organelles thus determines both the amplitude of mitochondrial Ca(2+) signals and the filling state of neighboring ER regions.     

Calreticulin modulates capacitative Ca2+ influx by controlling the extent of inositol 1,4,5-trisphosphate-induced Ca2+ store depletion

Calreticulin (CRT) is a highly conserved Ca(2+)-binding protein that resides in the lumen of the endoplasmic reticulum (ER). We overexpressed CRT in Xenopus oocytes to determine how it could modulate inositol 1,4,5-trisphosphate (InsP(3))-induced Ca(2+) influx. Under conditions where it did not affect the spatially complex elevations in free cytosolic Ca(2+) concentration ([Ca(2+)](i)) due to InsP(3)-induced Ca(2+) release, overexpressed CRT decreased by 46% the Ca(2+)-gated Cl(-) current due to Ca(2+) influx. Deletion mutants revealed that CRT requires its high capacity Ca(2+)-binding domain to reduce the elevations of [Ca(2+)](i) due to Ca(2+) influx. This functional domain was also required for CRT to attenuate the InsP(3)-induced decline in the free Ca(2+) concentration within the ER lumen ([Ca(2+)](ER)), as monitored with a "chameleon" indicator. Our data suggest that by buffering [Ca(2+)](ER) near resting levels, CRT may prevent InsP(3) from depleting the intracellular stores sufficiently to activate Ca(2+) influx.     

Capturing ER calcium dynamics

The lumen of the endoplasmic reticulum (ER) contributes to the dynamics of Ca(2+) signaling by acting as a source or sink of signal Ca(2+). Despite its relevance for the understanding of the cell biology and pathophysiology of the luminal calcium store, the direct measurement of luminal Ca(2+) release and uptake is still critical when Ca(2+) homeostasis is analyzed in neural cells. For the analysis of Ca(2+)-dependent signaling, synthetic Ca(2+) indicators have become popular. The properties of these indicators allow only limited targeting to subcellular structures such as the ER. Recently, we introduced a new strategy for the targeting of synthetic Ca(2+) indicators to the lumen of the ER. The method, termed Targeted-Esterase-induced Dye loading (TED) is based on the targeted recombinant expression of a high carboxylesterase (CES) activity in the lumen of the ER, which is needed to trap synthetic indicators. The method combines the selectivity of protein targeting with the biochemical advantages of low-affinity synthetic Ca(2+) indicators. TED permits direct and non-disruptive measurement and imaging of Ca(2+)-store dynamics. Here, we summarize major topics in the cell biology of ER Ca(2+) signaling and discuss the perspectives of the TED method for the morphological and physiological analysis of temporal and spatial Ca(2+)-dynamics in neural cells.     

Ca2+-calmodulin inhibits tail-anchored protein insertion into the mammalian endoplasmic reticulum membrane

Cytosolic components and pathways have been identified that are involved in inserting tail-anchored (TA) membrane proteins into the yeast or mammalian endoplasmic reticulum (ER) membrane. Searching for regulatory mechanisms of TA protein biogenesis, we found that Ca(2+)-calmodulin (CaM) inhibits the insertion of TA proteins into mammalian ER membranes and that this inhibition is prevented by trifluoperazine, a CaM antagonist that interferes with substrate binding of Ca(2+)-CaM. The effects of Ca(2+)-CaM on cytochrome b(5) and Synaptobrevin 2 suggest a direct interaction between Ca(2+)-CaM and TA proteins. Thus, CaM appears to regulate TA insertion into the ER membrane in a Ca(2+) dependent manner.     

Ca(2+) dynamics in the lumen of the endoplasmic reticulum in sensory neurons: direct visualization of Ca(2+)-induced Ca(2+) release triggered by physiological Ca(2+) entry

In cultured rat dorsal root ganglia neurons, we measured membrane currents, using the patch-clamp whole-cell technique, and the concentrations of free Ca(2+) in the cytosol ([Ca(2+)](i)) and in the lumen of the endoplasmic reticulum (ER) ([Ca(2+)](L)), using high- (Fluo-3) and low- (Mag-Fura-2) affinity Ca(2+)-sensitive fluorescent probes and video imaging. Resting [Ca(2+)](L) concentration varied between 60 and 270 microM. Activation of ryanodine receptors by caffeine triggered a rapid fall in [Ca(2+)](L) levels, which amounted to only 40--50% of the resting [Ca(2+)](L) value. Using electrophysiological depolarization, we directly demonstrate the process of Ca(2+)-induced Ca(2+) release triggered by Ca(2+) entry through voltage-gated Ca(2+) channels. The amplitude of Ca(2+) release from the ER lumen was linearly dependent on I(Ca).     

Involvement of the penta-EF-hand protein Pef1p in the Ca2+-dependent regulation of COPII subunit assembly in Saccharomyces cerevisiae

Although it is well established that the coat protein complex II (COPII) mediates the transport of proteins and lipids from the endoplasmic reticulum (ER) to the Golgi apparatus, the regulation of the vesicular transport event and the mechanisms that act to counterbalance the vesicle flow between the ER and Golgi are poorly understood. In this study, we present data indicating that the penta-EF-hand Ca(2+)-binding protein Pef1p directly interacts with the COPII coat subunit Sec31p and regulates COPII assembly in Saccharomyces cerevisiae. ALG-2, a mammalian homolog of Pef1p, has been shown to interact with Sec31A in a Ca(2+)-dependent manner and to have a role in stabilizing the association of the Sec13/31 complex with the membrane. However, Pef1p displayed reversed Ca(2+) dependence for Sec13/31p association; only the Ca(2+)-free form of Pef1p bound to the Sec13/31p complex. In addition, the influence on COPII coat assembly also appeared to be reversed; Pef1p binding acted as a kinetic inhibitor to delay Sec13/31p recruitment. Our results provide further evidence for a linkage between Ca(2+)-dependent signaling and ER-to-Golgi trafficking, but its mechanism of action in yeast seems to be different from the mechanism reported for its mammalian homolog ALG-2.     

Calreticulin differentially modulates calcium uptake and release in the endoplasmic reticulum and mitochondria

To study the role of calreticulin in Ca(2+) homeostasis and apoptosis, we generated cells inducible for full-length or truncated calreticulin and measured Ca(2+) signals within the cytosol, the endoplasmic reticulum (ER), and mitochondria with "cameleon" indicators. Induction of calreticulin increased the free Ca(2+) concentration within the ER lumen, [Ca(2+)](ER), from 306 +/- 31 to 595 +/- 53 microm, and doubled the rate of ER refilling. [Ca(2+)](ER) remained elevated in the presence of thapsigargin, an inhibitor of SERCA-type Ca(2+) ATPases. Under these conditions, store-operated Ca(2+) influx appeared inhibited but could be reactivated by decreasing [Ca(2+)](ER) with the low affinity Ca(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. In contrast, [Ca(2+)](ER) decreased much faster during stimulation with carbachol. The larger ER release was associated with a larger cytosolic Ca(2+) response and, surprisingly, with a shorter mitochondrial Ca(2+) response. The reduced mitochondrial signal was not associated with visible morphological alterations of mitochondria or with disruption of the contacts between mitochondria and the ER but correlated with a reduced mitochondrial membrane potential. Altered ER and mitochondrial Ca(2+) responses were also observed in cells expressing an N-truncated calreticulin but not in cells overexpressing calnexin, a P-domain containing chaperone, indicating that the effects were mediated by the unique C-domain of calreticulin. In conclusion, calreticulin overexpression increases Ca(2+) fluxes across the ER but decreases mitochondrial Ca(2+) and membrane potential. The increased Ca(2+) turnover between the two organelles might damage mitochondria, accounting for the increased susceptibility of cells expressing high levels of calreticulin to apoptotic stimuli.     

Measurements of the free luminal ER Ca(2+) concentration with targeted "cameleon" fluorescent proteins

The free ER Ca(2+) concentration, [Ca(2+)](ER), is a key parameter that determines both the spatio-temporal pattern of Ca(2+) signals as well as the activity of ER-resident enzymes. Obtaining accurate, time-resolved measurements of the Ca(2+) activity within the ER is thus critical for our understanding of cell signaling. Such measurements, however, are particularly challenging given the highly dynamic nature of Ca(2+) signals, the complex architecture of the ER, and the difficulty of addressing probes specifically into the ER lumen. Prompted by these challenges, a number of ingenious approaches have been developed over the last years to measure ER Ca(2+) by optical means. The two main strategies used to date are Ca(2+)-sensitive synthetic dyes trapped into organelles and genetically encoded probes, based either on the photoprotein aequorin or on the green fluorescent protein (GFP). The GFP-based Ca(2+) indicators comprise the camgaroo and pericam probes based on a circularly permutated GFP, and the cameleon probes, which rely on the fluorescence resonance energy transfer (FRET) between two GFP mutants of different colors. Each approach offers unique advantages and suffers from specific drawbacks. In this review, we will discuss the advantages and pitfalls of using the genetically encoded "cameleon" Ca(2+) indicators for ER Ca(2+) measurements.     

Intracellular Ca(2+) release triggers translocation of membrane marker FM1-43 from the extracellular leaflet of plasma membrane into endoplasmic reticulum in T lymphocytes

Stimulation of T cell receptor in lymphocytes enhances Ca(2+) signaling and accelerates membrane trafficking. The relationships between these processes are not well understood. We employed membrane-impermeable lipid marker FM1-43 to explore membrane trafficking upon mobilization of intracellular Ca(2+) in Jurkat T cells. We established that liberation of intracellular Ca(2+) with T cell receptor agonist phytohemagglutinin P or with Ca(2+)-mobilizing agents ionomycin or thapsigargin induced accumulation of FM1-43 within the lumen of the endoplasmic reticulum (ER), nuclear envelope (NE), and Golgi. FM1-43 loading into ER-NE and Golgi was not mediated via the cytosol because other organelles such as mitochondria and multivesicular bodies located in close proximity to the FM1-43-containing ER were free of dye. Intralumenal FM1-43 accumulation was observed even when Ca(2+) signaling in the cytosol was abolished by the removal of extracellular Ca(2+). Our findings strongly suggest that release of intracellular Ca(2+) may create continuity between the extracellular leaflet of the plasma membrane and the lumenal membrane leaflet of the ER by a mechanism that does not require global cytosolic Ca(2+) elevation.     

The Ca2+-binding protein ALG-2 is recruited to endoplasmic reticulum exit sites by Sec31A and stabilizes the localization of Sec31A

The formation of transport vesicles that bud from endoplasmic reticulum (ER) exit sites is dependent on the COPII coat made up of three components: the small GTPase Sar1, the Sec23/24 complex, and the Sec13/31 complex. Here, we provide evidence that apoptosis-linked gene 2 (ALG-2), a Ca(2+)-binding protein of unknown function, regulates the COPII function at ER exit sites in mammalian cells. ALG-2 bound to the Pro-rich region of Sec31A, a ubiquitously expressed mammalian orthologue of yeast Sec31, in a Ca(2+)-dependent manner and colocalized with Sec31A at ER exit sites. A Ca(2+) binding-deficient ALG-2 mutant, which did not bind Sec31A, lost the ability to localize to ER exit sites. Overexpression of the Pro-rich region of Sec31A or RNA interference-mediated Sec31A depletion also abolished the ALG-2 localization at these sites. In contrast, depletion of ALG-2 substantially reduced the level of Sec31A associated with the membrane at ER exit sites. Finally, treatment with a cell-permeable Ca(2+) chelator caused the mislocalization of ALG-2, which was accompanied by a reduced level of Sec31A at ER exit sites. We conclude that ALG-2 is recruited to ER exit sites via Ca(2+)-dependent interaction with Sec31A and in turn stabilizes the localization of Sec31A at these sites.     

Live cell calcium imaging combined with siRNA mediated gene silencing identifies Ca²⁺ leak channels in the ER membrane and their regulatory mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca(2+) leak channels. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca(2+) from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca(2+) leak channel and to characterize its putative regulatory mechanisms. This system combines siRNA mediated gene silencing and live cell Ca(2+) imaging. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca(2+)-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca(2+) concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca(2+) also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca(2+) leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca(2+) efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca(2+) efflux, and iii) study the effects of disease linked mutations in the relevant components.     

Stanniocalcin 2 is a negative modulator of store-operated calcium entry

The regulation of cellular Ca(2+) homeostasis is essential for innumerable physiological and pathological processes. Stanniocalcin 1, a secreted glycoprotein hormone originally described in fish, is a well-established endocrine regulator of gill Ca(2+) uptake during hypercalcemia. While there are two mammalian Stanniocalcin homologs (STC1 and STC2), their precise molecular functions remain unknown. Notably, STC2 is a prosurvival component of the unfolded protein response. Here, we demonstrate a cell-intrinsic role for STC2 in the regulation of store-operated Ca(2+) entry (SOCE). Fibroblasts cultured from Stc2 knockout mice accumulate higher levels of cytosolic Ca(2+) following endoplasmic reticulum (ER) Ca(2+) store depletion, specifically due to an increase in extracellular Ca(2+) influx through store-operated Ca(2+) channels (SOC). The knockdown of STC2 expression in a hippocampal cell line also potentiates SOCE, and the overexpression of STC2 attenuates SOCE. Moreover, STC2 interacts with the ER Ca(2+) sensor STIM1, which activates SOCs following ER store depletion. These results define a novel molecular function for STC2 as a negative modulator of SOCE and provide the first direct evidence for the regulation of Ca(2+) homeostasis by mammalian STC2. Furthermore, our findings implicate the modulation of SOCE through STC2 expression as one of the prosurvival measures of the unfolded protein response.     

Old players in a new role: mitochondria-associated membranes,VDAC, and ryanodine receptors as contributors to calcium signal propagation from endoplasmic reticulum to the mitochondria

In many cell types, IP(3) and ryanodine receptor (IP(3)R/RyR)-mediated Ca(2+) mobilization from the sarcoendoplasmic reticulum (ER/SR) results in an elevation of mitochondrial matrix [Ca(2+)]. Although delivery of the released Ca(2+) to the mitochondria has been established as a fundamental signaling process, the molecular mechanism underlying mitochondrial Ca(2+) uptake remains a challenge for future studies. The Ca(2+) uptake can be divided into the following three steps: (1) Ca(2+) movement from the IP(3)R/RyR to the outer mitochondrial membrane (OMM); (2) Ca(2+) transport through the OMM; and (3) Ca(2+) transport through the inner mitochondrial membrane (IMM). Evidence has been presented that Ca(2+) delivery to the OMM is facilitated by a local coupling between closely apposed regions of the ER/SR and mitochondria. Recent studies of the dynamic changes in mitochondrial morphology and visualization of the subcellular pattern of the calcium signal provide important clues to the organization of the ER/SR-mitochondrial interface. Interestingly, key steps of phospholipid synthesis and transfer to the mitochondria have also been confined to subdomains of the ER tightly associated with the mitochondria, referred as mitochondria-associated membranes (MAMs). Through the OMM, the voltage-dependent anion channels (VDAC, porin) have been thought to permit free passage of ions and other small molecules. However, recent studies suggest that the VDAC may represent a regulated step in Ca(2+) transport from IP(3)R/RyR to the IMM. A novel proposal regarding the IMM Ca(2+) uptake site is a mitochondrial RyR that would mediate rapid Ca(2+) uptake by mitochondria in excitable cells. An overview of the progress in these directions is described in the present paper.     

A thapsigargin-sensitive Ca(2+) pump is present in the pea Golgi apparatus membrane

The Golgi apparatus behaves as a bona fide Ca(2+) store in animal cells and yeast (Saccharomyces cerevisiae); however, it is not known whether this organelle plays a similar role in plant cells. In this work, we investigated the presence of an active Ca(2+) accumulation mechanism in the plant cell Golgi apparatus. Toward this end, we measured Ca(2+) uptake in subcellular fractions isolated from the elongating zone of etiolated pea (Pisum sativum) epicotyls. Separation of organelles using sucrose gradients showed a strong correlation between the distribution of an ATP-dependent Ca(2+) uptake activity and the Golgi apparatus marker enzyme, xyloglucan-fucosyltransferase. The kinetic parameters obtained for this activity were: the rate of maximum Ca(2+) uptake of 2.5 nmol mg min(-1) and an apparent K(m) for Ca(2+) of 209 nM. The ATP-dependent Ca(2+) uptake was strongly inhibited by vanadate (inhibitor concentration causing 50% inhibition [I(50)] = 126 microM) and cyclopiazonic acid (I(50) = 0.36 nmol mg protein(-1)) and was not stimulated by calmodulin (1 microM). Addition of Cd(2+) and Cu(2+) at nanomolar concentration inhibited the Ca(2+) uptake, whereas Mn(2+), Fe(2+), and Co(2+) had no significant effect. Interestingly, the active calcium uptake was inhibited by thapsigargin (apparent I(50) = 88 nM), a well-known inhibitor of the endoplasmic reticulum and Golgi sarco-endoplasmic reticulum Ca(2+) ATPase from mammalian cells. A thapsigargin-sensitive Ca(2+) uptake activity was also detected in a cauliflower (Brassica oleracea) Golgi-enriched fraction, suggesting that other plants may also possess thapsigargin-sensitive Golgi Ca(2+) pumps. To our knowledge, this is the first report of a plant Ca(2+) pump activity that shows sensitivity to low concentrations of thapsigargin.     

The C terminus of Bax inhibitor-1 forms a Ca2+-permeable channel pore

Bax inhibitor-1 (BI-1) is a multitransmembrane domain-spanning endoplasmic reticulum (ER)-located protein that is evolutionarily conserved and protects against apoptosis and ER stress. Furthermore, BI-1 is proposed to modulate ER Ca(2+) homeostasis by acting as a Ca(2+)-leak channel. Based on experimental determination of the BI-1 topology, we propose that its C terminus forms a Ca(2+) pore responsible for its Ca(2+)-leak properties. We utilized a set of C-terminal peptides to screen for Ca(2+) leak activity in unidirectional (45)Ca(2+)-flux experiments and identified an α-helical 20-amino acid peptide causing Ca(2+) leak from the ER. The Ca(2+) leak was independent of endogenous ER Ca(2+)-release channels or other Ca(2+)-leak mechanisms, namely translocons and presenilins. The Ca(2+)-permeating property of the peptide was confirmed in lipid-bilayer experiments. Using mutant peptides, we identified critical residues responsible for the Ca(2+)-leak properties of this BI-1 peptide, including a series of critical negatively charged aspartate residues. Using peptides corresponding to the equivalent BI-1 domain from various organisms, we found that the Ca(2+)-leak properties were conserved among animal, but not plant and yeast orthologs. By mutating one of the critical aspartate residues in the proposed Ca(2+)-channel pore in full-length BI-1, we found that Asp-213 was essential for BI-1-dependent ER Ca(2+) leak. Thus, we elucidated residues critically important for BI-1-mediated Ca(2+) leak and its potential channel pore. Remarkably, one of these residues was not conserved among plant and yeast BI-1 orthologs, indicating that the ER Ca(2+)-leak properties of BI-1 are an added function during evolution.     

An ATP transporter is required for protein translocation into the yeast endoplasmic reticulum.

The transfer of precursor proteins through the membrane of the rough endoplasmic reticulum (ER) in yeast is strictly dependent on the presence of ATP. Since Kar2p (the yeast homologue of mammalian BiP) is required for translocation, and is an ATP binding protein, an ATP transport system must be coupled to the translocation machinery of the ER. We report here the characterization of a transport system for ATP in vesicles derived from yeast ER. ATP uptake into vesicles was found to be saturable in the micromolar range with a Km of 1 x 10(-5) M. ATP transport into ER vesicles was specifically inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a stilbene derivative known to inhibit a number of other anion transporters, and by 3'-O-(4-benzoyl)benzoyl-ATP (Bz2-ATP). Inhibition of ATP uptake into yeast microsomes by DIDS and Bz2-ATP blocked protein translocation in vitro measured co- as well as post-translationally. The inhibitory effect of DIDS on translocation was prevented by coincubation with ATP. Moreover, selective membrane permeabilization, allowing ATP access to the lumen, restored translocation activity to DIDS-treated membranes. These results demonstrate that translocation requires a DIDS and Bz2-ATP-sensitive component whose function is to transport ATP to the lumen of the ER. These findings are consistent with current models of protein translocation in yeast which stipulate the participation of Kar2p in the translocation process.     

Analysis of Mn(2+)/Ca(2+) influx and release during Ca(2+) oscillations in mouse eggs injected with sperm extract

Repetitive Ca(2+) release from the endoplasmic reticulum (ER) is necessary for activation of mammalian eggs. Influx and release of Mn(2+) and Ca(2+) during Ca(2+) oscillations induced by injection of sperm extract (SE) into mouse eggs were investigated by Mn(2+)-quenching of intracellular Fura-2 after adding Mn(2+) to external medium. Mn(2+)/Ca(2+) influx was detected at the resting state. A marked Mn(2+)/Ca(2+) influx occurred during the first Ca(2+) release upon SE injection, and persistently facilitated Mn(2+)/Ca(2+) influx was observed during steady Ca(2+) oscillations. As intracellular Mn(2+) concentration ([Mn(2+)](i)) increased progressively, periodic [Mn(2+)](i) rises appeared, corresponding to each Ca(2+)transient but taking a slower time course. A numerical simulation based on continuous Mn(2+)/Ca(2+) influx-extrusion across the plasma membrane and release-uptake across the ER membrane in a competitive manner mimicked well the Mn(2+) oscillations calculated from experimental data, strongly suggesting that repetitive Mn(2+) release develops after Mn(2+) entry and uptake into the ER. In other experiments, a marked Mn(2+) influx occurred upon Mn(2+) addition to Ca(2+)-free medium after depletion of the ER using an ER Ca(2+) pump inhibitor plus repeated injection of inositol 1,4,5-trisphosphate (InsP(3)). No significant increase in Mn(2+) influx was induced by injection of SE, InsP(3), or Ca(2+), when Ca(2+) release was prevented by pre-injection of an antibody against the InsP(3) receptor. We concluded that Ca(2+) influx is activated during the initial large Ca(2+)release possibly by a capacitative mechanism and kept facilitated during steady Ca(2+) oscillations. The finding that repetitive Mn(2+) release is caused by continuous Mn(2+) entry suggests that continuous Ca(2+) influx may play a critical role in refilling the ER and, thereby, maintaining Ca(2+)oscillations in mammalian fertilization.     

Bcl-2 enhances Ca(2+) signaling to support the intrinsic regenerative capacity of CNS axons

At a certain point in development, axons in the mammalian CNS undergo a profound loss of intrinsic growth capacity, which leads to poor regeneration after injury. Overexpression of Bcl-2 prevents this loss, but the molecular basis of this effect remains unclear. Here, we report that Bcl-2 supports axonal growth by enhancing intracellular Ca(2+) signaling and activating cAMP response element binding protein (CREB) and extracellular-regulated kinase (Erk), which stimulate the regenerative response and neuritogenesis. Expression of Bcl-2 decreases endoplasmic reticulum (ER) Ca(2+) uptake and storage, and thereby leads to a larger intracellular Ca(2+) response induced by Ca(2+) influx or axotomy in Bcl-2-expressing neurons than in control neurons. Bcl-x(L), an antiapoptotic member of the Bcl-2 family that does not affect ER Ca(2+) uptake, supports neuronal survival but cannot activate CREB and Erk or promote axon regeneration. These results suggest a novel role for ER Ca(2+) in the regulation of neuronal response to injury and define a dedicated signaling event through which Bcl-2 supports CNS regeneration.