The Contractile Vacuole in Amoeba proteus: Temperature Effects

The influence of temperature on the various aspects of the contractile vacuole cycle of Amoeba proteus has been established. In the upper temperature range (20, 25 and 30 C) an increase in temperature results in shorter vacuolar cycles with greater systolic (final) volumes. The systole is rapid and always complete. At 35 C the vacuole shows the effect of heat stress, cycles are irregular in volume and duration with only partial systoles. In the lower temperature range (15, 10 and 5 C), a new phenomenon has been observed, the plateau. Instead of undergoing systole, after reaching a certain critical volume the vacuole abruptly ceases to grow in size and remains in a state of pause for a well defined period of time, ending at a comparatively slow but complete systole. The duration of this plateau as well as its inception and termination seem quite precisely controlled. Its effect, a decrease in the fluid output by the vacuole, is such as to adjust vacuolar output to near constant Q₁₀ kinetics over our temperature range. This is correlated with a single straight line fit in an Arrhenius plot. Available data do not permit a complete explanation of the nature of the plateau. It could represent a steady state between 2 opposing phenomena: active fluid influx into the vacuole and osmotic losses from the vacuole into the relatively hypertonic cytoplasm.     

A functional connection of Dictyostelium paracaspase with the contractile vacuole and a possible partner of the vacuolar proton ATPase

Dictyostelium discoideum possesses only one caspase family member, paracaspase (pcp). Two separate mutant cell lines were first analysed: one cell line was an over-expressed GFP-tagged Pcp (GFP-Pcp), while the other cell line was a pcp-null (pcp-). Microscopic analysis of cells expressing GFP-Pcp revealed that Pcp was associated with the contractile vacuole membrane consisting of bladder-like vacuoles. This association was disrupted when cells were exposed to osmotic stress conditions. Compared with wild-type cells, the GFP-Pcp-over-expressing cells were susceptible to osmotic stress and were seen to be very rounded in hypo-osmotic conditions and contained more abnormally swollen contractile vacuole. Cells with pcp- were also rounded but had few, if any, contractile vacuoles. These observations suggest that Pcp is essential for Dictyostelium osmotic regulation via its functioning in the contractile vacuole system. Subjecting these cells to selected contractile vacuole inhibitor provided additional support for these findings. Furthermore, yeast two-hybrid system identified vacuolar proton ATPase (VatM) as the protein interacting with Pcp. Taken together, this work gives evidence for an eukaryotic paracaspase to be associated with both localization in and regulation of the contractile vacuolar system, an organelle critical for maintaining the normal morphology of the cell.     

Structure and behavior of contractile vacuoles inChlamydomonas reinhardtii

Summary The contractile vacuole (CV) cycle ofChlamydomonas reinhardtii has been investigated by videomicroscopy and electron microscopy. Correlation of the two kinds of observation indicates that the total cycle (15 s under the hypo-osmotic conditions used for videomicroscopy) can be divided into early, middle, and late stages. In the early stage (early diastole, about 3 s long) numerous small vesicles about 70–120 nm in diameter are present. In the middle stage (mid-diastole, about 6 s long), the vesicles appear to fuse with one another to form the contractile vacuole proper. In the late stage (late diastole, also about 6 s long), the CV increases in diameter by the continued fusion of small vesicles with the vacuole, and makes contact with the plasma membrane. The CV then rapidly decreases in size (systole, about 0.2 s). In isosmotic media, CVs do not appear to be functioning; under these conditions, the CV regions contain numerous small vesicles typical of the earliest stage of diastole. Fine structure observations have provided no evidence for a two-component CV system such as has been observed in some other cell types. Electron microscopy of cryofixed and freeze-substituted cells suggests that the irregularity of the profiles of larger vesicles and vacuoles and some other morphological details seen in conventionally fixed cells may be shrinkage artefacts. This study thus defines some of the membrane events in the normal contractile vacuole cycle ofChlamydomonas, and provides a morphological and temporal basis for the study of membrane fusion and fluid transport across membranes in a cell favorable for genetic analysis.Abbrevations CV contractile vacuole - PM plasma membrane     

Membrane Dynamics of the Contractile Vacuole Complex of Paramecium1

ABSTRACT. Membrane dynamics of the contractile vacuole complex of Paramecium were investigated using conventional electron microscopy of cells so that the vacuoles were serial-sectioned longitudinally and transversely. During systole, vacuolar membrane collapses first into flattened cisternae which undergo further modification into a mass of interconnected small membrane tubules. These tubules retain their connections with the radiating microtubular ribbons; consequently they are found only in the poleward hemisphere. Permanent connections between ampullae and the collapsed vacuole membrane could not be verified nor was a sphincter-like mechanism for closing such a junction observed. Membranes of the ampullae and the collecting canals also collapse to varying extents into arrays of tubules that remain bound to microtubular ribbons during diastole. Thus vacuole, ampullae, and collecting canal membranes all assume tubular forms when internal volume is at a minimum. Having failed to observe a microfilamentous encasement of the vacuole, we suggest that an alternative mechanism for the “contractile” function should be sought. One such is based on fluid volume increase and fluid flow within transiently interconnected tubular membrane systems that cycle between a tubular and a planar membrane form as internal volume is periodically increased and reduced. The driving force for this mechanism might best be sought in the molecular structure of the membranes of the contractile vacuole complex.     

The Contractile Vacuole of Amoeba proteus. II. Numerical Analysis of the Steady-State Hypothesis*

SYNOPSIS. At temperatures below 15 C, the contractile vacuole cycle of Amoeba proteus includes a presystolic plateau. The hypothesis attributing this plateau to a steady-state equilibrium between active filling processes and osmotic losses of water from the vacuole into the cytoplasm has been expressed in an equation predicting vacuolar diameter as a function of time for the later part of the cycle. Computer-generated model cycles have been compared with actual recorded cycles at 15 C, 10 C and 5 C and conditions of best fit were determined. Statistical analysis shows that recorded cycles are quite compatible with the steady-state hypothesis.     

ELECTRON MICROSCOPY OF A CONTRACTILE-VACUOLE MUTANT OF CHLAMYDOMONAS MOEWUSII (CHLOROPHYTA) DEFECTIVE IN THE LATE STAGES OF DIASTOLE1

Electron microscopy of a “vacuole-less” mutant of Chlamydomonas moewusii Gerloff revealed the presence of small anterior vacuoles. These vacuoles behaved like contractile vacuoles in wild-type cells, but they were apparently unable to complete diastole and discharge their contents. When wild-type and mutant cells were incubated in hypertonic medium, small coated vacuoles persisted in the region where contractile vacuoles form. When these cells were transferred to hypotonic medium, the vacuoles appeared to fill and fuse to form larger vacuoles Shortly after the appearance of full expanded contractile vacuoles, collapsed vacuoles were observed in wild-type cells suggesting the completion of diastole and the onset of systole. In mutant cells, the initial steps of filling and fusion to form larger vacuoles apparent interactions of vacuoles with the plasma membrane were not observed. New contractile vacuoles accumulated around the nucleus. When fusion of the contractile vacuole with the plasma membrane was blocked by EGTA, a similar accumulation of large vacuoles occurred. Our observations suggest that the contractile-vacuole mutant of C. Moewusii produces vacuoles which can accumulate excess water as part of the mechanism of osmoregulation but which cannot complete diastole.     

An Electron Microscope Study of the Contractile Vacuole in Tokophrya infusionum

Contractile vacuoles are organelles that collect fluid from the cytoplasm and expel it to the outside. After each discharge (systole), they appear again and expand (diastole). They are widely distributed among Protozoa, and have been found also in some fresh water algae, sponges, and recently in some blood cells of the frog, guinea pig, and man. In spite of the extensive work on the contractile vacuole, very little is known concerning its mode of operation. An electron microscope study of a suctorian Tokophrya infusionum provided an opportunity to study thin sections of contractile vacuoles, and in these some structures were found which could be part of a mechanism for the systolic and diastolic motions the organelle displays. In Tokophrya, as in Suctoria and Ciliata in general, the contractile vacuole has a permanent canal connecting it with the outside. The canal appears to have a very elaborate structure and is composed of three parts: (1) a pore; (2) a channel; and (3) a narrow tubule located in a papilla protruding into the cavity of the contractile vacuole. Whereas the pore and channel have fixed dimensions and are permanently widely open, the tubule has a changeable diameter. At diastole it is so narrow (about 25 to 30 mµ in diameter) that it could be regarded as closed, while at systole it is widely open. It is assumed that the change in diameter is due to the contraction of numerous fine fibrils (about 180 A thick) which are radially disposed around the canal in form of a truncated cone, with its tip at the channel, and its base at the vacuolar membrane. It seems most probable that the broadening of the tubule results in discharge of the content of the contractile vacuole. In the vicinity of the very thin limiting vacuolar membrane, small vesicles and canaliculi of the endoplasmic reticulum, very small dense particles, and mitochondria may be found. In addition, rows of closely packed vesicles are present in this region, and in other parts of the cytoplasm. It is suggested that they might represent dictyosome-like bodies, responsible for withdrawing fluids from the cytoplasm and then conveying them to the contractile vacuole, contributing to its expansion at diastole.     

Osmotic stress enhances the competence of Beta vulgaris vacuoles to respond to inositol 1,4,5-trisphosphate

Isolated vacuoles from Beta vulgaris storage roots respond to the intracellular signalling molecule inositol 1,4,5-trisphosphate (InsP₃). Whole vacuole patch clamp enables measurement of an inward current (cytosol-directed) induced by cytosolic InsP₃ which is fully reversible upon removal of InsP₃. The reversal potentials of the InsP₃-induced whole vacuolar currents indicate a permeability ratio (P,Ca:P,K) of 200:1. Competence of vacuoles to respond to InsP₃ is dependent upon the root tissue undergoing hyperosmotic stress before vacuole isolation. The magnitude of the hyperosmotic stress and the density of InsP₃-induced current per unit membrane area are exponentially related. A standing osmotic gradient across the vacuolar membrane further enhances the InsP₃-induced current, the current being larger when there is net water flux from the cytosol to the vacuolar lumen. InsP₃-induced currents are not affected by the cytosolic free Ca²⁺ concentration. The conductance of InsP₃-induced single channel currents varied greatly between individual outside-out patches, but all showed a non-linear increase in single channel current at physiological potentials. The reversal potentials of these currents indicated a P_Ca:P_K of between 100:1 and 800:1. The significance of these findings is discussed in relation to technical aspects of monitoring InsP₃-induced currents in plant vacuoles and in the context of the physiological roles of InsP₃ and its receptor in cell water relations.     

Turgor regulation inValonia macrophysa following acute osmotic shock

Summary The marine algaValonia macrophysa an inhabitant of shallow subtropical waters, is subjected to sudden dilutions of external seawater during rain showers. This study describes the mechanisms involved in turgor pressure regulation following acute hyposmotic shock. Turgor regulation is 88% effective and complete within 4 hr following hyposmotic shocks of up to –10 bar. Loss of vacuolar K⁺, Na⁺ and Cl^– accounts for the decrease in vacuolar osmotic pressure associated with turgor regulation. A novel mechanism of turgor regulation is exhibited byValonia macrophysa given hyposmotic shocks greater than about –4 bar. Such an osmotic shock causes cell wall tension to increase above a critical value of about 6×10⁵ dyne/cm, whereupon the protoplasm ruptures and the cell wall stretches irreversibly at a localized site. The protoplasm rupture is suggested by (1) a large abrupt increase in K⁺ efflux (as measured by⁸⁶Rb⁺), (2) a rapid decrease in turgor pressure as measured with a pressure probe, and (3) sudden depolarization of the vacuole potential. Evidence for an increase in cell wall permeability includes efflux from the vacuole of dextran (mol wt 70,000), which normally has a very low cell wall permeability, and scanning electron micrographs which show a trabeculated scar area in the cell wall. This mechanism of turgor regulation is physiologically important because 98% of the cells regained normal growth rate and turgor following acute osmotic shock.     

Effect of alterations in the size of the vacuolar compartment on pinocytosis in J774.2 macrophages

J774.2 macrophages cultured in medium containing 10 mg/ml sucrose accumulate the sugar by pinocytosis and become highly vacuolated, due to the sugar's osmotic effect within the vacuolar compartment. When such cells are incubated in medium containing 0.5 mg/ml invertase, the enzyme reaches the sucrose vacuoles by pinocytosis, then cleaves the sugar to more permeant monosaccharides. Within 4 hours, the vacuoles shrink to smaller, phase-dense organelles (Cohn and Ehrenreich, 1969, J. Exp. Med., 129:201). We have used this reversible expansion of the lysosomal compartment to address two questions: (1) Does the increased size of the lysosomal compartment affect pinocytic accumulation of solute, and (2) what is the fate of the vacuolar membrane and its soluble content during invertase-induced vacuole shrinkage? Using lucifer yellow (LY) as a probe for pinocytic fluid influx and efflux, we found that vacuolated cells accumulated 30–50% less LY than controls and returned to higher rates of pinocytosis after invertase-induced vacuole shrinkage. A similar reduction in LY accumulation was achieved after feeding cells latex beads to increase the size of the lysosomal compartment. Thus, treatments that increased the size of the lysosomal compartment reduced solute accumulation via pinocytosis. A dramatic shrinkage of LY-containing sucrose vacuoles followed pinocytosis of invertase. Despite this reduction in size of the LY-containing vacuoles, the overall rate of LY efflux did not increase significantly during invertase-induced vacuole collapse. Electron microscopy revealed that during shrinkage, the excess vacuolar membrane was compressed into whorled membranous organelles (residual bodies), with fluid markers (colloidal gold and, by inference, LY) trapped inside. The trapping of LY inside lysosomes as J774.2 macrophages returned to their normal dimensions indicates that nearly all of the surplus membrane contents were removed from circulation as well.     

The Contractile Vacuole of Amoeba proteus. III. Vacuolar Response to Phagocytosis1

The reaction of the contractile vacuole of Amoeba proteus to single and multiple phagocytosis under controlled conditions has been studied. Fluid intake into the cytoplasm from the phagosomes induces secretion by the contractile vacuole of equivalent excess volumes:. Vacuolar response is rapid (200 sec) and may be initiated by increases of protoplasmic hydration of as little as 1%. Cytoplasmic uptake of fluid from the phagosome can occur against an osmotic gradient; thus some form of active transport is implied.     

A contractile vacuole complex is involved in osmoregulation in Trypanosoma cruzi

Acidocalcisomes are dense, acidic organelles with a high concentration of phosphorus present as pyrophosphate and polyphosphate complexed with calcium and other cations. Acidocalcisomes have been linked to the contractile vacuole complex in Chlamydomonas reinhardtii, Dictyostelium discoideum, and Trypanosoma cruzi. A microtubule- and cyclic AMP-mediated fusion of acidocalcisomes to the contractile vacuole complex in T. cruzi results in translocation of aquaporin and the resulting water movement which, in addition to swelling of acidocalcisomes, is responsible for the volume reversal not accounted for by efflux of osmolytes. Polyphosphate hydrolysis occurs during hyposmotic stress, probably increasing the osmotic pressure of the contractile vacuole and facilitating water movement.     

Behavior of the contractile vacuole of Tetrahymena pyriformis W: a redescription with comments on the terminology.

The behavior of the contractile vacuole of Tetrahymena pyriformis W has been recorded and analyzed quantitatively by cinephotography. The vacuole fills in a stepwise fashion by the confluence of ampullae which appear regularly at the beginning of systole and whose membranes are continuous with that of the contractile vacuole throughout the cycle. The vacuole may subsequently fill slowly by a means not discernible by light microscopy. The vacuole rounds up at the beginning of systole and shortly thereafter the ampullae reappear around the perimeter of the vacuole. They are expanded by fluid forced into them from the vacuole. Round-up and the mode of growth of the ampullae indicate that the contractile vacuole is truly contractile. Expulsion occurs soon after the appearance of the ampullae and terminates the cycle. Contraction is initiated at regular intervals by a timing mechanism which is independent of the size of the vacuole. Suitable terminology to describe the structure and behavior of the contractile vacuole is discussed.     

Behavior of the Contractile Vacuole of Tetrahymena pyriformis W: A Redescription with Comments on the Terminology

SYNOPSIS. The behavior of the contractile vacuole of Tetrahymena pyriformis W has been recorded and analyzed quantitatively by cinephotography. The vacuole fills in a stepwise fashion by the confluence of ampullae which appear regularly at the beginning of systole and whose membranes are continuous with that of the contractile vacuole throughout the cycle. The vacuole may subsequently fill slowly by a means not discernible by light microscopy. The vacuole rounds up at the beginning of systole and shortly thereafter the ampullae reappear around the perimeter of the vacuole. They are expanded by fluid forced into them from the vacuole. Round-up and the mode of growth of the ampullae indicate that the contractile vacuole is truly contractile. Expulsion occurs soon after the appearance of the ampullae and terminates the cycle. Contraction is initiated at regular intervals by a timing mechanism which is independent of the size of the vacuole. Suitable terminology to describe the structure and behavior of the contractile vacuole is discussed.     

Acidification of the young phagosomes ofParamecium is mediated by proton pumps derived from the acidosomes

Summary Although it is generally accepted that phagosome acidification is induced through the activity of a vacuolar proton pump (V-ATPase) present on the phagosome membrane, exactly how these pumps are delivered to the phagosomes is not well understood. To study this question inParamecium, it was necessary to first show that an authentic V-ATPase was present on their phagosomal membranes. Three antibodies raised against V-ATPases or their subunits were each found to label one or two large digestive vacuoles (DVs) inParamecium multimicronucleatum when immunofluorescence microscopy was used. Using horseradish peroxidase immunocytochemistry to increase sensitivity, about 10 DVs were shown to contain a V-ATPase. In high magnification images and cryoultramicrotomy these proton pumps were found to be located on the acidosomes, suggesting the vacuolar proton pumps on the DVs originate from the acidosomes. The authenticity of the V-ATPase was further confirmed by its sensitivity to cold temperature and to the V-ATPase specific inhibitor, concanamycin B, which at 10 nM doubled the t_1/2 for vacuole acidification. Thus, we conclude that (1) acidosomes and some DVs ofParamecium have a bona-fide concanamycin B-sensitive and cold-sensitive V-ATPase, (2) the V-ATPase is delivered to the young DVs during acidosome fusion, and (3) the V-ATPase is involved in vacuole acidification. Finally, we have now determined thatParamecium has two immunologically related V-ATPases that are involved in two very different functions, (1) the acidification of phagosomes and (2) fluid segregation in the contractile vacuole complexes.Abbreviations BS-FITC bovine serum albumin-fluorescein isothiocyanate - CVC contractile vacuole complex - DV-I to DV-IV digestive vacuole stages 1 to 4 - HRP horseradish peroxidase - V-ATPase vacuolar proton pump     

Effect of Osmotic Stress on Abscisic Acid Efflux and Compartmentation in the Roots of Two Maize Lines Differing in Drought Susceptibility

Roots of two Zea mays L. lines (drought-resistant Polj 17, and drought-susceptible F-2) were exposed to osmotic stress induced by sorbitol (osmotic potential –1.0 MPa). The following parameters were determined in cortex cells: membrane permeability for abscisic acid (ABA), ABA fluxes across membranes, pH values and ABA content in cytoplasm and vacuole. Osmotic stress induced different distribution of ABA within cell compartments in the investigated lines. ABA transport in the F-2 line occurred according to the intracellular pH gradient and the anion trap concept. In Polj 17, however, osmotic stress did not cause any significant effect on pH gradient and compartmental ABA content, but had a stimulating effect on ABA efflux from cytoplasm to apoplast and than via xylem to the leaf. These findings indicate different mechanisms of ABA transport between the investigated lines in response to osmotic stress.     

Fine Structure of the Contractile Vacuole Pore in Paramecium*

The pore through which a Paramecium contractile vacuole communicates with the external environment is a 1.2 μm long and 1 μm diameter cylindrical orifice in the pellicle. During diastole, the vacuole:pore junction is closed by a substantial diaphragm which parts to the side at systole. The diaphragm is composed of inner and outer membranes continuous with the vacuole and pore membranes, respectively, and an intervening cytoplasmic layer containing filaments and irregular membranous tubules and vesicles. Microtubules, organized into 2 sets, are an important component of the pore apparatus. One set of ～ 16 microtubules forms an annulus around the pore. These microtubules are organized into a right-handed helix with a pitch of 0.5-0.6 μm, and thus complete slightly more than 2 turns in their course from the level of the diaphragm to the pore outer lip. They appear to be embedded in a layer of dense material immediately adjacent to the pore membrane. The other set consists of 5 or more bands of 10–20 microtubules which radiate in a slight left-handed helix from an insertion at the pore out over the vacuole surface to the ampullae.     

Turgor pressure changes trigger characteristic changes in the electrical conductance of the tonoplast and the plasmalemma of the marine alga Valonia utricularis

The giant marine alga Valonia utricularis is capable of regulating its turgor pressure in response to changes in the osmotic pressure of the sea water. The turgor pressure response comprises two phases, a fast, exponential phase arising exclusively from water shifting between the vacuole and the external medium (time constant about 10 min) and a second very slow, almost exponential phase adjusting (but not always) the turgor pressure near to the original value by release or uptake of KCl (time constant about 5 h). The changes in the vacuolar membrane potential as well as in the individual conductances of the tonoplast and plasmalemma accompanying turgor pressure regulation were measured by using the vacuolar perfusion assembly (with integrated microelectrodes, pressure transducers and pressure‐regulating valves) as described by Wang et al. (J. Membrane Biology 157, 311–321, 1997). Measurements on pressure‐clamped cells gave strong evidence that the turgor pressure, but not effects related to water flow (i.e. electro‐osmosis or streaming potential) or changes in the internal osmotic pressure and in the osmotic gradients, triggers the cascade of osmotic and electrical events recorded after disturbance of the osmotic equilibrium. The findings definitely exclude the existence of osmosensors as postulated for other plant cells and bacteria. There was also evidence that turgor pressure signals were primarily sensed by ion transporters in the vacuolar membrane because conductance changes were first recorded in the many‐folded tonoplast and then significantly delayed in the plasmalemma independent of the direction of the osmotic challenge. Consistently, turgor pressure up‐regulation (but not down‐regulation) could be inhibited reversibly by external addition of the K⁺ transport inhibitor Ba²⁺ and/or by the Cl^– transport inhibitor 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS). Extensive studies under iso‐, hyper‐ and hypo‐osmotic conditions revealed that K⁺ and Cl^– contribute predominantly to the plasmalemma conductance. Addition of 0.3 mm NaCN showed further that part of the K⁺ and Cl^– transporters depended on ATP. These transporters are apparently up‐regulated upon hyper‐osmotic, but not hypo‐osmotic challenge. These findings explain the strong increase of the K⁺ influx upon lowering turgor pressure and the less pronounced pressure‐dependence of the Cl^– influx of V. utricularis reported in the literature. The data derived from the blockage experiments under hypo‐osmotic conditions were also equally consistent with the experimental findings that the K⁺ efflux is solely passive and progressively increases with increasing turgor pressure due to an increase of the volumetric elastic modulus of the cell wall. However, despite unravelling some of the sequences and other components involved in turgor pressure regulation of V. utricularis the co‐ordination between the ion transporters in the tonoplast and plasmalemma remains unresolved because of the failure to block the tonoplast transporters by addition of Ba²⁺ and DIDS from the vacuolar side.     

K+ currents through SV-type vacuolar channels are sensitive to elevated luminal sodium levels

Non-selective slow vacuolar (SV) channels mediate uptake of K+ and Na+ into vacuolar compartment. Under salt stress plant cells accumulate Na+ in the vacuole and release vacuolar K+ into the cytoplasm. It is, however, unclear how plants mediate transport of K+ from the vacuole without concomitant efflux of toxic Na+. Here we show by patch-clamp studies on isolated Arabidopsis thaliana cell culture vacuoles that SV channels do not mediate Na+ release from the vacuole as luminal Na+ blocks this channel. Gating of the SV channel is dependent on the K+ gradient across the vacuolar membrane. Under symmetrical K+ concentrations on both sides of the vacuolar membrane, SV channels mediate potassium uptake. When cytoplasmic K+ decreases, SV channels allow K+ release from the vacuole. In contrast to potassium, Na+ can be taken up by SV channels, but not released even in the presence of a 150-fold gradient (lumen to cytoplasm). Accumulation of Na+ in the vacuole shifts the activation potential of SV channels to more positive voltages and prevents gradient-driven efflux of K+. Similar to sodium, under physiological conditions, vacuolar Ca2+ is not released from vacuoles via SV channels. We suggest that a major Arabidopsis SV channel is equipped with a positively charged intrinsic gate located at the luminal side, which prevents release of Na+ and Ca2+, but permits efflux of K+. This property of the SV channel guarantees that K+ can shuttle across the vacuolar membrane while maintaining Na+ and Ca2+ stored in this organelle.