The <Emphasis Type="Italic">Arabidopsis</Emphasis> calmodulin-like proteins AtCML30 and AtCML3 are targeted to mitochondria and peroxisomes,respectively

Calmodulin (CaM) is a ubiquitous sensor/transducer of calcium signals in eukaryotic organisms. While CaM mediated calcium regulation of cytosolic processes is well established, there is growing evidence for the inclusion of organelles such as chloroplasts, mitochondria and peroxisomes into the calcium/calmodulin regulation network. A number of CaM-binding proteins have been identified in these organelles and processes such as protein import into chloroplasts and mitochondria have been shown to be governed by CaM regulation. What have been missing to date are the mediators of this regulation since no CaM or calmodulin-like protein (CML) has been identified in any of these organelles. Here we show that two Arabidopsis CMLs, AtCML3 and AtCML30, are localized in peroxisomes and mitochondria, respectively. AtCML3 is targeted via an unusual C-terminal PTS1-like tripeptide while AtCML30 utilizes an N-terminal, non-cleavable transit peptide. Both proteins possess the typical structure of CaMs, with two pairs of EF-hand motifs separated by a short linker domain. They furthermore display common characteristics, such as calcium-dependent alteration of gel mobility and calcium-dependent exposure of a hydrophobic surface. This indicates that they can function in a similar manner as canonical CaMs. The presence of close homologues to AtCML3 and AtCML30 in other plants further indicates that organellar targeting of these CMLs is not a specific feature of Arabidopsis. The identification of peroxisomal and mitochondrial CMLs is an important step in the understanding how these organelles are integrated into the cellular calcium/calmodulin signaling pathways.     

Calcium signaling in plant cell organelles delimited by a double membrane

Increases in the concentration of free calcium in the cytosol are one of the general events that relay an external stimulus to the internal cellular machinery and allow eukaryotic organisms, including plants, to mount a specific biological response. Different lines of evidence have shown that other intracellular organelles contribute to the regulation of free calcium homeostasis in the cytosol. The vacuoles, the endoplasmic reticulum and the cell wall constitute storage compartments for mobilizable calcium. In contrast, the role of organelles surrounded by a double membrane (e.g. mitochondria, chloroplasts and nuclei) is more complex. Here, we review experimental data showing that these organelles harbor calcium-dependent biological processes. Mitochondria, chloroplasts as well as nuclei are equipped to generate calcium signal on their own. Changes in free calcium in a given organelle may also favor the relocalization of proteins and regulatory components and therefore have a profound influence on the integrated functioning of the cell. Studying, in time and space, the dynamics of different components of calcium signaling pathway will certainly give clues to understand the extraordinary flexibility of plants to respond to stimuli and mount adaptive responses. The availability of technical and biological resources should allow breaking new grounds by unveiling the contribution of signaling networks in integrative plant biology.     

Arabidopsis calcium-binding mitochondrial carrier proteins as potential facilitators of mitochondrial ATP-import and plastid SAM-import

Chloroplasts and mitochondria are central to crucial cellular processes in plants and contribute to a whole range of metabolic pathways. The use of calcium ions as a secondary messenger in and around organelles is increasingly appreciated as an important mediator of plant cell signaling, enabling plants to develop or to acclimatize to changing environmental conditions. Here, we have studied the four calcium-dependent mitochondrial carriers that are encoded in the Arabidopsis genome. An unknown substrate carrier, which was previously found to localize to chloroplasts, is proposed to present a calcium-dependent S-adenosyl methionine carrier. For three predicted ATP/phosphate carriers, we present experimental evidence that they can function as mitochondrial ATP-importers.     

Phenothiazine inhibition of calmodulin stimulates calcium-dependent potassium efflux in human red blood cells

Elevation of red blood cell calcium increases the efflux of potassium. The active extrusion of calcium from the red cell is regulated by calmodulin. Phenothiazines bind to calmodulin in a calcium-dependent manner preventing the calmodulin from activating a wide variety of cellular processes. The present study shows that phenothiazines increase the efflux of potassium from red cells incubated with the calcium ionophore A23187. The dose dependent effect of trifluoperazine on potassium efflux correlates with its inhibition of Ca-ATPase activity. The phenothiazine effects are dependent upon ATP in that increases in potassium efflux are not observed in energy depleted cells. In calcium buffered ghosts no direct effect of calmodulin or an antibody to calmodulin can be shown. These data suggest that phenothiazines stimulate calcium-dependent potassium loss indirectly by a drug-induced blockage of the calmodulin-activated Ca-ATPase.     

Calcium depletion and calmodulin inhibition affect the import of nuclear-encoded proteins into plant mitochondria

Many metabolic processes essential for plant viability take place in mitochondria. Therefore, mitochondrial function has to be carefully balanced in accordance with the developmental stage and metabolic requirements of the cell. One way to adapt organellar function is the alteration of protein composition. Since most mitochondrial proteins are nuclear encoded, fine-tuning of mitochondrial protein content could be achieved by the regulation of protein translocation. Here we present evidence that the import of nuclear-encoded mitochondrial proteins into plant mitochondria is influenced by calcium and calmodulin. In pea mitochondria, the calmodulin inhibitor ophiobolin A as well as the calcium ionophores A23187 and ionomycin inhibit translocation of nuclear-encoded proteins in a concentration-dependent manner, an effect that can be countered by the addition of external calmodulin or calcium, respectively. Inhibition was observed exclusively for proteins translocating into or across the inner membrane but not for proteins residing in the outer membrane or the intermembrane space. Ophiobolin A and the calcium ionophores further inhibit translocation into mitochondria with disrupted outer membranes, but their effect is not mediated via a change in the membrane potential across the inner mitochondrial membrane. Together, our results suggest that calcium/calmodulin influences the import of a subset of mitochondrial proteins at the inner membrane. Interestingly, we could not observe any influence of ophiobolin A or the calcium ionophores on protein translocation into mitochondria of yeast, indicating that the effect of calcium/calmodulin on mitochondrial protein import might be a plant-specific trait.     

The regulatory role of mitochondria in capacitative calcium entry

Capacitative regulation of calcium entry is a major mechanism of Ca2+ influx into electrically non-excitable cells, but it also operates in some excitable ones. It participates in the refilling of intracellular calcium stores and in the generation of Ca2+ signals in excited cells. The mechanism which couples depletion of intracellular calcium stores located in the endoplasmic reticulum with opening of store-operated calcium channels in the plasma membrane is not clearly understood. Mitochondria located in close proximity to Ca2+ channels are exposed to high Ca2+ concentration, and therefore, they are able to accumulate this cation effectively. This decreases local Ca2+ concentration and thereby affects calcium-dependent processes, such as depletion and refilling of the intracellular calcium stores and opening of the store-operated channels. Finally, mitochondria modulate the intensity and the duration of calcium signals induced by extracellular stimuli. Ca2+ uptake by mitochondria requires these organelles to be in the energized state. On the other hand, Ca2+ flux into mitochondria stimulates energy metabolism. To sum up, mitochondria couple cellular metabolism with calcium homeostasis and signaling.     

Cytoplasmic inheritance of mitochondria and chloroplasts in the anisogamous brown alga Mutimo cylindricus (Phaeophyceae)

Based on the morphology of gametes, sexual reproduction in brown algae is usually classified into three types: isogamy, anisogamy, and oogamy. In isogamy, chloroplasts and chloroplast DNA (chlDNA) in the sporophyte cells are inherited biparentally, while mitochondria (or mitochondrial DNA, mtDNA) is inherited maternally. In oogamy, chloroplasts and mitochondria are inherited maternally. However, the patterns of mitochondrial and chloroplast inheritance in anisogamy have not been clarified. Here, we examined derivation of mtDNA and chlDNA in the zygotes through strain-specific PCR analysis using primers based on single nucleotide polymorphism in the anisogamous brown alga Mutimo cylindricus. In 20-day-old sporophytes after fertilization, mtDNA and chlDNA derived from female gametes were detected, thus confirming the maternal inheritance of both organelles. Additionally, the behavior of mitochondria and chloroplasts in the zygotes was analyzed by examining the consecutive serial sections using transmission electron microscopy. Male mitochondria were isolated or compartmentalized by a double-membrane and then completely digested into a multivesicular structure 2 h after fertilization. Meanwhile, male chloroplasts with eyespots were observed even in 4-day-old, seven-celled sporophytes. The final fate of male chloroplasts could not be traced. Organelle DNA copy number was also examined in female and male gametes. The DNA copy number per chloroplast and mitochondria in male gametes was lower compared with female organelles. The degree of difference is bigger in mtDNA. Thus, changes in different morphology and DNA amount indicate that maternal inheritance of mitochondria and chloroplasts in this species may be based on different processes and timing after fertilization.

     

Intracellular compartmentation of CTP synthase in Drosophila

Compartmentation is essential for the localization of biological processes within a eukaryotic cell.ATP synthase localizes to organelles such as mitochondria and chloroplasts.By contrast,little is known about the subcellular distribution of CTP synthase,the critical enzyme in the production of CTP,a high-energy molecule similar to ATP.Here I describe the identification of a novel intracellular structure con-taining CTP synthase,termed the cytoophidium,in Drosophila cells.I find that cytoophidia are present ...     

Direct Targeting of Proteins from the Cytosol to Organelles: The ER versus Endosymbiotic Organelles

In eukaryotic cells consisting of many different types of organelles, targeting of organellar proteins is one of the most fundamental cellular processes. Proteins belonging to the endoplasmic reticulum (ER), chloroplasts and mitochondria are targeted individually from the cytosol to their cognate organelles. As the targeting to these organelles occurs in the cytosol during or after translation, the most crucial aspect is how specific targeting to these three organelles can be achieved without interfering with other targeting pathways. For these organelles, multiple mechanisms are used for targeting proteins, but the exact mechanism used depends on the type of protein and organelle, the location of targeting signals in the protein and the location of the protein in the organelle. In this review, we discuss the various mechanisms involved in protein targeting to the ER, chloroplasts and mitochondria, and how the targeting specificity is determined for these organelles in plant cells .     

Active Calcium Transport by Plant Cell Membranes

The cytosolic free calcium concentration of higher plant cellsis maintained at about 01 µM by the action of membranecalcium transporters. These act to remove calcium from the cytosoland expel it to the apoplast or accumulate it in intracellularstores. In this review, the properties and subcellular localizationsof these systems are described. The major calcium transporterof the plasma membrane is a calcium pumping ATPase which showsmany similarities to its equivalent in mammalian cells. Thetransporter has been purified from maize coleoptiles and isof M_r 140 000, binds (and is activated by) calmodulin and showscommon antigenicity with the mammalian protein. Higher plantendoplasmic reticulum also contains a calcium pumping ATPasewhich transports calcium from the cytoplasm and its role andproperties, together with those of the tonoplast calcium/protonantiporter are presented. Evidence for calcium accumulationby chloroplasts and mitochondria is considered. The review alsodeals with the regulation of plant cell membrane calcium transportand its role in providing intracellular pools of calcium forsignal transduction. Key words: Plant, calcium transport, ATPase, cell membrane, calmodulin     

Effects of Al3+ on Ca2+-ATPase Activity on Chloroplasts of Rice and Relation to Calmodulin

The relationship between aluminium phytotoxicity and calmodulin has been studied with. calcium-dependent ATPase in chloroplasts of rice. This enzyme could be activated by extrinsic calmodulin. It showed that the activity of Ca2+-ATPase in chloroplasts was regulated by calmodulin. The activation of calmodulin to the enzyme might be inhibited by calmodulin antagonists, TFP and CPZ. The effects of A13+ on the activation of calmodulin was similar to that of the calmodulin antagonists. Calcium could reduce the inhibition of aluminiutn. It seems that there is a model of toxic responses in plants to aluminium: Al3+→calmodulin→target enzy mes→metabolism.     

The role of calcium in chloroplasts—an intriguing and unresolved puzzle

More than 70?years of studies have indicated that chloroplasts contain a significant amount of calcium, are a potential storage compartment for this ion, and might themselves be prone to calcium regulation. Many of these studies have been performed on the photosynthetic light reaction as well as CO(2) fixation via the Calvin-Benson-Bassham cycle, and they showed that calcium is required in several steps of these processes. Further studies have indicated that calcium is involved in other chloroplast functions that are not directly related to photosynthesis and that there is a calcium-dependent regulation similar to cytoplasmic calcium signal transduction. Nevertheless, the precise role that calcium has as a functional and regulatory component of chloroplast processes remains enigmatic. Calcium concentrations in different chloroplast subcompartments have been measured, but the extent and direction of intra-plastidal calcium fluxes or calcium transport into and from the cytosol are not yet very well understood. In this review we want to give an overview over the current knowledge on the relationship between chloroplasts and calcium and discuss questions that need to be addressed in future research.     

Programmed cell death. Cytochemical evidence for accumulation of calcium in mitochondria and its translocation into lysosomes: X-ray microanalysis in metamorphosing insect muscles

Summary The intersegmental muscles in the metamorphosing silkmothAntheraea polyphemus were examined by two electron cytochemical procedures for demonstration of calcium compartmentation during the two-day period of degeneration after emergence. Muscle fibres were treated with either oxalate—pyroantimonate, or phosphate—pyroantimonate procedures. The elemental composition of the reaction product arising from the oxalate procedure was determined with electron probe X-ray microanalysis of unstained thin sections by energy dispersive spectrometry and wavelength dispersive spectrometry. The wavelength dispersive data revealed high peaks of calcium and antimony in the electron-dense precipitates. No reaction was obtained in muscles after treatment with the phosphate—pyroantimonate method.Shortly after the emergence of the moth, very few calcium deposits were found in the mitochondria, which also contained amorphous matrix densities. During the rapid lytic phase (17 and 30 h after ecdysis), the mitochondria, autophagic vacuoles sequestering mitochondria, and lysosomal dense bodies issuing from the latter were highly reactive in each muscle fibre.These results demonstrate that the collapse of tracheae (hypoxic conditions) is correlated with the calcium overload of mitochondria when the cell calcium homeostasis is apparently lost. Such calcium overload of the mitochondria appears to cause irreversible damage to these organelles which are then sequestered in autophagic vacuoles. This mitochondrial autophagic process leads to calcium translocation into a lysosomal compartment. We suggest that the calcium lysosomal stores may have a transient function of cell detoxification and stimulation of calcium-dependent degradative processes prior to the final muscle collapse.     

Regulation of cytoplasmic free calcium by plant cell membranes

The free calcium concentration in the cytoplasm of higher plant cells is believed to be about 10⁻⁷M. The role of various membrane-borne calcium transporters present in plant tissues, together with chloroplasts and mitochondria, in maintaining this calcium concentration is reviewed, together with the role of various organelles in providing transient calcium fluxes upon stimulation.     

Interference Microscopy in Cell Biophysics. 1. Principles and Methodological Aspects of Coherent Phase Microscopy

The coherent phase microscopy (CPM) provides a convenient and non-invasive tool for imaging cells and intracellular organelles. In this article, we consider the applications of the CPM method to imaging different cells and energy-transducing intracellular organelles (mitochondria and chloroplasts). Experimental data presented below demonstrate that the optical path length difference of the object, which is the basic optical parameter measured by the CPM method, can serve as an indicator of metabolic states of different biological objects at cellular and subcellular levels of structural organization.     

Interference Microscopy in Cell Biophysics. 2. Visualization of Individual Cells and Energy-Transducing Organelles

The coherent phase microscopy (CPM) provides a convenient and non-invasive tool for imaging cells and intracellular organelles. In this article, we consider the applications of the CPM method to imaging different cells and energy-transducing intracellular organelles (mitochondria and chloroplasts). Experimental data presented below demonstrate that the optical path length difference of the object, which is the basic optical parameter measured by the CPM method, can serve as an indicator of metabolic states of different biological objects at cellular and subcellular levels of structural organization.     

Vacuolar digestion of entire damaged chloroplasts in Arabidopsis thaliana is accomplished by chlorophagy

In yeast and mammals, selective vacuolar delivery and degradation of whole mitochondria, or mitophagy, represents an important quality control system and is achieved by a cargo recognition mechanism enabling selective elimination of dysfunctional mitochondria. As photosynthetic organelles that need light for energy production, plant chloroplasts accumulate sunlight-induced damage. Plants have evolved multiple mechanisms to avoid, relieve, or repair chloroplast photodamage. Our recent study showed that vacuolar degradation of entire chloroplasts, termed chlorophagy, is induced to degrade chloroplasts that are collapsed due to photodamage. Our results underscore the involvement of autophagy in the quality control of endosymbiotic, energy-converting organelles in eukaryotes.     

Endosymbiosis and the design of eukaryotic electron transport

The bioenergetic organelles of eukaryotic cells, mitochondria and chloroplasts, are derived from endosymbiotic bacteria. Their electron transport chains (ETCs) resemble those of free-living bacteria, but were tailored for energy transformation within the host cell. Parallel evolutionary processes in mitochondria and chloroplasts include reductive as well as expansive events: On one hand, bacterial complexes were lost in eukaryotes with a concomitant loss of metabolic flexibility. On the other hand, new subunits have been added to the remaining bacterial complexes, new complexes have been introduced, and elaborate folding patterns of the thylakoid and mitochondrial inner membranes have emerged. Some bacterial pathways were reinvented independently by eukaryotes, such as parallel routes for quinol oxidation or the use of various anaerobic electron acceptors. Multicellular organization and ontogenetic cycles in eukaryotes gave rise to further modifications of the bioenergetic organelles. Besides mitochondria and chloroplasts, eukaryotes have ETCs in other membranes, such as the plasma membrane (PM) redox system, or the cytochrome P450 (CYP) system. These systems have fewer complexes and simpler branching patterns than those in energy-transforming organelles, and they are often adapted to non-bioenergetic functions such as detoxification or cellular defense.     

Activation of an islet cell plasma membrane (Ca2+ + Mg2+)-ATPase by calmodulin and Ca-EGTA

Islet cell plasma membranes contain a calcium-stimulated and magnesium-dependent ATPase (Ca2+ + Mg2+)-ATPase) which requires calmodulin for maximum enzyme activity (Kotagal, N., Patke, C., Landt, M., McDonald, J., Colca, J., Lacy, P., and McDaniel, M. (1982) FEBS Lett. 137, 249-252). Investigations indicated that exogenously added calmodulin increases the velocity and decreases the Km for Ca2+ of the high affinity (Ca2+ + Mg2+)-ATPase. These studies routinely employed the chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to maintain Ca2+ concentrations in the submicromolar range. During the course of these investigations, it was found unexpectedly that increasing the concentrations of EGTA (0.1-4 mM) and total calcium in the media, while maintaining constant free Ca2+ levels, increased the velocity of the high affinity (Ca2+ + Mg2+)-ATPase. The free calcium concentrations under these conditions were verified by a calcium-sensitive electrode. The (Ca2+ + Mg2+)-ATPase maximally activated by 2-4 mM EGTA was not further stimulated by calmodulin, whereas camodulin stimulation increased as the concentration of EGTA in the media was decreased. A similar enhancement by Ca-EGTA was observed on active calcium transport by the plasma membrane-enriched fraction. Moreover, Ca-EGTA had a negligible effect on both active calcium transport as well as Ca2+-stimulated ATPase activity by the islet cell endoplasmic reticulum, processes which are not stimulated by calmodulin. The results indicate that stimulation by Ca-EGTA may be used to differentiate calcium transport systems by these subcellular organelles. Furthermore, the concentration of EGTA routinely employed to maintain free Ca2+ levels may itself obscure effects of calmodulin and other physiological agents on calcium-dependent activities.