Jeanne S. Peters

Chloroplast Inner-Envelope ATPase Acts as a Primary H+ Pump'

The stromal pH of the chloroplast must be maintained higher than that of the surrounding cytosol for photosynthetic carbon assimilation to occur. Experimental evidence demonstrating how this is accomplished in the plant cell is lacking. In the experiments reported here, we studied H+ and K+ flux across membranes of purified chloroplast inner-envelope vesicles. We were able to demonstrate ATP-dependent transport of both cations across the membranes of these vesicles. The data presented document the presence of an H+-pump ATPase in the chloroplast envelope. Energy-dependent K+ flux across these membranes occurs as a consequence of primary H+ pumping. The H+-pumping activity demonstrated in this report is consistent with a model involving the activity of this envelope ATPase as a primary mechanism facilitating a stroma:cytosol [delta]pH.

Characterization of a Chloroplast Inner Envelope K+ Channel

A K+-conducting protein of the chloroplast inner envelope was characterized as a K+ channel. Studies of this transport protein in the native membrane documented its sensitivity to K+ channel blockers. Further studies of native membranes demonstrated a sensitivity of K+ conductance to divalent cations such as Mg2+, which modulate ion conduction through interaction with negative surface charges on the inner-envelope membrane. Purified chloroplast inner-envelope vesicles were fused into an artificial planar lipid bilayer to facilitate recording of single-channel K+ currents. These single-channel K+ currents had a slope conductance of 160 picosiemens. Antibodies generated against the conserved amino acid sequence that serves as a selectivity filter in the pore of K+ channels immunoreacted with a 62-kD polypeptide derived from the chloroplast inner envelope. This polypeptide was fractionated using density gradient centrifugation. Comigration of this immunoreactive polypeptide and K+ channel activity in sucrose density gradients further suggested that this polypeptide is the protein facilitating K+ conductance across the chloroplast inner envelope.

Evidence for cytochrome f involvement in eggplant cell death induced by palmitoleic acid.

In response to certain stimuli, animal and plant cells activate genetic and biochemical programs resulting in cell death, a process known as PCD. A large volume of information on animal PCD is now available. In contrast, less is known about plant PCD. It has been shown that there are certain morphological and biochemical similarities between plant and animal PCD. Thus, it is useful to compare the PCD of plants and animals in developing strategies to study PCD in plants. One of the features found in certain PCD is the release of cyt c from mitochondria to the cytosol. This initiates a process leading to DNA fragmentation, and eventually to biochemical execution of the cell. The evolutionary and biological significance of the involvement of the mitochondrion, an organelle known for its life-sustaining role with its energy conversion ability, in PCD is not clear. Plant cells have another energy conversion organelle, that is, the chloroplast. While the mitochondrion converts one form of chemical energy to another, the chloroplast converts light energy to chemical energy. In spite of this difference, both organelles utilize electron transport chains in their energy conversion. There are similarities as well as variations among the components of the electron transport chains of these two organelles. For example, cyt f is found in chloroplasts but not in mitochondria. Cyt f is a c-type cytochrome characterized by the covalent attachment of the heme to the polypeptide chain via thioether linkages, which are formed from two vinyl groups of heme and two cysteine residues provided by the CXXCH motif sequence (X denotes any amino acid). We have previously found that cyt f release from eggplant chloroplasts could be induced by palmitoleic acid (16:1). We have also observed that 16:1 induces death of eggplant cells. While the chloroplast has been found to be associated with certain forms of cell death we are not aware of any information on the involvement of cyt f in the process. Here, we report our investigation on whether cyt f release plays a role in the death of eggplant cells. Cultured eggplant cells treated with 16:1 underwent cytoplasmic shrinkage and condensation (data not shown). 16:1 also caused shrinkage and collapse of eggplant protoplasts (Figure 1a). As it was easier to monitor protoplasts than irregularly shaped cells and cell colonies, we used protoplasts in this study. Death of protoplasts was accompanied by changes in their nuclei. Nuclei of untreated protoplasts stained with DAPI (4,6-diamidino-2-phenylindole) exhibited bright blue fluorescence which stayed unchanged throughout the experiment (Figure 1b), but the fluorescence of the nuclei of the treated protoplasts gradually became diffuse, dispersed, and eventually disappeared (Figure 1a). These changes were concurrent with protoplast shrinkage and collapse. DNA damage was also revealed by the TUNEL assay for nicked DNA and DNA breaks as 16:1 treatment rendered the protoplasts TUNEL positive (Figure 1c). Release of cyt f was probed with an antiserum raised against a synthetic peptide corresponding to the first 21 amino acids of the mature N-terminus of the cyt f protein. Two cyt f proteins (38 and the 39 kDa) were found in the membrane fraction and none in the cytosolic fraction. 16:1 treatment led to the disappearance of the 38 kDa cyt f from the membranes and its appearance in the cytosol, suggesting its release from the membranes into the cytosol (Figure 1d). The release was detected within 10 min of treatment (Figure 1e). Thus, cyt f release apparently preceded protoplast collapse and nuclear disintegration, which was not evident until 20 min after treatment. 16:1 could directly affect chloroplasts (Figure 1f). Intact chloroplasts possess three membranes (outer, inner, and thylakoid). Under a phase contrast microscope an isolated chloroplast is seen to possess a halo, which is dependent on the integrity of the outer and the inner envelopes. As such, the halo is a convenient marker for chloroplast integrity. 16:1 treatment was found to cause shrinkage of the outer membrane, but swelling of the area surrounding the thylakoid membranes, leading to the rupture of the chloroplast as manifested by the disappearance of its halo within approximately 7.5 min (Figure 1f). Electron microscopy study showed that the in vivo chloroplasts within a protoplast treated with 16:1 also collapsed but it took over 12 min (Figure 1g, h). The release of cyt c from mitochondria has been found to be associated with the swelling and rupture of mitochondria. Chloroplast rupture was also associated with cyt f release (Figure 1i). In untreated chloroplasts, cyt f proteins (38 and 39 kDa) were only found in the thylakoid membranes (Figure 1i). Following 16:1 treatment, while the 39-kDa cyt f remained in the thylakoids, the 38-kDa cyt f now was found in the stromal extract (Figure 1j). It has been demonstrated that Ba2þ is able to block both cyt c release and death of Hela cells treated with apoptotic-inducing agents. Ba2þ was also found to prevent the release of cyt f and provide relatively effective protection to the protoplasts from collapse and nuclear damage induced by 16:1 (Figure 1k, 1l). Spermine (Spm) also prevented cyt f release, but was less effective in protecting the protoplasts from collapse (Figure 1l, Cell Death and Differentiation (2005) 12, 405–407 & 2005 Nature Publishing Group All rights reserved 1350-9047/05

Characterization of a chloroplast inner envelope P-ATPase proton pump

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Pectin Methylesterase Regulates Methanol and Ethanol Accumulation in Ripening Tomato (Lycopersicon esculentum) Fruit

P-ATPases such as the plasma membrane proton pump are known to generate a phosphorylated intermediate as a step in their reaction mechanism; phosphoenzyme formation is a basis for classification of an ATPase as a member of this subfamily of ion pumps. The chloroplast inner envelope is known to contain a H+-ATPase which acts to maintain an alkaline stroma and, thus, optimal photosynthesis. Our characterization of this chloroplast envelope proton pump described in this report focused on determining whether purified chloroplast inner envelope membrane protein preparations containing this ATPase form a phosphorylated intermediate. Incubation of envelope membranes with [γ-32P]ATP documented the formation of P-type ATPase phosphoenzyme intermediates by these membrane protein preparations. Our work cannot discount the possibility that more than one chloroplast inner envelope ATPase contributes to this phosphoenzyme formation. However, the kinetics of this phosphoenzyme formation, along with the sensitivity of phosphoenzyme formation to inhibitors and other assay conditions suggested that one of the envelope membrane proteins which is covalently radiolabeled by [γ-32P]ATP is a P-type H+-ATPase. Autoradiography of chloroplast envelope membrane proteins size fractionated on lithium dodecyl sulfate-PAGE indicated that the phosphoenzyme intermediate corresponds to a 103 kDa polypeptide. P-type proton pumps are known to be comprised of a single type of ∼100 kDa subunit. Experimental evidence presented in this report is consistent with the classification of a chloroplast inner envelope H+-ATPase as a P-type proton pump.