Development and Use of Chlorotetracycline Fluorescence as a Measurement Assay of Chloroplast Envelope-Bound Mg<sup>2+</sup>

Gupta, Apoorva; Berkowitz, Gerald A.

doi:10.1104/pp.89.3.753

Cited by 13 publications

(20 citation statements)

References 22 publications

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“…As shown in Table I, this Mg2+-wash treatment resulted in both stromal acidification and photosynthetic inhibition, mimicking the effect of high reaction medium Mg2+ (15). The Mg2+-induced photosynthetic inhibition has been previously shown to be reversed by high K+ in the reaction medium (22). This effect of K4 is likely not due to a displacement of Mg2+ from binding sites on the chloroplast envelope (15,22).…”

Section: Chloroplast Isolationmentioning

confidence: 63%

“…(As discussed by Sen Gupta and Berkowitz [22], this grind medium contains approximately 45 nm free EDTA and Mg2+, and essentially no free Mn2+.) The homogenate was filtered through four layers ofcheesecloth and two layers ofMiracloth, and then centrifuged (SS34 rotor and RC5B centrifuge; Sorvall, Wilmington, DE) for 50 s at 750g.…”

Section: Chloroplast Isolationmentioning

confidence: 87%

“…Data presented in Table I confirm this assertion. Incubation of intact plastids during the isolation protocol in solutions containing free Mg2+, and then quantitative removal of the chloroplasts from these solutions, results in relatively high levels of envelope-bound Mg2+ (22). As shown in Table I, this Mg2+-wash treatment resulted in both stromal acidification and photosynthetic inhibition, mimicking the effect of high reaction medium Mg2+ (15).…”

Section: Chloroplast Isolationmentioning

confidence: 97%

“…resulted in photosynthetic inhibition of isolated chloroplasts. Previous work in this laboratory (22) using chlorotetracycline fluorescence as a monitor of chloroplast envelope-bound Mg2+ led to the speculation that it was not the presence of millimolar concentrations of unchelated Mg2+ in the reaction medium, but rather a high extent of envelope-bound Mg2+ that affected chloroplast metabolism. Data presented in Table I confirm this assertion.…”

Section: Chloroplast Isolationmentioning

confidence: 99%

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Studies on the System Regulating Proton Movement across the Chloroplast Envelope

Peters¹,

Berkowitz²

1991

Plant Physiol.

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Studies were undertaken to further characterize the spinach (Spinacea oleracea) chloroplast envelope system, which facilitates H+ movement into and out of the stroma, and, hence, modulates photosynthetic activity by regulating stromal pH. It was demonstrated that high envelope-bound Mg2+ causes stromal acidification and photosynthetic inhibition. High envelope-bound Mg2+ was also found to necessitate the activity of a digitoxinand oligomycin-sensitive ATPase for the maintenance of high stromal pH and photosynthesis in the illuminated chloroplast. In chloroplasts that had high envelope Mg2+ and inhibited envelope ATPase activity, 2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide was found to raise stromal pH and stimulate photosynthesis. 2-(Diethylamino)-N-(2,6-dimethylphenyl)acetamide is an amine anesthetic that is known to act as a monovalent cation channel blocker in mammalian systems. We postulate that the system regulating cation and Ho fluxes across the plastid envelope includes a monovalent cation channel in the envelope, some degree of (envelope-bound Mg2 modulated) H+ flux linked to monovalent cation antiport, and ATPase-dependent H+ efflux.Upon illumination, the pH of the spinach chloroplast stroma rises by approximately 0.4 to 0.5 pH unit (11). This stromal alkalinization is a regulatory mechanism that facilitates optimal photosynthesis in the light (24), primarily due to the high pH optima ofthe photosynthetic carbon reduction cycle enzymes fructose 1,6-bisphosphatase and sedoheptulose 1,7-bisphosphatase (1, 8). For quite some time, the rise in stromal pH in the light has been primarily attributed to H+ pumping into the thylakoid lumen (1 1, 24). However, optimal photosynthetic capacity, both ofisolated chloroplasts ( 11) and in situ in illuminated leaves (17) (7), Huber and co-workers (13, 15) concluded that a K+/H+ antiporter facilitated this exchange. This hypothesized antiport mechanism was thought to result in either stromal acidification or alkalinization, depending on the extrachloroplastic monovalent cation concentration. However, in later studies, Demmig and Gimmler (6, 7) speculated that H+ pumping into the thylakoid during illumination is not entirely chargebalanced, and the resultant membrane potential developed between the stroma and external solution (inside negative) drives K+ and/or H+ movement into the stroma. K+ efflux was thought to result in H+ influx due to changes in this membrane potential. They concluded, in contrast to Huber, that K+/H+ exchange across the envelope was not facilitated by specific intrinsic envelope proteins acting as "antiporters."The studies of 15)

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Section: Chloroplast Isolationmentioning

confidence: 63%

Section: Chloroplast Isolationmentioning

confidence: 87%

Section: Chloroplast Isolationmentioning

confidence: 97%

Section: Chloroplast Isolationmentioning

confidence: 99%

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Studies on the System Regulating Proton Movement across the Chloroplast Envelope

Peters¹,

Berkowitz²

1991

Plant Physiol.

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“…These glycolipids could provide the negative surface charges on the native membrane that interact with Mg2+, resulting in the screening effect that impacts K+ conductance through the channel protein. The effect Mg2+ has on K+ flux through the channel is quite profound and may be an important regulatory mechanism affecting photosynthetic capacity of the chloroplast (Huber and Maury, 1980;Sen Gupta and Berkowitz 1989;Berkowitz and Wu, 1993).…”

Section: Results and Discussion Lnhibitor Effects On K+ Flux Across Tmentioning

confidence: 99%

Characterization of a Chloroplast Inner Envelope K+ Channel

Peters

Berkowitz

1994

Plant Physiol.

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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 MgZ+, 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.Studies with intact chloroplasts (Wu and Berkowitz, 1992) indicate that K+ flux across the inner envelope occurs through a well-regulated transport pathway that is likely a K+-conducting ion channel. K' flux into or out of the chloroplast stroma is indirectly linked to H+ counterexchange (Maury et al., 1981; Wu and Berkowitz, 1992). Movement of K+ through this channel protein, therefore, has a profound effect on stromal pH and, hence, photosynthesis (Werdan et al., 1975;Maury et al., 1981; Wu and Berkowitz, 1992). Previous work from this laboratory has demonstrated that a K'-conducting protein can be successfully detergent-solubilized from preparations of purified inner-envelope membrane vesicles and functionally reconstituted into artificial liposomes (Wang et al., 1993). This putative K+ channel protein has not previously been characterized. In the work reported here, we used severa1 approaches to characterize the nature of this K+ transport protein. Results of these experiments are consistent with the presence of a K+ channel in the inner envelope that shares some structural, functional, and regulatory properties with K' channels from other membrane systems ( e g Rudy et al., 1991; Anderson et al., 1992). 955 MATERIALS AND METHODS Preparation of Chloroplast Inner-Envelope Membrane VesiclesInner-envelope vesicles were prepared from spinach (Spinacia oleracea L.) as described previously (Berkowitz and Peters, 1993). Briefly, intact chloroplasts (isolated from 5-10 kg of spinach leaves using Perco11 step gradients) were exposed to freeze/thaw cycles in hyperosmotic medium, and the thylakoids were removed before the crude membrane fraction was loaded on a discontinuous SUC step gradient. Inner-envelope vesicles were removed from the 0.8 ~/ 0 . 4 6 M Suc interface of the gradient, washed in envelope medium (0.2 M SUC, 2 mM Na2EDTA, 2 mM DTT, and 10 m~ TricineNaOH, pH 7.5), a...

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A unique protein-kinase activity is responsible for the phosphorylation of the 26- and 14-kDa proteins but not of the 67-kDa protein in the chloroplast envelope membranes of spinach

Siegenthaler

Bovet

1993

Planta

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Abstract. Protein-phosphorylation activity has been reported in chloroplast envelope membranes of several species. In spinach (Spinacia oteraeea L.), we found three major phosphoproteins after incubation in vitro of envelope membranes in the presence of [?-3Zp]ATP. A 67-kDa phosphoprotein was associated with both inner and outer envelope membranes whereas 26-and 14-kDa proteins were observed in the inner membrane. Although the phosphorylation of the 67-kDa protein is likely to take place via its phosphoglucomutase activity (Salvucci et al., 1990, Plant Physiol. 93, 105-109), the mechanism by which 32p is incorporated into the 26-and 14-kDa proteins remains to be elucidated. To this aim, we have compared the conditions under which phosphorylation occurs in these three proteins. The effects of Mg 2+, Ca 2+, pH, ATP and H7 [l-(5-isoquinolinesulfonyl)-2-methylpiperazine], a specific inhibitor of protein-kinase C, as well as pulse-chase experiments with cold ATP, showed that the phosphorylation mechanism was identical for the 26-and 14-kDa proteins but quite different for the 67-kDa one. The protein kinase involved in the phosphorylation of the 26-and 14-kDa proteins was Ca 2+-dependent, which was not the case of the 67-kDa protein. In addition, the use of a Triton X-114 phase-separation treatment indicated that both the 26-and 14-kDa proteins exhibited strong hydrophobic properties, in contrast to the hydrophilic character of the 67-kDa phosphoprotein. As indicated by analyses of phosphoamino acids, the three proteins were exclusively phosphorylated on serine residues. Furthermore, a treatment of envelopes by phospholipase C prior to the phosphorylation process inhibited 32p incorporation into the three phosphoproteins to different extents (61%, 50% and 29% inhibition for the 67-, 14-and 26-kDa proteins, respectively). These results show that phosphatidylcholine and -or phosphatidylglycerol but not phosphatidylinositol were involved in this phosphorylation process.

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Development and Use of Chlorotetracycline Fluorescence as a Measurement Assay of Chloroplast Envelope-Bound Mg²⁺

Cited by 13 publications

References 22 publications

Studies on the System Regulating Proton Movement across the Chloroplast Envelope

Studies on the System Regulating Proton Movement across the Chloroplast Envelope

Characterization of a Chloroplast Inner Envelope K+ Channel

A unique protein-kinase activity is responsible for the phosphorylation of the 26- and 14-kDa proteins but not of the 67-kDa protein in the chloroplast envelope membranes of spinach

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Development and Use of Chlorotetracycline Fluorescence as a Measurement Assay of Chloroplast Envelope-Bound Mg2+

Cited by 13 publications

References 22 publications

Studies on the System Regulating Proton Movement across the Chloroplast Envelope

Studies on the System Regulating Proton Movement across the Chloroplast Envelope

Characterization of a Chloroplast Inner Envelope K+ Channel

A unique protein-kinase activity is responsible for the phosphorylation of the 26- and 14-kDa proteins but not of the 67-kDa protein in the chloroplast envelope membranes of spinach

Contact Info

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Development and Use of Chlorotetracycline Fluorescence as a Measurement Assay of Chloroplast Envelope-Bound Mg²⁺