Spatial coordination of chloroplast and plasma membrane activities in Chara cells and its disruption through inactivation of 14-3-3 proteins.

In Chara corallina cells exposed to continuous light, external pH (pH(o)) and photosystem II (PSII) photochemical yield show correlated banding patterns. Photosynthetic activity is low in cell regions producing alkaline zones and high in the acid regions. We addressed the question whether (and how) photosynthetic activity and plasma membrane (PM) H+-pumping and H+-conductance are coupled in the different bands. First, PM H+-pump activity was stimulated with fusicoccin. This resulted in a more acidic pH in the acid bands without disturbing the correlation of photosynthetic electron transport and H+ fluxes across the PM. Next, H+-pump activity was reduced through microinjection of a phosphorylated peptide matching the canonical 14-3-3 binding motif RSTpSTP in the acid cell region. Microinjection induced a rapid (~5 min) rise in pH(o) by ca. 1.0 unit near the injection site, whereas the injection of the non-phosphorylated peptide had no effect. This pH rise confirms the supposed inhibition of the H+-pump upon the detachment of 14-3-3 proteins from the H+-ATPase. However, the PSII yield in the cell regions corresponding to the new alkaline peak remained high, which violated the normal inverse relations between the pH(o) and PSII photochemical yield. We conclude that the injection of the competitive inhibitor of the H+-ATPase disrupts the balanced operation of PM H+-transport and photosynthetic electron flow and promotes electron flow through alternative pathways.

Slow vacuolar channels from barley mesophyll cells are regulated by 14-3-3 proteins.

The conductance of the vacuolar membrane at elevated cytosolic Ca(2+) levels is dominated by the slow activating cation selective (SV) channel. At physiological, submicromolar Ca(2+) concentrations the SV currents are very small. Only recently has the role of 14-3-3 proteins in the regulation of voltage-gated and Ca(2+)-activated plasma membrane ion channels been investigated in Drosophila, Xenopus and plants. Here we report the first evidence that plant 14-3-3 proteins are involved in the down-regulation of ion channels in the vacuolar membrane as well. Using the patch-clamp technique we have demonstrated that 14-3-3 protein drastically reduces the current carried by SV channels. The current decline amounted to 80% and half-maximal reduction was reached within 5 s after 14-3-3-addition to the bath. The voltage sensitivity of the channel was not affected by 14-3-3. A coordinating role for 14-3-3 proteins in the regulation of plasma membrane and tonoplast ion transporters is discussed.

Further analysis of the involvement of the envelope anion channel PIRAC in chloroplast protein import.

The ability of preferredoxin to inactivate a 50-pS anion channel of the chloroplast inner membrane in the presence of an energy source was investigated using single-channel recordings. It was found that preferredoxin cannot inactivate the channel when GTP is the only energy source present. From this it is concluded that the precursor has to interact with the, translocon of the inner membrane of chloroplasts (Tic) complex to be able to inactivate the 50-pS anion channel. The ability of two mutants of preferredoxin with deletions in their transit sequence to inactivate the channel was also tested. Both mutants have been shown to have a similar binding affinity for the chloroplast envelope, but only one is able to fully translocate. The mutants were both able to inactivate the channel in a similar manner. From this it is concluded that full translocation is not necessary for the inactivation of the channel. It is also shown that preferredoxin is capable of inactivating the 50-pS anion channel in the chloroplast-attached configuration as was previously found in the inside-out configuration. From this it is concluded that stromal factors do not influence the protein-import-induced inactivation of the 50-pS anion channel of the chloroplast inner membrane. Finally the effect of the anion channel blocker 4, 4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) on the channel activity and on protein import was investigated. It was found that DIDS blocked the channel. Furthermore the addition of the channel blocker reduces the efficiency of import to 52%. This leads to the conclusion that correct functioning of the channel is important for protein import.

The mechanism of inactivation of a 50-pS envelope anion channel during chloroplast protein import.

The mechanism of import-competent precursor protein-induced inactivation of a 50-pS anion channel of the chloroplast envelope is investigated using single-channel recordings. The inactivation by precursor protein is the result of the induction of a long-lived closed state of the channel. The mean duration of this state does not depend on precursor concentration. From this it can be concluded that the protein import related anion channel enters the inactive state less frequently when the precursor concentration is lowered, but that the time spent in this state remains the same. Furthermore, it was found that the presence of precursor protein also decreases the mean durations of preexisting open and closed states of the channel. This decrease is found to be dependent on the precursor concentration. From this it is concluded that there is a direct interaction between the precursor protein and a protein complex of which the channel is a constituent. The mean duration of the precursor-induced long-lived closed state does not depend on the length of the translocation-competent precursor. This suggests that the duration of import is independent of precursor length.

The C terminus of a chloroplast precursor modulates its interaction with the translocation apparatus and PIRAC.

The import of proteins into chloroplasts involves a cleavable, N-terminal targeting sequence known as the transit peptide. Although the transit peptide is both necessary and sufficient to direct precursor import into chloroplasts, the mature domain of some precursors has been shown to modulate targeting and translocation efficiency. To test the influence of the mature domain of the small subunit of Rubisco during import in vitro, the precursor (prSSU), the mature domain (mSSU), the transit peptide (SS-tp), and three C-terminal deletion mutants (Delta52, Delta67, and Delta74) of prSSU were expressed and purified from Escherichia coli. Activity was then evaluated by competitive import of (35)S-prSSU. Both IC(50) and K(i) values consistently suggest that removal of C-terminal prSSU sequences inhibits its interaction with the translocation apparatus. Non-competitive import studies demonstrated that prSSU and Delta52 were properly processed and accumulated within the chloroplast, whereas Delta67 and Delta74 were rapidly degraded via a plastid-localized protease. The ability of prSSU-derived proteins to induce inactivation of the protein-import-related anion channel was also evaluated. Although the C-terminal deletion mutants were less effective at inducing channel closure upon import, they did not effect the mean duration of channel closure. Possible mechanisms by which C-terminal residues of prSSU modulate chloroplast targeting are discussed.

The envelope anion channel involved in chloroplast protein import is associated with Tic110.

An anion channel of the chloroplast envelope was previously shown to be involved in protein import. Some gating characteristics of the channel are presented. The pore size of the channel is estimated to be around 6.5 A. Antibodies raised to Tic110 completely inactivate the protein import-related channel. These observations suggest that the channel is associated with the Tic machinery and can function as the protein conducting channel of the inner envelope membrane.

The tachykinin NK1 receptor antagonist, RP67580, inhibits the bradykinin-induced rise in intracellular Ca2+ concentration in bovine pulmonary artery endothelial cells.

The bradykinin-induced rise in intracellular Ca2+ concentration ([Ca2+]i) and the bradykinin receptor involved in this response were characterized in bovine pulmonary artery endothelial cells. It was found that bradykinin induces an intracellular biphasic Ca2+ response, consisting of a transient peak followed by an elevated plateau phase. Both bradykinin and the bradykinin B1 receptor agonist, des-Arg9-bradykinin, induced a concentration-dependent increase in [Ca2+]i, but the bradykinin-induced rise was much greater. Moreover, the bradykinin-induced [Ca2+]i rise could be inhibited by the bradykinin B2 receptor antagonists, D-Arg0[Hyp3, Thi(5,8), D-Phe7]bradykinin and Hoe 140 (D-Arg[Hyp3, Thi5, D-Tic7, Oic8]bradykinin), but not by the bradykinin B1 receptor antagonist, des-Arg9-[Leu8]bradykinin. From these results it can be concluded that a bradykinin B2 receptor is involved in this response. Furthermore, we found that the tachykinin NK1 receptor antagonist, RP67580 ([imino 1 (methoxy-2-phenyl)-2 ethyl]-2 diphenyl 7,7 perhydroisoindolone-4 (3aR, 7aR)), and its negative enantiomer, RP68651 (2-[1-imino 2-(2 methoxy phenyl) ethyl] 7,7 diphenyl 4-perhydroisoindolone (3aS-7aS)), could inhibit the bradykinin-induced [Ca2+]i response, although no functional tachykinin NK1 receptors were found. Binding studies evidenced no binding of RP67580 or RP68651 to the bradykinin receptor. We conclude that RP67580 inhibits the bradykinin-induced rise in [Ca2+]i via a bradykinin B2 receptor-independent mechanism.

A 50-picosiemens anion channel of the chloroplast envelope is involved in chloroplast protein import.

Single channel recordings were used to investigate the changes on the pea chloroplast envelope during protein import. In the inside-out patch configuration a 50-picosiemens (pS) anion channel of the chloroplast envelope membrane was identified. The open time probability of the channel was decreased by the addition of the wild type precursor protein of ferredoxin (wt-prefd) to the pipette-filling solution in the presence of 0.5 mM ATP. In the absence of ATP or in the presence of 50 microM ATP, wt-prefd did not affect the open time probability of the channel. A deletion mutant of prefd, Delta6-14-prefd, which is inactive in in vitro import, was also unable to affect the open time probability of the 50-pS anion channel. In the presence of 100 microM ATP, wt-prefd decreased the open time probability of the channel to a lesser extent, as did the transit peptide alone. It is concluded that the 50-pS anion channel could be part of the protein import machinery of the inner membrane. In addition the precursor protein under import conditions induced burst-like increases of the envelope conductivity. The implication of both responses for the chloroplast protein import process are discussed.

Anionic phospholipids can mediate membrane insertion of the anionic part of a bound peptide.

The effect of anionic lipids on the membrane insertion of a carboxyl group on a specially designed palmitoylated peptide was studied, using tryptophan fluorescence. It is demonstrated that the negatively charged membrane surface of mixed phosphatidylcholine/phosphatidylglycerol small unilamellar vesicles enhances the protonation of the C-terminal carboxyl group, and the subsequent insertion of that part of the peptide.