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 (⌬52, ⌬67, and ⌬74) 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 ⌬52 were properly processed and accumulated within the chloroplast, whereas ⌬67 and ⌬74 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.

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 M ATP, wt-prefd did not affect the open time probability of the channel. A deletion mutant of prefd, ⌬6 -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 M 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.A large part of the chloroplast proteins is nuclear encoded. These proteins are synthesized in the cytosol and have to be imported into the chloroplast. Nuclear encoded chloroplast proteins are therefore synthesized with an N-terminal extension called the transit sequence. The transit sequence is both necessary and sufficient to target a protein to the chloroplast (1). Several components of the chloroplast import machinery have been identified (2, 3). A role for chloroplast envelope channels has been proposed in protein import (2-4). However, little is known about the function of channels in the import process and the response of the envelope during protein import. An increase in envelope conductivity during protein import is shown to occur in Peperomia metallica chloroplasts (4). This increase was identified using electrophysiological measurements in the whole chloroplast configuration. It was suggested that the opening of protein translocation channels is the cause of the observed response of the envelope.Ion channels in the chloroplast envelope are also thought to be involved in osmoregulation during photosynthesis. Several ion channels of the inner as well as the outer envelope membrane have been identified by reconstitution of the channels in giant liposomes or in planar lipid bilayers (5-8). Direct electrophysiological measurements on isolated chloroplasts have also revealed a number of envelope ion channels (9, 10).In this report the use of single channel recordings in investigating the role of chloroplast envelope components in protein import is described for the first time. The involvement of a chloroplast envelope anion channel in protein import is identified. A hitherto unidentified electrical response of the chloroplast envelope associated with protein import is also described. The implications of both responses for the chloroplast protei...

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 halfmaximal 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. ß

Summary Germination of seeds proceeds in general in two phases, an initial imbibition phase and a subsequent growth phase. In grasses like barley, the latter phase is evident as the emergence of the embryonic root (radicle). The hormone abscisic acid (ABA) inhibits germination because it prevents the embryo from entering and completing the growth phase. Genetic and physiological studies have identified many steps in the ABA signal transduction cascade, but how it prevents radicle elongation is still not clear. For elongation growth to proceed, uptake of osmotically active substances (mainly K+) is essential. Therefore, we have addressed the question of how the activity of K+ permeable ion channels in the plasma membrane of radicle cells is regulated under conditions of slow (+ABA) and rapid germination (+fusicoccin). We found that ABA arrests radicle growth, inhibits net K+ uptake and reduces the activity of K+in channels as measured with the patch‐clamp technique. In contrast, fusicoccin (FC), a well‐known stimulator of germination, stimulates radicle growth, net K+ uptake and reduces the activity of K+out channels. Both types of channels are under the control of 14‐3‐3 proteins, known as integral components of signal transduction pathways and instrumental in FC action. Intriguingly, 14‐3‐3 affected both channels in an opposite fashion: whereas K+in channel activity was fully dependent upon 14‐3‐3 proteins, K+out channel activity was reduced by 14‐3‐3 proteins by 60%. Together with previous data showing that 14‐3‐3 proteins control the activity of the plasma membrane H+‐ATPase, this makes 14‐3‐3 a prime candidate for molecular master regulator of the cellular osmo‐pump. Regulation of the osmo‐pump activity by ABA and FC is an important mechanism in controlling the growth of the embryonic root during seed germination.