The electrophysiology of chloroplast protein import : the involvement of an anion channel in protein translocation across the inner membrane

The electrophysiological response of the chloroplast envelope during protein import is investigated by using single channel recordings of the chloroplast envelope. An anion channel localised in the inner envelope membrane is identified. The channel has a single channel conductance of 50 pS (chapter 2 and 5). It is found that this channel is inactivated by precursor protein. The precursor induced inactivation of the channel is dependent on the presence of ATP and a functional transit sequence. A deletion mutant of preferredoxin (preFd), that is defective in in vitro import and initial binding, is unable to inactivate the channel (chapter 2). From this it is concluded that the 50 pS anion channel is involved in protein import. The channel is therefore termed P rotein I mport R elated A nion C hannel (PIRAC).Inactivation of PIRAC is shown to require the translocation of precursor protein across the outer membrane and association of the precursor with Tic. In the presence of GTP preFd is unable to inactivate PIRAC (chapter 3). A deletion mutant of preFd (Δ15-25-preFd) is shown to inactivate PIRAC (chapter 3). This deletion mutant can compete with the wild-type precursor for import, but is not imported into the stroma. Furthermore evidence has been presented before that Δ15-25-preFd does interact with Tic. It is therefore concluded that translocation of the precursor across the inner envelope membrane is not required for PIRAC inactivation.Blocking PIRAC activity with the anion channel blocker DIDS leads to a decrease in import efficiency of isolated pea chloroplasts (chapter 3). This indicates that normal functioning of the channel is required for protein translocation.It is shown that the precursor protein induces a long-lived closed state of PIRAC, which is not observed in the absence of precursor (chapter 4). The mean duration of this inactive state is found to be independent of the overall precursor length. In the absence of precursor protein PIRAC is observed to have two distinct open and three distinct closed states (chapter 4). These states can be distinguished on the basis of there mean durations. It is found that the addition of precursor protein decreases the mean duration of one of the open states and two of the closed states (chapter 4). The only states of PIRAC that are unaffected by the addition of precursor are of very short duration (below 1 ms). The precursor induced decrease of the mean duration of PIRAC states is found to be concentration dependent. This leads to the conclusion that there is a direct interaction between the translocating precursor and PIRAC, leading to the switching of the channel to the long-lived inactive state.It is observed that addition of antibodies to Tic110 at the stromal side of an excised inside out patch irreversibly inactivated PIRAC activity (chapter 5). Tic110 is a known component of the translocon of the inner membrane of the chloroplast envelope. It is an integral membrane protein with a large hydrophilic loop facing the stroma. It is therefore concluded that PIRAC is associated with the translocon of the inner membrane of the chloroplast envelope.