Energy-transducing thylakoid membranes remain highly impermeable to ions during protein translocation

Abstract

We investigated the operation of a posttranslational protein translocation pathway to determine whether ions are excluded from the translocase during protein transport. The membrane capacitance during protein translocation across chloroplast thylakoid membranes was monitored via electric-field-indicating carotenoid electrochromic bandshift measurements. Evidence is presented that shows that the membrane ion conductance is not increased during the complete cycle of binding, transport, and substrate release by the ⌬pH-dependent translocase; i.e., the membrane remains iontight during protein translocation. We further demonstrate that a synthetic targeting peptide that directs proteins across this membrane does not gate translocation pores. We conclude that protein transport across the thylakoid membrane does not compromise its ability to maintain ion gradients and is, thus, unlikely to affect its functions in energy transduction.Although a number of experiments indicate that many proteins traverse membranes through aqueous pores, passing from N terminus to C terminus in an extended conformation (1), it is becoming increasingly apparent that some translocases are capable of transporting proteins possessing a variety of structures. Mitochondria and bacterial membrane transporters can accommodate branched (2) and disulfide-bridged (3) proteins, respectively. Chloroplasts have recently been shown to import fully folded polypeptides (4, 5) and the import of oligomerized proteins and even gold particles has been demonstrated in peroxisomes (6-8). In addition, it has been shown for several translocases that the same machinery mediates the transport of both soluble and integral membrane proteins (9, 10). These observations indicate that protein translocases are dynamic and versatile in their ability to transport substrates with different structures into diverse environments. Because proteins have irregular surfaces and translocators have broad specificities, the question arises as to whether ion leakage accompanies protein transport through a pore, thereby compromising the ion permeability barrier of the membrane. This issue has been examined by using artificially induced membrane-spanning translocation intermediates, with some researchers describing an attendant ion leakage and others reporting no accompanying change in ion permeability (11)(12)(13). In addition, the presumed interactions of different targeting peptides with translocators have been reported to alter ion flux, in some cases by opening ion-conducting channels and in other cases by transiently blocking ion movement through active channels (14 -18). In one such report, substratedependent proton flux through the bacterial protein secretion machinery was observed, albeit only when some of the translocon components were overproduced (19). In the present study, we used a native precursor to examine the total ion conductance of the energy-transducing chloroplast thylakoid membrane during uninterrupted translocation cycles of a protein transporter. MATERIAL...

Role of Vesicle‐Inducing Protein in Plastids 1 in cpTat transport at the thylakoid

“…Therefore, it is possible that VIPP1 acts on cpTat transport via the Δψ component of the protonmotive force. To test this hypothesis, we measured the electrochromic shift of carotenoids, a phenomenon in which the pigments undergo an absorption red shift in response to the charge separation across the thylakoid membrane, and that serves as an indicator of the Δψ (Junge and Witt, 1968; Junge, 1982; Teter and Theg, 1998). In order to fully investigate the possible effect of VIPP1 on Δψ, we performed electrochromic shift measurements with untreated thylakoids as well as with thylakoids in which the Δψ had been dissipated through addition of ionophores.…”

Component Specificity for the Thylakoidal Sec and Delta Ph–Dependent Protein Transport Pathways

“…This is interesting because our estimate of the number of active Delta pH translocation sites in pea thylakoids is ‫000,8ف‬ per chloroplast. This was estimated from the V max for transport of iOE23 in an intact chloroplast (8,000/chloroplast per minute; Cline et al, 1993) and from the minimum time required to observe a fully translocated protein on the Delta pH pathway (1 min; Cline, K., unpublished results; Teter and Theg, 1998). This observation suggests that Hcf106 and Tha4 are present in a great excess over the functional translocation sites.…”

Current concepts in nuclear pore electrophysiologyThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

“…We were very fortunate to later find that our discovery of the phenomenon of ion channel plugging by macromolecules and (or) nanoparticles resembled the observation made for protein-conducting ion channels in other intracellular structures (Juin et al 1997;Pelleschi et al 1997;Teter and Theg 1998;Mazzanti et al 2001).…”