The presequence of a chimeric construct dictates which of two mechanisms are utilized for translocation across the thylakoid membrane: evidence for the existence of two distinct translocation systems.

Abstract

The translocation of plastocyanin across the thylakoid membrane in Pisum sativum has been studied in reconstitution assays and using chimeric constructs. The reconstitution assays demonstrate that plastocyanin translocation is absolutely dependent on the presence of a stromal factor(s) and nucleotide triphosphates (NTPs), whereas neither element is required for the translocation of the 23 or 16 kDa proteins of the oxygen-evolving complex. Previous studies had revealed that the transthylakoidal delta pH is essential for translocation of the 23 and 16 kDa proteins but unnecessary for plastocyanin translocation. The basis for these mechanistic differences has been tested by analysing the translocation of a chimeric construct consistng of the presequence of the 23 kDa protein linked to the mature plastocyanin sequence. This construct is efficiently imported into thylakoids in the absence of stromal extracts or NTPs and translocation across the thylakoid membrane within intact chloroplasts is totally inhibited by the uncoupler nigericin: the translocation requirements are thus identical to those of the pre-23 kDa protein and diametrically opposite to those of preplastocyanin. Transport across the thylakoid membrane of a second fusion protein, consisting of the presequence of the 16 kDa protein linked to mature plastocyanin, is also dependent on a delta pH. The data suggest that two distinct systems are involved in the translocation of proteins across the thylakoid membrane, with each system recognizing specific signals within the presequences of a subset of lumenal protein precursors.

Chloroplast SRP54 Interacts with a Specific Subset of Thylakoid Precursor Proteins

“…Interactions between nascent polypeptides and other stromal components, for example, SecA, may require longer chains than those used in this study and no evidence of any interaction between short nascent polypeptides and E. coli SecA was obtained in a recent cross-linking study (57). In contrast to the other thylakoid proteins used in this study, the OE23 protein has been shown to be transported across the thylakoid membrane by a distinct, ⌬pH-dependent, route (26,27,32). As with OE33, we found no evidence of 54CP being cross-linked to a truncated OE23 polypeptide.…”

“…Particular precursors have been proposed to utilize one of the following pathways: 1) Sec A-dependent (20,21); 2) SRP-dependent (22); 3) ⌬pH-dependent (23, 24); and 4) "spontaneous integration" (25). Pathway selection by a thylakoid lumen-resident precursor seems to be a function of the thylakoid-targeting signal that it bears (26,27). A well characterized example is the twinarginine motif which has been shown to be a critical determinant for entry into the ⌬pH-dependent pathway (28).…”

Analysis of the Import of Carboxyl-Terminal Truncations of the 23-Kilodalton Subunit of the Oxygen-Evolving Complex Suggests That Its Structure Is an Important Determinant for Thylakoid Transport

“…A difficulty is encountered with the interpretation that the pH gradient-dependent transporter responsible for iOE23 translocation into the thylakoid lumen requires the native conformation of the substrate polypeptide when one considers that other passenger proteins can be made to follow this pathway by attachment of the appropriate transit peptide (Henry et al, 1994;Robinson et al, 1994). This difficulty can be met by relaxing the requirement for particular structures to allow only a bias toward specific structural components.…”

Transport of CtpA Protein from the Cyanobacterium Synechocystis 6803 Across the Thylakoid Membrane in Chloroplasts

“…249) 1995; Laidler et al, 1995;Nohara et al, 1995;Berghofer and Klosgen, 1996), and is assumed to be simlar in mechanistic terms also to the Sec pathway of bacteria, the main secretion route for proteins in prokaryotes (Pugsley, 1993). This pathway is specific for plastocyanin, the 33-kDa subunit of the oxygenevolving system and the photosystem I subunit F (Theg et al, 1989;Mould et al, 1991 ;Hulford et al, 1994;Kamauchov et al, 1994;Mant et al, 1994;Robinson et al, 1994;. The second thylakoid transport pathway for proteins carrying bipartite transit peptides, used for example by the 16-kDa and 23-kDa subunits of the oxygen-evolving system, photosystem I subunit N and photosystem 11 subunit T, does not require nucleoside triphosphates nor soluble factors (Mould et al, 1991;Cline et al, 1992;Klosgen et al, 1992;., 1994; Kapazoglou et al, 1995).…”