Targeting of proteins to the thylakoids by bipartite presequences: CFoII is imported by a novel, third pathway.

Abstract

The CFoII subunit of the ATP synthase is an integral component of the thylakoid membrane which is synthesized in the cytosol with a bipartite, lumen‐targeting presequence similar in structural terms to those of imported lumenal proteins such as plastocyanin. This presequence is shown to possess a terminal cleavage site for the thylakoidal processing peptidase, but no intermediate site for the stromal processing peptidase. The integration of CFoII into the thylakoid membrane of Pisum sativum has been analysed using in vitro assays for the import of proteins into intact chloroplasts or isolated thylakoids. Efficient integration into thylakoids is observed in the light and dark, and the integration process does not require the presence of either stromal extracts or nucleoside triphosphates. The uncoupler nigericin inhibits integration only very slightly, indicating that the thylakoidal delta pH does not play a significant role in the integration mechanism. In each of these respects, the requirements for CFoII integration differ notably from those determined for integration of the light‐harvesting chlorophyll‐binding protein of photosystem II. The integration mechanism also differs significantly from the two mechanisms involved in the translocation of lumenal proteins across the thylakoid membrane, since one of these processes requires the presence of stromal protein factors and ATP, and the other mechanism is dependent on the thylakoidal delta pH. This conclusion is reinforced by the finding that saturation of the translocation system for the precursor to the lumenal 23 kDa oxygen‐evolving complex protein does not affect integration of CFoII into thylakoids.(ABSTRACT TRUNCATED AT 250 WORDS)

Sec-independent Insertion of Thylakoid Membrane Proteins

“…1, the three thylakoid proteins share additional common features with procoat: each contains a single transmembrane span in the mature protein and the translocated regions are all negatively charged. It was thus unsurprising that initial models for the Sec-independent insertion of CF o II (25) were based on the procoat insertion model, and this study has confirmed that in one fundamental respect the insertion of PSII-W does indeed resemble that of procoat. Both proteins adopt a loop intermediate during the insertion process, strongly suggesting that the function of the PSII-W signal peptide is to provide a second hydrophobic domain which, in concert with the transmembrane segment in the mature protein, is then able to drive the translocation of the hydrophilic intervening region into the thylakoid lumen.…”

“…Gallusser and Kuhn (32) have shown that electrostatic interactions play a critical role in the insertion of procoat, in which basic residues in both the extreme N and C termini bind to the negatively charged membrane surface; removal of either set of basic residues renders procoat wholly insertion incompetent. Like procoat, pre-CF o II contains positively charged N and C termini and a similar mechanism was considered possible for this protein (25). However, the C-terminal region of pre-PSII-W is extremely negatively charged (a series of five acidic residues follows the transmembrane section), precluding any electrostatic binding between this region and the membrane surface.…”

“…Most Sec-dependent bacterial proteins are at least partially dependent on the transmembrane potential for efficient insertion, but the best characterized Sec-independent proteins (M13 procoat and Pf3 coat) are entirely dependent on the ⌬H ϩ (10, 33) In both cases this has been attributed to an essential role of the membrane potential in driving the translocation of negatively charged regions through the E. coli plasma membrane by an electrophoretic mechanism (34). In contrast, dissipation of the thylakoidal ⌬H ϩ has essentially no effect on the insertion of CF o II, PSII-X, or PSII-W (23,25,26). In the absence of any apparent role for electrophoretic forces, we propose that hydrophobic interactions between the H-domains in pre-PSII-W and the membrane interior are the primary driving force for membrane insertion.…”

“…oside triphosphates, or a ⌬pH (25,26). Furthermore, mild proteolysis of thylakoids blocks the Sec-, SRP-, and ⌬pH-dependent mechanisms but has no effect on the insertion of CF o II, PSII-W, and PSII-X (26,27).…”

Chloroplast SecA and Escherichia coli SecA Have Distinct Lipid and Signal Peptide Preferences

“…These routes include the spontaneous insertion of proteins (18), and signal recognition particle-dependent (9), ⌬pH-dependent (6, 21), and SecA-dependent pathways (22,33). A twin-arginine motif is specifically recognized by components of the ⌬pH-dependent pathway, and hydrophobic sequences are favored by the signal recognition particle-dependent pathway (12,13).…”