Protease‐sensitive thylakoidal import machinery for the Sec‐, ΔpH‐ and signal recognition particle‐dependent protein targeting pathways, but not for CF_oII integration

Abstract

SummaryNuclear-encoded proteins are targeted into and across the thylakoid membrane by a surprising variety of mechanisms. Distinct Sec-and ~pH-dependent mechanisms have been shown to operate for lumenal proteins, and an integral membrane protein, LHCP, has been shown to insert via a signal recognition particle-dependent route. Integration of a further membrane protein, CFolI, requires neither soluble factors nor energy sources, prompting speculation of a spontaneous insertion mechanism. Although the requirements for soluble factors and energy sources have been determined in some detail, much less is known of the events taking place at the membrane surface. This report examines whether thylakoid proteins are involved in each of these pathways, by testing the effects of trypsin on the capacity of isolated thylakoids to import proteins. Because all of the pathways rely to some extent on the thylakoidal ApH, and a light-induced ApH is easily destroyed by proteolysis, the conditions under which reverse action of the ATP synthase in the dark generates a high ~pH even after proteolysis of thylakoids have been established. This system is used to show that protease-sensitive thylakoidal import machinery is crucial for the ~pH-, Sec-and signal recognition particle-dependent pathways, but not for integration of CFolI.

Dual Signal Peptides Mediate the Signal Recognition Particle/Sec-independent Insertion of a Thylakoid Membrane Polyprotein, PsbY

“…Stromal extract, when present, was at a concentration equivalent to 1.3ϫ the chlorophyll concentration. The import/insertion incubation was carried out under a green safelight, for 30 min at 26°C, and the prevailing ⌬pH was measured by 9-aminoacridine fluorescence quenching as described (17); this was found to be invariably over 2 units.…”

“…However, it should be noted that an RNA molecule has yet to be identified in chloroplast SRP, and certain aspects of the insertion process may therefore differ. Proteolysis of thylakoids destroys their ability to integrate LHCP (17), indicating that membrane-bound protein transport apparatus is required (probably the thylakoidal Sec machinery, although this remains to be confirmed).…”

“…A very different insertion process has been demonstrated for However, CF o II, PsbW, and PsbX have all been shown to insert into thylakoids in the absence of SecA, SRP, or NTPs, and proteolysis of thylakoids has been shown to block Sec-dependent transport but have no effect on the insertion of the above proteins (17)(18)(19)(20)(21). These findings represent strong evidence that neither SRP nor the Sec apparatus is required, and it has been proposed that these proteins may insert spontaneously into the thylakoid membrane.…”

Functional assembly of thylakoid ΔpH‐dependent/Tat protein transport pathway components in vitro

“…Another characteristic of spontaneous integration is the ability of proteins to insert into protease pretreated thylakoids [26]. Tha4 and Hcf106 integrated into thermolysin-treated membranes (Fig.…”

Sec-independent Insertion of Thylakoid Membrane Proteins

“…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). It has therefore been proposed that they insert spontaneously into the thylakoid membrane.…”