The delta pH‐driven and Sec‐related thylakoidal protein translocases recognise distinct types of thylakoid transfer signal, yet all transfer signals resemble bacterial signal peptides in structural terms. Comparison of known transfer signals reveals a single concrete difference: signals for the delta pH‐dependent system contain a common twin‐arginine motif immediately before the hydrophobic region. We show that this motif is critical for the delta pH‐driven translocation process; substitution of the arg‐arg by gln‐gln or even arg‐lys totally blocks translocation across the thylakoid membrane, and replacement by lys‐arg reduces the rate of translocation by > 100‐fold. The targeting information in this type of signal thus differs fundamentally from that of bacterial signal peptides, where the required positive charge can be supplied by any basic amino acid. Insertion of a twin‐arg motif into a Sec‐dependent substrate does not alter the pathway followed but reduces translocation efficiency, suggesting that the motif may also repel the Sec‐type system. Other information must help to specify the choice of translocation mechanism, but this information is unlikely to reside in the hydrophobic region because substitution by a hydrophobic section from an integral membrane protein does not affect the translocation pathway.

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.

The topology of the Rieske protein of the cytochrome b 6 lf complex in thylakoids from spinach chloroplasts was examined by protease protection experiments as well as polypeptide extraction assays using solutions of chaotropic salts or alkaline pH. While neither thermolysin nor trypsin cleave any of the Rieske protein when added to the stromal side of the thylakoid membrane, proteinase K is capable of removing approximately four residues from its NH 2 -terminus. The protein is resistant to membrane extraction by 0.1 M Na 2 C0 3 or 2 M NaBr but is quantitatively released by 0.1 M NaOH. Treatment of thylakoids with 2 M NaSCN leads to extraction of variable amounts of the protein, depending on the presence or absence of sucrose in the medium which apparently stabilizes the cytochrome complex. From these results we conclude that the Rieske protein is an integral component of the cytochrome complex which spans the thylakoid membrane with a single hydrophobic segment and is anchored predominantly by electrostatic interactions.

The Rieske Fe/S protein, a nuclear-encoded subunit of the cytochrome b 6 /f complex in chloroplasts, is retarded in the stromal space after import into the chloroplast and only slowly translocated further into the thylakoid membrane system. As shown by the sensitivity to nigericin and to specific competitor proteins, thylakoid transport takes place by the ⌬pH-dependent TAT pathway. The Rieske protein is an untypical TAT substrate, however. It is only the second integral membrane protein shown to utilize this pathway, and it is the first authentic substrate without a cleavable signal peptide. Transport is instead mediated by the NH 2 -terminal membrane anchor, which lacks, however, the twin-arginine motif indicative of ⌬pH/TAT-dependent transport signals. Furthermore, transport is affected by sodium azide as well as by competitor proteins for the Sec pathway in chloroplasts, demonstrating for the first time some cross-talk of the two pathways. This might take place in the stroma where the Rieske protein accumulates after import in several complexes of high molecular mass, among which the cpn60 complex is the most prominent. These untypical features suggest that the Rieske protein represents an intermediate or early state in the evolution of the thylakoidal protein transport pathways.