The Thylakoid ΔpH-dependent Pathway Machinery Facilitates RR-independent N-Tail Protein Integration

Abstract

The thylakoidal ⌬pH-dependent and bacterial twin arginine transport systems are structurally and functionally related protein export machineries. These recently discovered systems have been shown to transport folded proteins but are not known to assemble integral membrane proteins. We determined the translocation pathway of a thylakoidal FtsH homologue, plastid fusion/ protein translocation factor, which is synthesized with a chloroplast-targeting peptide, a hydrophobic signal peptide, and a hydrophobic membrane anchor. The twin arginine motif in its signal peptide and its sole integration requirement of a ⌬pH suggested that plastid fusion/ protein translocation factor employs the ⌬pH pathway. Surprisingly, changing the twin arginine to twin lysine or deleting the signal peptide did not abrogate integration capability or characteristics. Nevertheless, three criteria argue that all three forms require the ⌬pH pathway for integration. First, integration was competed by an authentic ⌬pH pathway precursor. Second, antibodies to ⌬pH pathway component Hcf106 specifically inhibited integration. Finally, chloroplasts from the hcf106 null mutant were unable to integrate Pftf into their thylakoids. Thus, ⌬pH pathway machinery facilitates both signal peptide-directed and N-tail-mediated membrane integration and does not strictly require the twin arginine motif.Export type pathways target proteins to the bacterial plasma membrane, the endoplasmic reticulum, the mitochondrial inner membrane, and the chloroplast thylakoid membrane (1). Where known, component and mechanistic similarity support the hypothesis that the organelle pathways are descendent from those of the prokaryotic endosymbiont. This is most evident when comparing export pathways of chloroplasts and bacteria. Chloroplasts share with bacteria at least four distinct pathways: a Sec pathway, a ⌬pH/Tat 1 pathway, a signal recognition particle (SRP) pathway, and a spontaneous insertion pathway (2-4). Chloroplasts and bacteria also appear to employ homologues of the mitochondrial Oxa1p export protein (5).The ⌬pH/Tat pathway in thylakoids and bacteria is the most recently recognized protein translocation pathway. The ⌬pH pathway was first described as a system that transported a subset of thylakoid lumenal proteins using only the thylakoidal ⌬pH as an energy source (6). Substrates of this pathway possess cleavable, amino-terminal signal peptides with an invariant twin arginine motif amino-terminal to the hydrophobic core (7). Where examined, substitution of one or both arginines abolishes ⌬pH pathway transport (7,8). Identification of a component of the ⌬pH pathway, Hcf106 (9), and the recognition that a subset of bacterial periplasmic proteins possesses twin arginine-containing signal peptides (10, 11) led to the description of a homologous bacterial pathway. Disruption or mutations of Hcf106 homologues impaired transport of a range of twin arginine-bearing precursors (12, 13). In addition, disruption of a Tat operon gene for the multispanning membrane protein TatC al...

Toc, Tic, Tat et al.: structure and function of protein transport machineries in chloroplasts

“…The other two protein transport pathways operating at the thylakoid membrane, notably the Secand DpH/Tat-dependent pathways, are responsible for the translocation of hydrophilic proteins into the thylakoid lumen, although it should be mentioned that for both pathways also the transport of a few integral membrane proteins has been described (Karnauchov et al, 1994;Summer et al, 2000;Molik et al, 2001). Nuclear-encoded proteins targeted by either of the two pathways are synthesized in the cytosol with bipartite transit peptides carrying two translocation signals in tandem, an NH 2 -terminal envelope transit signal for the import of the protein into the chloroplast stroma followed by a second transport signal which mediates translocation across the thylakoid membrane and is generally removed after transport by a membrane-bound endopeptidase executing its function on the lumenal side of the membrane (thylakoidal processing peptidase, TPP).…”

The Bacterial Twin-Arginine Translocation Pathway

“…In addition to C-terminal TM α-helices, other means of membrane tethering have been found in Tat substrates of the cpTat pathway but so far have not been observed in bacteria (84,124). For example, the Rieske protein of the cytochrome b 6 /f complex that is exported via the cpTat pathway uses an un-cleaved signal sequence rather than a C-tail for membrane anchoring (84).…”

Central Functions of the Lumenal and Peripheral Thylakoid Proteome of Arabidopsis Determined by Experimentation and Genome-Wide Prediction

“…Proteins translocated via the ⌬ pH or twin-arginine translocation (TAT) pathway require neither soluble factors nor GTP or ATP, but they do require a transthylakoid proton gradient and several TAT proteins. The TAT substrates also require a specific TAT motif, RRx-h-h, where R is arginine and h is a hydrophobic residue (Walker et al, 1999;Robinson and Bolhuis, 2001; for an exception, see Summer et al, 2000).…”