Evidence for a loop mechanism of protein transport by the thylakoid Delta pH pathway

Abstract

The thylakoid Delta pH pathway is a protein transport system with unprecedented characteristics. To investigate its mechanism, the topology of precursor insertion was determined. A fusion protein comprising a large polypeptide domain fused to the amino terminus of pOE17 (a Delta pH pathway precursor) was efficiently processed by thylakoid membranes. The amino terminus, including the targeting peptide, remained on the cis side of the membrane. Mature OE17 was transported to the lumen. These experiments demonstrate that Delta pH directed precursors enter the thylakoid membrane in a loop, implying that the Delta pH pathway has evolved from an export-type protein translocation system. z 1998 Federation of European Biochemical Societies.

Unassisted Membrane Insertion as the Initial Step in ΔpH/Tat-dependent Protein Transport

“…24 In line with that, the size of corresponding translocation intermediates obtained after treatment of the thylakoids with protease in the in thylakoido and in organello assays is indistinguishable ( Figure 1(a), lanes C), although the actual substrates for thylakoid transport in these two assays have different N termini. In in thylakoido experiments, it is the full precursor protein that binds to the thylakoid membrane, whereas in in organello assays the chloroplast import domain is removed in the stroma from the N terminus prior to thylakoid transport, yielding a polypeptide intermediate in size between precursor and mature protein (compare lanesKin Figure 1(a)).…”

Translocation of a Phycoerythrin α Subunit across Five Biological Membranes

“…This appears surprising at first glance but similar observations have been described already earlier (23). It is further support for the recently proposed model that after direct loop insertion of the Tat substrate into the lipid bilayer with both, the N and C terminus, exposed to the stroma, the C-terminally located passenger protein is translocated across the thylakoid membrane, while the N terminus of the precursor protein remains on the stromal side of the membrane (15) until the thylakoid targeting transport signal is proteolytically removed (24).…”

Mapping Precursor-binding Site on TatC Subunit of Twin Arginine-specific Protein Translocase by Site-specific Photo Cross-linking

“…In addition, clear precursor adducts were obtained for Tyr-100 and Lys-101 located within the first predicted cytosolic loop of TatC. Consistent with these cytosolically exposed domains of TatC interacting with RR precursors, the signal sequences of membrane-bound Tat substrates are still accessible to proteases and can be detached by disrupting electrostatic interactions (15,33).…”