Assembly of the components of the thylakoid ΔpH‐dependent/Tat protein transport machinery was analyzed in vitro. Upon incubation with intact chloroplasts, precursors to all three components, Hcf106, cpTatC and Tha4, were imported into the organelle and assembled into characteristic endogenous complexes. In particular, all of the imported cpTatC and approximately two‐thirds of the imported Hcf106 functionally assembled into 700 kDa complexes capable of binding Tat pathway precursor proteins. The amounts assembled into thylakoids by this procedure were moderate. However, physiological quantities of mature forms of Tha4 and Hcf106 were integrated into isolated thylakoids and a significant percentage of the Hcf106 so integrated was assembled into the 700 kDa complex. Interestingly, a mutant form of Hcf106 in which an invariant transmembrane glutamate was changed to glutamine integrated into the membrane but did not assemble into the receptor complex. Analysis of energy and known pathway component requirements indicated that Hcf106 and Tha4 integrate by an unassisted or ‘spontaneous’ mechanism. The functionality of in vitro integrated Tha4 was verified by its ability to restore transport to thylakoid membranes from the maize tha4 mutant, which lacks the Tha4 protein. Development of this functional in vitro assembly assay will facilitate structure–function studies of the thylakoid Tat pathway translocation machinery.

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.