Sec-independent insertion of thylakoid membrane proteins: Analysis of insertion forces and identification of a loop intermediate involving the signal peptide

A group of membrane proteins are synthesized with cleavable signal sequences but inserted into the thylakoid membrane by an unusual Sec/SRP- independent mechanism. In this report we describe a key intermediate in the insertion of one such protein, photosystem II subunit W (PSII-W). A single mutation in the terminal cleavage site partially blocks processing and leads to the formation of an intermediate-size protein in the thylakoid membrane during chloroplast import assays. This protein is in the form of a loop structure: the N and C termini are exposed on the stromal face, whereas the cleavage site has been translocated into the lumen. In this respect the insertion of this protein resembles that of M13 procoat, which also adopts a loop structure during insertion, and we present preliminary evidence that a similar mechanism is used by another thylakoid protein, PSII-X. However, whereas the negatively charged region of procoat is translocated by an apparently electrophoretic mechanism using the ΔμH⁺, the corresponding region of PSII-W is equally acidic but insertion is ΔμH⁺ independent. We furthermore show that neutralization of this region has no apparent effect on the insertion process. We propose that a central element in this insertion mechanism is a loop structure whose formation is driven by hydrophobic interactions.