Sec-independent Insertion of Thylakoid Membrane Proteins

Abstract

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 ⌬H ؉ , the corresponding region of PSII-W is equally acidic but insertion is ⌬H ؉ 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.

Dual Signal Peptides Mediate the Signal Recognition Particle/Sec-independent Insertion of a Thylakoid Membrane Polyprotein, PsbY

“…Thereafter, we propose that two distinct cleavable signal peptides are used to assist the insertion of the A1 and A2 proteins. The evidence is overwhelmingly in favor of this premise: these sequences are certainly removed and then degraded, the sequences bear the typical hallmarks of thylakoid signal peptides (a hydrophobic region followed by a helix-breaking Pro or Gly residue and then an Ala-X-Ala motif) and the substitution of the Ϫ1 alanine residues results in the almost complete inhibition of TPP activity as found in other studies using thylakoid signal peptides (22,30). The psbY gene therefore encodes the first chloroplasttargeted polyprotein to be characterized in higher plants.…”

“…Finally, we addressed the possibility that the presence of a Thr residue might affect the translocation of the intervening regions as well their removal by TPP. This was, in our view, extremely unlikely because Thr residues had no detectable effect on the rate of insertion insertion of PsbX or PsbW into the the thylakoid membrane or on the translocation of the hydrophilic section into the lumen (22). Nevertheless, we tested this possibility directly by altering the Ϫ1 Ala at the first predicted cleavage site to Leu on the basis that this would similarly prevent cleavage by TPP, while having no predicted effect on insertion (if anything, the more hydrophobic Leu residue should enhance insertion).…”

“…However, the precise topologies of the intermediates are difficult to determine. Studies on PsbW have shown that the precursor protein inserts as a loop intermediate prior to cleavage by TPP in the lumen (22), and the two signal peptides in PsbY probably form similar loops with their cognate mature proteins. It is, however, difficult to determine whether both loops have formed in the inserted PsbY/1 or PsbY/2 intermediate forms.…”

“…Construct pre-A2 was synthesized by amplification of the coding region for the C-terminal region of PsbY and the introduction of a start codon in place of the Gly indicated by an arrow (see under "Experimental Procedures"). would furthermore help to define these cleavable peptides as substrates for TPP because only minor, single substitutions were made at each site, and identical substitutions had no effect on the insertion of loop structures in pre-PsbW or prePsbX (22). Both terminal Ala residues were therefore altered to Thr by site-specific mutagenesis of the cDNA clone (the relevant residues are shown in Fig.…”

“…Cleavage by the thylakoidal processing peptidase (TPP) on the trans side of the membrane then yields the mature protein. Inhibition of the processing step has been shown to generate such a loop intermediate (22) akin to that involved in the insertion of M13 procoat (10). To date, only simple, single-span proteins have been definitively shown to insert into bacterial or thylakoid membranes by SRP/Sec-independent mechanisms, raising the possibility that these factors tend to be recruited for more complex proteins and that the biogenesis of multispanning proteins may thus be rather more involved in general terms.…”