Multiple pathways for protein transport into or across the thylakoid membrane.

Abstract

Many thylakoid proteins are cytosolically synthesized and have to cross the two chloroplast envelope membranes as well as the thylakoid membrane en route to their functional locations. In order to investigate the localization pathways of these proteins, we over‐expressed precursor proteins in Escherichia coli and used them in competition studies. Competition was conducted for import into the chloroplast and for transport into or across isolated thylakoids. We also developed a novel in organello method whereby competition for thylakoid transport occurred within intact chloroplasts. Import of all precursors into chloroplasts was similarly inhibited by saturating concentrations of the precursor to the OE23 protein. In contrast, competition for thylakoid transport revealed three distinct precursor specificity groups. Lumen‐resident proteins OE23 and OE17 constitute one group, lumenal proteins plastocyanin and OE33 a second, and the membrane protein LHCP a third. The specificity determined by competition correlates with previously determined protein‐specific energy requirements for thylakoid transport. Taken together, these results suggest that thylakoid precursor proteins are imported into chloroplasts on a common import apparatus, whereupon they enter one of several precursor‐specific thylakoid transport pathways.

A folded protein can be transported across the chloroplast envelope and thylakoid membranes.

“…This made possible the later chemical attachment of BPTI. Overexpression of this altered protein in E. coli in the presence of tritiated leucine resulted in sequestration in inclusion bodies of radiolabeled prOE17, which was then purified, solubilized in urea, and imported into chloroplasts and thylakoids following the protocols of Cline et al (1993). Cleavage of the transit peptide by the thylakoid processing peptidase during import into the lumen was observed, and the resultant mature form was found as a soluble protein in the thylakoid lumen.…”

“…These proteins are transported by a mechanism that is supported solely by a ApH (Cline et al, 1992). The separation of these pathways has been elegantly demonstrated by in organello competition studies showing that high levels of iOE33 and iOE23 inhibit the transthylakoid transport of iPC and iOE17, respectively, but that these two groups do not crosscompete (Cline et al, 1993). For a precursor protein to be translocated across the membrane of many organelles, it is thought to at least temporarily assume a conformation that differs from that of the stably folded mature protein (Schatz and Dobberstein, 1996).…”

Functional assembly of thylakoid ΔpH‐dependent/Tat protein transport pathway components in vitro

“…Maize chloroplasts were isolated as described [14]. Chloroplast lysate, washed thylakoids and stromal extract were prepared from isolated chloroplasts [18]. Chlorophyll concentrations were determined according to Arnon [19].…”

“…Assays were terminated by transfer to 0°C. Where indicated, recovered chloroplasts or thylakoids were protease post-treated with thermolysin [18]. Chloroplasts were repurified on Percoll cushions and washed in import buffer.…”

Molecular chaperones are present in the thylakoid lumen of pea chloroplasts

“…Antisera against OEE33 from pea [18] and SSU from tobacco [19] were raised in chicken. OEE33 and SSU were synthesized by overexpression in E. coli using the pET-vector system (Novagen, Madison/ USA) and purified as insoluble inclusion bodies [20].…”