title: A folded protein can be transported across the chloroplast envelope and thylakoid membranes.
abstract: Many thylakoid lumenal proteins are nuclear encoded, cytosolically synthesized, and reach their functional location after posttranslational targeting across two chloroplast envelope membranes and the thylakoid membrane via proteinaceous transport systems. To study whether these transmembrane transport machineries can translocate folded structures, we overexpressed the 17-kDa subunit of the oxygen-evolving complex of photosystem II (prOE17) that had been modified to contain a unique C-terminal cysteine. This allowed us to chemically link a terminal 6.5-kDa bovine pancreatic trypsin inhibitor (BPTI) moiety to prOE17 to create the chimeric protein prOE17-BPTI. Redox reagents and an irreversible sulfhydryl-specific cross-linker, bis-maleimidohexane, were used to manipulate the structure of BPTI. Import of prOE17-BPTI into isolated chloroplasts and thylakoids demonstrates that the small tightly folded BPTI domain is carried across both the chloroplast envelopes and the delta pH-dependent transmembrane transporter of the thylakoid membrane when linked to the correctly targeted OE17 precursor. Transport proceeded even when the BPTI moiety was internally cross-linked into a protease-resistant form. These data indicate that unfolding is not a ubiquitous requirement for protein translocation and that at least some domains of targeted proteins can maintain a nonlinear structure during their translocation into and within chloroplasts.
authors.affiliation: Division of Biological Sciences, University of California, Davis 95616, USA.
authors.forename: S A
authors.initials: SA
authors.lastname: Clark
authors.forename: S M
authors.initials: SM
authors.lastname: Theg
mesh_terms: D000818:Animals; D007611:Aprotinin / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D002417:Cattle; D002736:Chloroplasts / Q000378:metabolism*; D003432:Cross-Linking Reagents; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000235:genetics / Q000378:metabolism*; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D010456:Peptides, Cyclic; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D011993:Recombinant Fusion Proteins; C046916:circular bovine pancreatic trypsin inhibitor; D007611:Aprotinin