The thylakoid-targeting domain of the chloroplast Rieske iron-sulfur protein is located in the N-terminal hydrophobic region of the mature protein

F Madueño,S.A Bradshaw,J.C Gray

doi:10.1016/s0021-9258(17)32462-6

F Madueño, S.A Bradshaw + Show 1 more

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https://doi.org/10.1016/s0021-9258(17)32462-6

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Abstract

The thylakoid-targeting domain of the Rieske FeS protein has been located in the N-terminal 55 amino acids of the mature protein by importing chimeric and truncated precursor proteins into isolated pea chloroplasts. A chimeric protein consisting of the presequence of ribulose-bisphosphate carboxylase/oxygenase (Rubisco) small subunit fused to the mature Rieske protein sequence was imported by chloroplasts, processed in the stroma, and translocated into the thylakoids, indicating that the thylakoid-targeting information was located within the mature Rieske protein. A truncated Rieske protein precursor consisting of the presequence and the N-terminal 55 amino acids of the mature protein was imported by chloroplasts and routed to the thylakoids, indicating that the thylakoid-targeting domain of the Rieske protein is located within the predominantly hydrophobic N-terminal region of the mature protein. Chimeric proteins consisting of this truncated Rieske protein precursor fused to the mature Rubisco small subunit and to plastocyanin were efficiently imported by chloroplasts and translocated across the thylakoid membrane. A proton-motive force was necessary for thylakoid translocation of the mature Rieske protein, the truncated Rieske protein, and the fusion protein consisting of the truncated Rieske protein and the mature Rubisco small subunit. In contrast, plastocyanin and the fusion of the truncated Rieske protein with plastocyanin were translocated across the thylakoid membrane in the presence of nigericin and valinomycin, indicating that the energy requirement for thylakoid translocation was conferred by the passenger protein and not by the thylakoid-targeting sequence.

Highlights

From the Department of Plant Sciences and Cambridge Center for Molecular Recognition, Universityof Cambridge, Downing Street, Cambridge CB2 3EA, United Kingdom
Rieske protein contains an extended hydrophobic region near small subunit fused to the mature Rieske protein se- the N terminus and the liganfdosr the Fe,S, cluster arelocated quence was importedby chloroplasts, processedin the stroma, and translocated into the thylakoids, indicating that the thylakoid-targeting informationwaslocated within the mature Rieske protein
A truncated Rieske protein precursor consisting of the presequence and the N-terminal 55 amino acids of the mature protein was in the C-terminarlegion of the protein [4,5,10]

Summary

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UnitedKingdom.Tel.: 44-223-333925 (direct line) or 44-223-333900 a / b-binding protein; Rubiscor,ibulose-bisphosphatecarboxylase/. Truncated Rieske protein-small subunit (pRiTSS), were synthesized from transcripts produced by SP6 polymerase on cDNA sequences in for the spinachRieske protein by chloroplast import of chimeric proteins inwhich the presequences and maturepolypeptides of the Rieske protein and plastocyanin were exchanged [8]. The consisting of the presequence of pea Rubisco small subunit and the thylakoid membranes were separated from the lumenal contents by mature pea Rieske protein sequence, was constructed as follows. DNA sequence encoding the mature pea Rieske protein was amplified mal, wash, or thylakoid lumen fraction were precipitbayteidncubation using the polymerase chain reaction with twoligonucleotide primers, with 10% trichloroacetic acid on ice for 30 min.

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DISCUSSION