Toc159- and Toc75-independent import of a transit sequence-less precursor into the inner envelope of chloroplasts.
Chloroplast envelope quinone oxidoreductase (ceQORH) is an inner plastid envelope protein that is synthesized without cleavable chloroplast transit sequence for import. In the present work, we studied the in vitro-import characteristics of Arabidopsis ceQORH. We demonstrate that ceQORH import requires ATP and is dependent on proteinaceous receptor components exposed at the outer plastid surface. Competition experiments using small subunit precursor of ribulose-bisphosphate carboxylase/oxygenase and precursor of ferredoxin, as well as antibody blocking experiments, revealed that ceQORH import does not involve the main receptor and translocation channel proteins Toc159 and Toc75, respectively, which operate in import of proteins into the chloroplast. Molecular dissection of the ceQORH amino acid sequence by site-directed mutagenesis and subsequent import experiments in planta and in vitro highlighted that ceQORH consists of different domains that act concertedly in regulating import. Collectively, our results provide unprecedented evidence for the existence of a specific import pathway for transit sequence-less inner plastid envelope membrane proteins into chloroplasts.
Miras
Stéphane
S
Laboratoire de Physiologie Cellulaire Végétale, CNRS Unité Mixte de Recherche (UMR) (5168), Grenoble 38054 cedex 9, France.
Salvi
Daniel
D
Piette
Laurie
L
Seigneurin-Berny
Daphné
D
Grunwald
Didier
D
Reinbothe
Christiane
C
Joyard
Jacques
J
Reinbothe
Steffen
S
Rolland
Norbert
N
D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000378:metabolism*; D001667:Binding, Competitive; D002736:Chloroplasts / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D005288:Ferredoxins / Q000737:chemistry; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D001467:Hordeum / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D018087:Plastids / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013347:Subcellular Fractions / Q000378:metabolism
D029681:Arabidopsis Proteins; D003432:Cross-Linking Reagents; D005288:Ferredoxins; D008565:Membrane Proteins; D011498:Protein Precursors; C403444:TOC159 protein, Arabidopsis; C491244:TOC75 protein, Arabidopsis; D020558:GTP Phosphohydrolases