A substrate-independent, 14:3:3 protein-mediated plastid import pathway of NADPH:protochlorophyllide oxidoreductase A

doi:10.1073/pnas.0702058104

A substrate-independent, 14:3:3 protein-mediated plastid import pathway of NADPH:protochlorophyllide oxidoreductase A

^†Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität Bochum, Universitätsstrasse 150, Gebäude ND, D-44801 Bochum, Germany;
^‡Lehrstuhl für Pflanzenphysiologie, Universität Bayreuth, Universitätsstrasse 30, D-95447 Bayreuth, Germany; and
^§Unité Mixte de Recherche 5575, Université Joseph Fourier et Centre National de la Recherche Scientifique, BP53, F-38041 Grenoble Cedex 9, France

Communicated by Diter von Wettstein, Washington State University, Pullman, WA, March 10, 2007 (received for review January 21, 2007)

Abstract

Plastids are semiautonomous organelles that contain only limited coding information in their own DNA. Because most of their genome was transferred to the nucleus after their endosymbiotic origin, plastids must import the major part of their protein constituents from the cytosol. The exact role of cytosolic targeting factors in the regulation of plastid protein import has not been determined. Here, we report that the nucleus-encoded NADPH:protochlorophyllide (Pchlide) oxidoreductase A plastid precursor (pPORA) can use two different plastid import pathways that differ by the requirements for cytosolic 14:3:3 proteins and Hsp70. pPORA synthesized in a wheat germ lysate segregated into different precursor fractions. While import of free pPORA and only Hsp70-complexed pPORA was Pchlide-dependent and involved the previously identified Pchlide-dependent translocon, 14:3:3 protein- and Hsp70-complexed pPORA was transported into Pchlide-free chloroplasts through the Toc75-containing standard translocon at the outer chloroplast membrane/translocon at the inner chloroplast membrane machinery. A 14:3:3 protein binding site was identified in the mature region of the ³⁵S-pPORA, which governed 14:3:3 protein- and Hsp70-mediated, Pchlide-independent plastid import. Collectively, our results reveal that the import of pPORA into the plastids is tightly regulated and involves different cytosolic targeting factors and plastid envelope translocon complexes.

Footnotes

^¶To whom correspondence should be addressed. E-mail: steffen.reinbothe{at}ujf-grenoble.fr
Author contributions: C.R. and S.R. designed research; A.S., S.P., C.R., and S.R. performed research; A.S., S.P., C.R., and S.R. analyzed data; and S.R. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0702058104/DC1.
Abbreviations:

5-ALA,

5-aminolevulinic acid;

APDP,

N-[4[(p-azidosalicylamido)butyl]-3′(2-pyridyldithio) propion amid;

CAO,

chlorophyllide a oxygenase;

pFd,

precursor ferredoxin;

Pchlide,

protochlorophyllide;

pPORA,

Pchlide oxidoreductase A;

pPORB,

Pchlide oxidoreductase B;

pSSU,

presmall subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase;

Ptc,

Pchlide-dependent translocon;

Toc,

translocon at the outer chloroplast membrane.