The Chloroplast Import Receptor Toc90 Partially Restores the Accumulation of Toc159 Client Proteins in the Arabidopsis thaliana ppi2 Mutant

Abstract Iron–sulfur (Fe-S) proteins have critical functions in plastids, notably participating in photosynthetic electron transfer, sulfur and nitrogen assimilation, chlorophyll metabolism, and vitamin or amino acid biosynthesis. Their maturation relies on the so-called SUF (sulfur mobilization) assembly machinery. Fe-S clusters are synthesized de novo on a scaffold protein complex and then delivered to client proteins via several transfer proteins. However, the maturation pathways of most client proteins and their specificities for transfer proteins are mostly unknown. In order to decipher the proteins interacting with the Fe-S cluster transfer protein NFU2, one of the three plastidial representatives found in Arabidopsis thaliana, we performed a quantitative proteomic analysis of shoots, roots, and seedlings of nfu2 plants, combined with NFU2 co-immunoprecipitation and binary yeast two-hybrid experiments. We identified 14 new targets, among which nine were validated in planta using a binary bimolecular fluorescence complementation assay. These analyses also revealed a possible role for NFU2 in the plant response to desiccation. Altogether, this study better delineates the maturation pathways of many chloroplast Fe-S proteins, considerably extending the number of NFU2 clients. It also helps to clarify the respective roles of the three NFU paralogs NFU1, NFU2, and NFU3.

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Importin [alpha] from Arabidopsis thaliana Is a Nuclear Import Receptor That Recognizes Three Classes of Import Signals

PLANT PHYSIOLOGY ◽

10.1104/pp.114.2.411 ◽

1997 ◽

Vol 114 (2) ◽

pp. 411-417 ◽

Cited By ~ 50

Author(s):

HMS. Smith ◽

G. R. Hicks ◽

N. V. Raikhel

Keyword(s):

Arabidopsis Thaliana ◽

Nuclear Import ◽

Importin Alpha ◽

Import Receptor

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The Chloroplast Import Receptor Toc34 Functions as Preprotein-Regulated GTPase

Biological Chemistry ◽

10.1515/bc.2002.211 ◽

2002 ◽

Vol 383 (12) ◽

Cited By ~ 51

Author(s):

M. Jelic ◽

N. Sveshnikova ◽

M. Motzkus ◽

P. Hörth ◽

J. Soll ◽

...

Keyword(s):

Chloroplast Import ◽

Import Receptor

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Plastocyanin of Arabidopsis thaliana; isolation and characterization of the gene and chloroplast import of the precursor protein

Gene ◽

10.1016/0378-1119(88)90417-9 ◽

1988 ◽

Vol 65 (1) ◽

pp. 59-69 ◽

Cited By ~ 37

Author(s):

Oscar Vorst ◽

Renske Oosterhoff-Teertstra ◽

Pierre Vankan ◽

Sjef Smeekens ◽

Peter Weisbeek

Keyword(s):

Arabidopsis Thaliana ◽

Precursor Protein ◽

Chloroplast Import ◽

Isolation And Characterization

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In vivo lipid ‘tag and track’ approach shows acyl editing of plastid lipids and chloroplast import of phosphatidylglycerol precursors in Arabidopsis thaliana

The Plant Journal ◽

10.1111/tpj.13999 ◽

2018 ◽

Vol 95 (6) ◽

pp. 1129-1139 ◽

Cited By ~ 7

Author(s):

Anna K. Hurlock ◽

Kun Wang ◽

Tomomi Takeuchi ◽

Patrick J. Horn ◽

Christoph Benning

Keyword(s):

Arabidopsis Thaliana ◽

Chloroplast Import

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Modifications at the A-domain of the chloroplast import receptor Toc159

Plant Signaling & Behavior ◽

10.4161/psb.5.11.13707 ◽

2010 ◽

Vol 5 (11) ◽

pp. 1513-1516 ◽

Cited By ~ 2

Author(s):

Birgit Agne ◽

Felix Kessler

Keyword(s):

Chloroplast Import ◽

Import Receptor

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The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64

The EMBO Journal ◽

10.1038/sj.emboj.7601091 ◽

2006 ◽

Vol 25 (9) ◽

pp. 1836-1847 ◽

Cited By ~ 116

Author(s):

Soumya Qbadou ◽

Thomas Becker ◽

Oliver Mirus ◽

Ivo Tews ◽

Jürgen Soll ◽

...

Keyword(s):

Molecular Chaperone ◽

Chloroplast Import ◽

Precursor Proteins ◽

Import Receptor

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The chloroplast import receptor is an integral membrane protein of chloroplast envelope contact sites.

The Journal of Cell Biology ◽

10.1083/jcb.111.5.1825 ◽

1990 ◽

Vol 111 (5) ◽

pp. 1825-1838 ◽

Cited By ~ 56

Author(s):

D J Schnell ◽

G Blobel ◽

D Pain

Keyword(s):

Membrane Protein ◽

Protein Import ◽

Integral Membrane Protein ◽

Chloroplast Envelope ◽

Bisphosphate Carboxylase ◽

Chloroplast Import ◽

Chloroplast Membrane ◽

Phosphate Translocator ◽

Triton X ◽

Import Receptor

A chloroplast import receptor from pea, previously identified by antiidiotypic antibodies was purified and its primary structure deduced from its cDNA sequence. The protein is a 36-kD integral membrane protein (p36) with eight potential transmembrane segments. Fab prepared from monospecific anti-p36 IgG inhibits the import of the ribulose-1,5-bisphosphate carboxylase small subunit precursor (pS) by interfering with pS binding at the chloroplast surface. Anti-p36 IgGs are able to immunoprecipitate a Triton X-100 soluble p36-pS complex, suggesting a direct interaction between p36 and pS. This immunoprecipitation was specific as it was abolished by a pS synthetic transit peptide, consistent with the transit sequence receptor function of p36. Immunoelectron microscopy localized p36 to regions of the outer chloroplast membrane that are in close contact with the inner chloroplast membrane. Comparison of the deduced sequence of pea p36 to that of other known proteins indicates a striking homology to a protein from spinach chloroplasts that was previously suggested to be the triose phosphate-3-phosphoglycerate-phosphate translocator (phosphate translocator) (Flügge, U. I., K. Fischer, A. Gross, W. Sebald, F. Lottspeich, and C. Eckerskorn. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:39-46). However, incubation of Triton X-100 solubilized chloroplast envelope material with hydroxylapatite indicated that p36 was quantitatively absorbed, whereas previous reports have shown that phosphate translocator activity does not bind to hydroxylapatite (Flügge, U. I., and H. W. Heldt. 1981. Biochim. Biophys. Acta. 638:296-304. These data, in addition to the topology and import inhibition data presented in this report support the assignment of p36 as a receptor for chloroplast protein import, and argue against the assignment of the spinach homologue of this protein as the chloroplast phosphate translocator.

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The plastidial Arabidopsis thaliana NFU1 protein binds and delivers [4Fe-4S] clusters to specific client proteins

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011034 ◽

2020 ◽

Vol 295 (6) ◽

pp. 1727-1742 ◽

Cited By ~ 6

Author(s):

Mélanie Roland ◽

Jonathan Przybyla-Toscano ◽

Florence Vignols ◽

Nathalie Berger ◽

Tamanna Azam ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

De Novo ◽

Acid Synthesis ◽

Bimolecular Fluorescence Complementation ◽

Cluster Assembly ◽

Amino Acid Synthesis ◽

Cluster Transfer ◽

Client Proteins ◽

Branched Chain

Proteins incorporating iron–sulfur (Fe-S) co-factors are required for a plethora of metabolic processes. Their maturation depends on three Fe-S cluster assembly machineries in plants, located in the cytosol, mitochondria, and chloroplasts. After de novo formation on scaffold proteins, transfer proteins load Fe-S clusters onto client proteins. Among the plastidial representatives of these transfer proteins, NFU2 and NFU3 are required for the maturation of the [4Fe-4S] clusters present in photosystem I subunits, acting upstream of the high-chlorophyll fluorescence 101 (HCF101) protein. NFU2 is also required for the maturation of the [2Fe-2S]-containing dihydroxyacid dehydratase, important for branched-chain amino acid synthesis. Here, we report that recombinant Arabidopsis thaliana NFU1 assembles one [4Fe-4S] cluster per homodimer. Performing co-immunoprecipitation experiments and assessing physical interactions of NFU1 with many [4Fe-4S]-containing plastidial proteins in binary yeast two-hybrid assays, we also gained insights into the specificity of NFU1 for the maturation of chloroplastic Fe-S proteins. Using bimolecular fluorescence complementation and in vitro Fe-S cluster transfer experiments, we confirmed interactions with two proteins involved in isoprenoid and thiamine biosynthesis, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase and 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase, respectively. An additional interaction detected with the scaffold protein SUFD enabled us to build a model in which NFU1 receives its Fe-S cluster from the SUFBC2D scaffold complex and serves in the maturation of specific [4Fe-4S] client proteins. The identification of the NFU1 partner proteins reported here more clearly defines the role of NFU1 in Fe-S client protein maturation in Arabidopsis chloroplasts among other SUF components.

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