The Arabidopsis ppi1 Mutant Is Specifically Defective in the Expression, Chloroplast Import, and Accumulation of Photosynthetic Proteins Online version contains Web-only data. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.012955.

The import of nucleus-encoded proteins into chloroplasts is mediated by translocon complexes in the envelope membranes. A component of the translocon in the outer envelope membrane, Toc34, is encoded in Arabidopsis by two homologous genes, atTOC33 and atTOC34. Whereas atTOC34 displays relatively uniform expression throughout development, atTOC33 is strongly upregulated in rapidly growing, photosynthetic tissues. To understand the reason for the existence of these two related genes, we characterized the atTOC33 knockout mutant ppi1. Immunoblotting and proteomics revealed that components of the photosynthetic apparatus are deficient in ppi1 chloroplasts and that nonphotosynthetic chloroplast proteins are unchanged or enriched slightly. Furthermore, DNA array analysis of 3292 transcripts revealed that photosynthetic genes are moderately, but specifically, downregulated in ppi1. Proteome differences in ppi1 could be correlated with protein import rates: ppi1 chloroplasts imported the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit and 33-kD oxygen-evolving complex precursors at significantly reduced rates, but the import of a 50S ribosomal subunit precursor was largely unaffected. The ppi1 import defect occurred at the level of preprotein binding, which is consistent with a role for atToc33 during preprotein recognition. The data suggest that atToc33 is involved preferentially in the import of photosynthetic proteins and, by extension, that atToc34 is involved in the import of nonphotosynthetic chloroplast proteins.

[1]  B. Bruce,et al.  The paradox of plastid transit peptides: conservation of function despite divergence in primary structure. , 2001, Biochimica et biophysica acta.

[2]  C. Tsai,et al.  A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[3]  K. Keegstra,et al.  ATP is required for the binding of precursor proteins to chloroplasts. , 1989, The Journal of biological chemistry.

[4]  K. Keegstra,et al.  Protein Import and Routing Systems of Chloroplasts , 1999, Plant Cell.

[5]  G. Blobel,et al.  Isolation of components of the chloroplast protein import machinery. , 1994, Science.

[6]  K. Keegstra,et al.  The binding of precursor proteins to chloroplasts requires nucleoside triphosphates in the intermembrane space. , 1992, The Journal of biological chemistry.

[7]  C. Subramanian,et al.  Structural and guanosine triphosphate/diphosphate requirements for transit peptide recognition by the cytosolic domain of the chloroplast outer envelope receptor, Toc34. , 2002, Biochemistry.

[8]  M. Ünlü,et al.  Difference gel electrophoresis. A single gel method for detecting changes in protein extracts , 1997, Electrophoresis.

[9]  K. Keegstra,et al.  Arabidopsis genes encoding components of the chloroplastic protein import apparatus. , 2001, Plant physiology.

[10]  A. Feinberg,et al.  A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. , 1983, Analytical biochemistry.

[11]  P. Jarvis Intracellular Signalling: The Language of the Chloroplast , 2003, Current Biology.

[12]  N. Pfanner,et al.  Versatility of the mitochondrial protein import machinery , 2001, Nature Reviews Molecular Cell Biology.

[13]  S. E. Perry,et al.  Envelope membrane proteins that interact with chloroplastic precursor proteins. , 1994, The Plant Cell.

[14]  J. Soll,et al.  A protein import receptor in pea chloroplasts, Toc86, is only a proteolytic fragment of a larger polypeptide , 1998, FEBS letters.

[15]  M. Gutensohn,et al.  Functional analysis of the two Arabidopsis homologues of Toc34, a component of the chloroplast protein import apparatus. , 2000, The Plant journal : for cell and molecular biology.

[16]  Hsou-min Li,et al.  Chloroplast protein translocon components atToc159 and atToc33 are not essential for chloroplast biogenesis in guard cells and root cells. , 2001, Plant physiology.

[17]  K. Keegstra,et al.  Stable association of chloroplastic precursors with protein translocation complexes that contain proteins from both envelope membranes and a stromal Hsp100 molecular chaperone , 1997, The EMBO journal.

[18]  Joachim Kurth,et al.  Gene-sequence-tag expression analyses of 1,800 genes related to chloroplast functions , 2002, Planta.

[19]  M. Hohwy,et al.  Targeting of an abundant cytosolic form of the protein import receptor at Toc159 to the outer chloroplast membrane , 2001, The Journal of cell biology.

[20]  J. Chory,et al.  Signal Transduction between the Chloroplast and the Nucleus Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010446. , 2002, The Plant Cell Online.

[21]  J. Ecker,et al.  Chloroplast to nucleus communication triggered by accumulation of Mg-protoporphyrinIX , 2003, Nature.

[22]  D. Leister,et al.  Covariations in the nuclear chloroplast transcriptome reveal a regulatory master‐switch , 2003, EMBO reports.

[23]  F. Kessler,et al.  Protein translocon at the Arabidopsis outer chloroplast membrane. , 2001, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[24]  R. Stroud,et al.  The signal recognition particle. , 2001, Annual review of biochemistry.

[25]  A. Kouranov,et al.  Two components of the chloroplast protein import apparatus, IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of precursor proteins at the outer envelope , 1996, The Journal of cell biology.

[26]  S. Brunak,et al.  Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. , 2000, Journal of molecular biology.

[27]  C. Hsiao,et al.  Crystal structure of pea Toc34, a novel GTPase of the chloroplast protein translocon , 2002, Nature Structural Biology.

[28]  J. Soll,et al.  Erratum to: “Toc, Tic, and chloroplast protein import” [Biochim. Biophys. Acta 1541 (2001) 64–79] , 2002 .

[29]  D. Leister,et al.  GST-PRIME: a genome-wide primer design software for the generation of gene sequence tags. , 2001, Nucleic acids research.

[30]  D. Schnell,et al.  Mechanism of protein import across the chloroplast envelope. , 2000, Biochemical Society transactions.

[31]  Jörg Bauer,et al.  The major protein import receptor of plastids is essential for chloroplast biogenesis , 2000, Nature.

[32]  J. Soll,et al.  The chloroplast protein import channel Toc75: pore properties and interaction with transit peptides. , 2002, Biophysical journal.

[33]  E. Schleiff,et al.  Toc34 is a preprotein receptor regulated by GTP and phosphorylation. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[34]  D. Leister,et al.  Knock-out of the plastid ribosomal protein L11 in Arabidopsis: effects on mRNA translation and photosynthesis. , 2001, The Plant journal : for cell and molecular biology.

[35]  J. Soll,et al.  A constituent of the chloroplast import complex represents a new type of GTP-binding protein. , 1995, The Plant journal : for cell and molecular biology.

[36]  J. Soll,et al.  Toc, Tic, and chloroplast protein import. , 2001, Biochimica et biophysica acta.

[37]  Dario Leister,et al.  Chloroplast research in the genomic age. , 2003, Trends in genetics : TIG.

[38]  P. Jarvis,et al.  A simple method for isolating import‐competent Arabidopsis chloroplasts , 2002, FEBS letters.

[39]  G. von Heijne,et al.  A receptor component of the chloroplast protein translocation machinery. , 1994, Science.

[40]  J. Chory,et al.  An Arabidopsis mutant defective in the plastid general protein import apparatus. , 1998, Science.

[41]  G. Blobel,et al.  Identification of two GTP-binding proteins in the chloroplast protein import machinery. , 1994, Science.

[42]  A. Kouranov,et al.  Analysis of the Interactions of Preproteins with the Import Machinery over the Course of Protein Import into Chloroplasts , 1997, The Journal of cell biology.