Abstract
The precursors of RNP29 and Ferredoxin (Fd2) were previously identified in the cytosol of ppi2 plant cells with their N-terminal amino acid acetylated. Here, we explore whether precursor accumulation in ppi2 is characteristic for Toc159 client proteins, by characterizing the import properties of the RNP29 precursor in comparison to Fd2 and other Toc159-dependent or independent substrates. We find specific accumulation of the RNP29 precursor in ppi2 but not in wild type or ppi1 protoplasts. With the exception of Lhcb4, precursor accumulation is also detected with all other tested constructs in ppi2. However, RNP29 is clearly different from the other proteins because only precursor but almost no mature protein is detectable in protoplast extracts. Co-transformation of RNP29 with Toc159 complements its plastid import, supporting the hypothesis that RNP29 is a Toc159-dependent substrate. Exchange of the second amino acid in the RNP29 transit peptide to Glu or Asn prevents methionine excision but not N-terminal acetylation, suggesting that different N-acetyltransferases may act on chloroplast precursor proteins in vivo. All different RNP29 constructs are efficiently imported into wild type but not into ppi2 plastids, arguing for a minor impact of the N-terminal amino acid on the import process.
Highlights
Most chloroplast localized proteins are encoded in the nuclear genome and synthesized at cytosolic ribosomes as precursor proteins with N-terminal transit peptides
PLANT MATERIAL After 2 days of stratification at 4◦C Arabidopsis thaliana (Columbia-0) and ppi2 (Toc159, CS11072 introgressed into the Columbia-0 ecotype) (Kubis et al, 2004) were grown on halfstrength Murashige and Skoog (M&S) medium supplemented with 0.8% (w/v) plant agar (Duchefa) and 3% (w/v) sucrose under short day conditions at 21◦C for 5 weeks before harvesting the seedlings and directly preparing protoplast
Here we establish RNP29 as Toc159-dependent client protein, which is in line with the original interpretation of RNP29 precursor accumulation in ppi2 plastids (Bischof et al, 2011)
Summary
Most chloroplast localized proteins are encoded in the nuclear genome and synthesized at cytosolic ribosomes as precursor proteins with N-terminal transit peptides. Based on training sets with known and established chloroplast proteins, software tools were developed that predict for individual proteins their subcellular localization. Sequence features that mediate chloroplast protein import specificity are currently not known (Agne and Kessler, 2009). Recognition and selection of chloroplast-imported proteins are mediated by GTPbinding proteins that belong to two small families: Toc34/33 and Toc159/132/120/90. The Toc159 family members possess a GTP-binding domain (G domain) and a membrane anchoring domain (M domain). They differ by the length of an acidic domain (A domain) that is located N-terminal to the G- and M-domains. Depletion of the major Toc receptors usually results in a defect in photosynthetic growth as demonstrated by decreased accumulation of photosynthetic proteins in ppi and ppi (Jarvis, 2008)
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