Abstract
Post-translational import of nucleus-encoded chloroplast pre-proteins is critical for chloroplast biogenesis, and the Toc159 family of proteins serve as receptors for the process. Toc159 shares with other members of the family (e.g. Toc132), homologous GTPase (G−) and Membrane (M−) domains, but a highly dissimilar N-terminal acidic (A−) domain. Although there is good evidence that atToc159 and atToc132 from Arabidopsis mediate the initial sorting step, preferentially recognizing photosynthetic and non-photosynthetic preproteins, respectively, relatively few chloroplast preproteins have been assigned as substrates for particular members of the Toc159 family, which has limited the proof for the hypothesis. The current study expands the number of known preprotein substrates for members of the Arabidopsis Toc159 receptor family using a split-ubiquitin membrane-based yeast two-hybrid system using the atToc159 G-domain (Toc159G), atToc132 G-domain (Toc132G) and atToc132 A- plus G-domains (Toc132AG) as baits. cDNA library screening with all three baits followed by pairwise interaction assays involving the 81 chloroplast preproteins identified show that although G-domains of the Toc159 family are sufficient for preprotein recognition, they alone do not confer specificity for preprotein subclasses. The presence of the A-domain fused to atToc132G (Toc132AG) not only positively influences its specificity for non-photosynthetic preproteins, but also negatively regulates the ability of this receptor to interact with a subset of photosynthetic preproteins. Our study not only substantiates the fact that atToc132 can serve as a receptor by directly binding to chloroplast preproteins but also proposes the existence of subsets of preproteins with different but overlapping affinities for more than one member of the Toc159 receptor family.
Highlights
Chloroplasts are specialized plastids, which are the site of photosynthesis, and play crucial roles in other essential processes such as lipid, phytohormone and amino acid biosynthesis [1,2], nitrogen and sulphur metabolism [3], and chloroplast-tonucleus retrograde signaling [4]
In order to maximise the complexity of the preproteins being screened, both random- and oligo(dT)primed Arabidopsis cDNA libraries suitable for split-ubiquitin membrane-based yeast two-hybrid screening were prepared
Based on the previously hypothesized alternate import pathways for photosynthetic and non-photosynthetic preproteins and recent evidence that the A-domain of the atToc159 family of receptors contributes to the regulatory role in their preprotein recognition and/or selectivity [37], we examined the relative binding properties of atToc159AG and atToc132AG with transit peptides of representative photosynthetic preproteins, namely, LightHarvesting Chlorophyll-Protein Complex I Subunit A4 (LHCA4) and Ferredoxin-NADP(+)-oxidoreductase 1 (FNR1)
Summary
Chloroplasts are specialized plastids, which are the site of photosynthesis, and play crucial roles in other essential processes such as lipid, phytohormone and amino acid biosynthesis [1,2], nitrogen and sulphur metabolism [3], and chloroplast-tonucleus retrograde signaling [4]. Because of their semi-autonomous nature [5], the biogenesis of chloroplasts requires a well coordinated and tightly regulated interaction between the nuclear and chloroplast genomes [4]. Extensive studies on the Toc159 and Toc families of GTPase receptors and the import of photosynthetic and constitutive non-photosynthetic pre-proteins have given rise to the hypothesis that there are multiple import pathways in Arabidopsis, which are responsible for maintaining chloroplast homeostasis by supporting proportionate import of variably expressed nucleusencoded chloroplast proteins at different stages of chloroplast biogenesis [21,22,23,24]
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