title: A split-ubiquitin yeast two-hybrid screen to examine the substrate specificity of atToc159 and atToc132, two Arabidopsis chloroplast preprotein import receptors.
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.
authors.affiliation: Department of Biology, Wilfrid Laurier University, Waterloo, Ontario, Canada.
authors.forename: Siddhartha
authors.initials: S
authors.lastname: Dutta
authors.affiliation: Department of Biology, Wilfrid Laurier University, Waterloo, Ontario, Canada.
authors.forename: Howard J
authors.initials: HJ
authors.lastname: Teresinski
authors.affiliation: Department of Biology, Wilfrid Laurier University, Waterloo, Ontario, Canada.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Smith
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D015723:Gene Library; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques
chemical_list: C000594370:AT2G16640 protein, Arabidopsis; D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D008565:Membrane Proteins; D010455:Peptides; D018160:Receptors, Cytoplasmic and Nuclear; C403444:TOC159 protein, Arabidopsis; D020558:GTP Phosphohydrolases
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