Recombinant Arabidopsis thaliana Translocase of chloroplast 90, chloroplastic (TOC90)

Basic Research Questions

• What is the function of TOC90 in the chloroplast protein import machinery?

  TOC90 is a GTP-binding receptor protein that forms part of the Translocon at the Outer Chloroplast membrane (TOC) complex in Arabidopsis thaliana. It functions primarily in the recognition and import of nucleus-encoded photosynthetic proteins into the chloroplast . As one of four Toc159 homologs (alongside atToc159, atToc132, and atToc120), TOC90 exhibits a characteristic tripartite structure consisting of an N-terminal acidic domain (A-domain, though greatly reduced in TOC90), a central GTP binding domain (G-domain), and a C-terminal membrane-anchor domain (M-domain) . Experimental evidence indicates that TOC90 specifically contributes to the accumulation of photosynthetic proteins in plastids but is not required for import of several constitutive proteins .

• How does TOC90 relate structurally and functionally to other members of the Toc159 family?

  TOC90 shares significant structural homology with other members of the Toc159 family, particularly within the conserved G- and M-domains. Sequence analysis reveals that the G-domain of TOC90 shares 44.3% identity with atToc159, while full-length sequence identity between these proteins is approximately 30.5% . Unlike other family members, TOC90 has a greatly reduced A-domain.

  Phylogenetic relationships among Toc159 family members show distinct clustering patterns:

  TOC Protein Pairs	G-domain Identity	Full-length Identity
  atToc132-atToc120	93.4%	68.9%
  atToc159-atToc120	58.1%	36.7%
  atToc159-atToc90	44.3%	30.5%

  Functionally, TOC90 appears to share substrate specificity with atToc159, both preferentially facilitating the import of photosynthetic proteins, while atToc132 and atToc120 show redundancy in importing non-photosynthetic proteins .

• What phenotypes are observed in toc90 knockout mutants?

  Single toc90 knockout mutants do not display obvious visible phenotypes throughout development, appearing indistinguishable from wild-type plants . Multiple independent alleles (toc90-1, toc90-2, and toc90-3) confirm this lack of visible phenotype. Chlorophyll measurements in toc90 plants show wild-type levels of chlorophyll per unit of fresh weight . This suggests functional redundancy with other TOC receptors, particularly atToc159. The absence of a visible phenotype contrasts with the severe albino phenotype of toc159 (ppi2) mutants and the mild yellow-green, somewhat reticulate phenotype observed in mature toc132 plants .

• How is TOC90 gene expression regulated in different tissues and developmental stages?

  RNA gel blot analysis reveals that atTOC90 is expressed at a uniformly high level throughout development, unlike other TOC159 family members that show more tissue-specific expression patterns . The expression profile of atTOC90 differs notably from atTOC159, which is strongly expressed in young, photosynthetic tissues, and from atTOC132/atTOC120, which are expressed at uniformly low levels and relatively more prominent in non-photosynthetic tissues . This uniform high expression pattern suggests that atToc90 may not exhibit the same substrate specificity limitations as other family members and might serve a more general function across different tissue types and developmental stages.

• What experimental approaches are typically used to study TOC90 function?

  Several complementary experimental approaches are commonly employed to study TOC90 function:

  • Genetic knockout studies: T-DNA insertion mutants (toc90-1, toc90-2, toc90-3) allow phenotypic characterization .

  • Complementation assays: Overexpression of TOC90 in toc159 (ppi2) mutants to assess functional overlap .

  • Proteome profiling: Assessment of protein accumulation patterns in wild-type, mutants, and complemented lines .

  • Protein-protein interaction studies: Co-immunoprecipitation assays to identify interaction partners .

  • Bimolecular fluorescence complementation (BiFC): To visualize protein-protein interactions in vivo .

  • RNA expression analysis: RNA gel blot analysis to determine expression patterns .

  • Phylogenetic analysis: Comparative sequence analysis of G-domains to establish evolutionary relationships .

Advanced Research Questions