Date of Award
Master of Science
Barry D. Bruce
Albrecht von Arnim, Beth Mullin, Todd Reynolds
The majority of plastid proteins are nuclear-encoded and imported post-translationally. A cleavable N-terminal extension, the transit peptide, targets these preproteins to the plastid. Transit peptides show very little primary sequence homology, yet are able to direct the precursor protein to interact with the protein components of the translocation complexes located within the inner and outer membranes of the chloroplast. In this study, a semi-conserved motif of the transit peptide, (F/W)(P/G)h(R/K) has been targeted for deletion in order to probe its importance. Two corresponding regions were deleted in the transit peptide of the precursor to the small subunit of Rubisco (prSSU) from tobacco: FTGLK and FPVSR. Both full and partial deletions were made in each region, resulting in the following 6 deletions: FTGLK, FTG, GLK, FPVSR, FPV, and VSR. The mutant preproteins as well as WT precursor (prSSU) and mature protein (mSSU) were overexpressed and purified in E. coli and the import competence evaluated by in vitro chloroplast import competition assays. Both of the full deletions caused a marked decrease in ability to compete for import, whereas the partial deletions had less of an effect. One aspect of import that could be compromised is transit peptide interactions with the Toc GTPase components of the translocon, Toc34 & Toc159. Recent work has shown a complex interaction between these GTPases and their substrates: preproteins/transit peptides and GTP/GDP. Although the Toc GTPases have been proposed to function as “gate-keepers”, neither the psToc34 crystal structure nor the low- resolution Toc complex structure reveal the dynamics of Toc-Toc or Toc-preprotein interactions during preprotein binding and translocation. In order to investigate the interactions between the soluble cytosolic domain of the Toc GTPase psToc34 and the transit peptide, analytical ultracentrifugation (AUC) was used. Using sedimentation velocity centrifugation, the presence of a homodimer of psToc34 was observed. The population of the homodimer increased in the presence of the transit peptide substrate, and decreased in the presence of nucleotide substrate. AUC of the mutant transit peptides with psToc34 showed that they were unable to influence the oligomeric state of psToc34 to the extent of WT transit peptide.
Wright, Sarah Jean, "Probing Precursor Interactions with the Chloroplast Import Apparatus. " Master's Thesis, University of Tennessee, 2006.