Date of Award

8-2011

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Dr. Barry Bruce

Committee Members

Gladys Alexandre, Nitin Jain

Abstract

During chloroplast formation via endosymbiosis most of the plastid genome was transferred to the host nuclear genome. Genomic and proteomic analysis suggests that >95% of the original plastid proteome is now encoded in the nucleus, and these now cytosolically fabricated proteins require a post-translational transport pathway back into the organelle. This process is not well understood, yet it has been shown to involve translocons at the outer and inner envelope of the chloroplast membranes (TOC & TIC). These translocons interact with a cleavable N-terminal extension of between 20 and 100 residues on chloroplast-bound precursor proteins known as the transit-peptide. Precursor proteins pass through the outer membrane via the outer chloroplast membrane beta-barrel, Toc75. In addition to containing a transmembrane β-barrel, Toc75 also contains three polypeptide transport (POTRA) domain repeats at the N-terminus. Despite widespread occurrence the role of POTRAs is poorly understood. One possibility is that they function to promote either homo- or heterotypic protein:protein interactions.

To investigate these possibilities, we modeled the psToc75 POTRA domains and purified recombinant POTRA domains. POTRA1, POTRA3, and POTRA1-3 have been used to investigate interactions. Homotypic POTRA interactions have been supported by crosslinking experiments and analytical ultra centrifugation (AUC). Crosslinking data shows POTRA1 and POTRA3 undergo oligimerization. AUC suggests that POTRA1 may homodimerize. Heterotypic interactions have been studied via pull-down assays, crosslinking, and AUC and demonstrate that POTRA1 and POTRA3 interact with transit peptide. Soluble POTRA1-3 seems to stimulate precursor protein import into isolated chloroplasts in an import assay. The role of POTRAs in guiding TOC assembly by homodimerization is being investigated, and experiments to establish how POTRAs aggregate are underway.

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