Dynamics of the Toc GTPases: Modulation by Nucleotides and Transit Peptides Reveal a Mechanism for Chloroplast Protein Import

The chloroplast is the green organelle in the plant cell responsible for harvesting energy from sunlight and converting it into sugars and ATP. Origins of this organelle can be traced back to an endosymbiotic event in which a primitive eukaryotic cell capable of oxidative phosphorylation engulfed a free-living cyanobacterium capable of photosynthetic respiration (1). Immediately following this event the details are not clear, however what is known is that over the course of evolution, the engulfed cyanobacteria relinquished approximately 97% of its protein coding sequences to the host cell nucleus, thus making the newly formed chloroplast reliant on its host cell (2). This resulted in the requirement of a post-translational import mechanism (3,4). Accomplishing posttranslational import are Translocons of the Outer and Inner Chloroplast membranes, or TOC and TIC complexes (5). These complexes are comprised of multiple proteins whose function is the efficient and robust recognition of chloroplast-destined preproteins and their subsequent import. Preproteins are synthesized in the cytosol with a cleavable Nterminal extension of approximately 50-150 amino acids known as a transit peptide (6-8). It is the transit peptide that is recognized by the Toc complex which facilitates the import of the preprotein (9). It is this transit peptide mediated chloroplast protein import mechanism that will be the subject of this dissertation. Presented in Chapter II is an analysis of the basal enzymology of the isolated, soluble forms of the Toc GTPases. Chapter III analyzes the homo- and heterodimeric interaction between Toc proteins and how this oligomerization can be modulated. Chapter IV presents evidence that the transit 2 peptide interacts with the Toc proteins in such a way as to increase enzymatic activity as well as bias the dimeric equilibria. Analysis of the data presented in Chapters II, III and IV allow the creation of a chloroplast protein import model, Chapter V, to potentially explain the observed phenomenon. Finally, Chapter VI presents potential future directions for this research.