Studies into the Molecular Basis of Chloroplast Division

Abstract

Chloroplasts are the powerhouses of plants and also perform important storage functions. Chloroplast division is an essential process that involves proteins that are conserved from prokaryotic fission and proteins evolved in eukaryotes. Due to their endosymbiotic origin, the division machineries of chloroplasts and all plastids share some core similarities with the bacterial division apparatus, but during evolution some prokaryotic components of the division machinery were not conserved and some novel components evolved to fulfill new functions. The components of the division apparatus and their interactions are being elucidated, but relatively little is known about the mechanism and dynamics of the first protein families to localize to the division site, FtsZ1 and FtsZ2. This work details a thorough investigation of the biochemical characterization of Arabidopsis thaliana FtsZ proteins and begins to determine the mechanism of FtsZ assembly. To achieve these ends a number of techniques were incorporated including: electron microscopy, protein purification, sedimentation and image processing. Following expression of FtsZ and subsequent purification, experiments aimed at assessing the activity were conducted. These included determining whether the protein was an active GTPase and capable of self-assembly as the bacterial FtsZ homolog displayed these characteristics. The recombinant protein displayed both of these activities and this result allowed for further characterization. The co-assembly critical concentration and assembly efficiency were determined by sedimentation and were 82.75 μg/ml and 33.4 ± 0.9%, respectively. Bacterial FtsZ assemblies have been reported to be in dynamic exchange with a soluble pool of FtsZ and the existence of a similar pool in plants has been discussed in the literature. Chapter III of this work investigates the composition of the soluble pool in Arabidopsis chloroplasts. Gel chromatography revealed that prior to FtsZ assembly initiation the pool consists solely of dimers. Image processing and native PAGE results suggest that at least one assembly intermediate exists between the dimer and mature filamentous assemblies. The most common intermediate observed in assembly reactions is a tetramer. Three-dimensional renderings of the dimer and tetramer are presented in chapter III and suggest that these oligomeric forms may represent consecutive steps in the assembly mechanism of Arabidopsis FtsZ.