Master of Science (MSc)
Faculty of Science
Chloroplasts are organelles that are unique to plant and algal cells and are the site of photosynthesis. Though chloroplasts contain their own genome, an estimated 95% of chloroplast proteins are encoded in the nucleus, and therefore rely on post-translational targeting to the organelle. The majority of known chloroplast proteins are targeted to the chloroplast interior by cleavable signals at the N-terminal end of preproteins known as transit peptides. The translocon at the outer envelope membrane of chloroplasts (Toc) is a multimeric complex that recognizes and binds N-terminal transit peptides at the cytosolic surface of chloroplasts. Though transit peptides are necessary and sufficient for guiding nuclear-encoded preproteins into the chloroplast interior, the nature of sequence information of transit peptides is not fully understood due to their high divergence in length and composition. Over the last nine years, the number of proteins known or predicted to reside in the chloroplast outer envelope membrane of Arabidopsis has tripled to one hundred and seventeen. Although the functions for some of these outer envelope proteins (OEPs) have been characterized, the precise mechanism of their targeting to the chloroplast outer membrane has not been fully elucidated. Besides Toc75, the targeting mechanisms used by OEPs that have been characterized do not involve an N-terminal transit peptide. The bioinformatics tool ChloroP can be used to predict if amino acid sequences contain an N-terminal transit peptide. Recently, ChloroP analysis and protoplast transient expression assays were used to identify a novel chloroplast targeting signal in the C-terminus of the chloroplast preprotein receptor Toc159 in Bienertia sinuspersici (Lung and Chuong, 2012). Toc159 was also shown to lack a canonical transmembrane domain typically present in OEPs. While the unique C-terminal targeting sequence has been partially characterized in Toc159 (Lung et al., 2014), it left open the question of whether this type of signal is unique to Toc159, or if it is used by other OEPs as well. In the current study, to determine if other OEPs use this novel targeting pathway, ChloroP analysis identified eight potential candidates possessing the putative C-terminal targeting signal in Arabidopsis. Transient protoplast expression assays have been performed on OEP18, the protein predicted with the highest ChloroP score, to determine its subcellular localization and the sequences required for its targeting to chloroplasts. The primary purpose of the current study was to establish whether chloroplast outer membrane proteins other than Toc159 use a similar C-terminal targeting signal. Overall, the data in this thesis suggest that some OEPs other than Toc159, such as OEP18, may use this novel targeting pathway.
Grimberg, Nicholas A., "Characterizing an Alternative Chloroplast Outer Membrane Targeting Signal in Arabidopsis thaliana" (2016). Theses and Dissertations (Comprehensive). 1891.