OEP15-1 and OEP7.3 Localize to the Outer Envelope of Chloroplasts Using a Novel and Uncharacterized Targeting Pathway

Abstract

Chloroplasts originated from an endosymbiotic event where a Gram-negative cyanobacterium was engulfed by an ancestral eukaryotic cell. The genome of the endosymbiont has since gone through extensive gene transfer to the host cell nucleus during the evolutionary transition to an organelle. The majority of the plastid proteome is now encoded in the nucleus of plant cells. This means that plastid targeted proteins are translated in the host cytosol and must be translocated across the two membranes surrounding the plastids before they can perform their function. Stroma-targeted proteins posses transit peptides that are recognized for translocation by the TOC and TIC complexes. The proteins which make up the TOC complex are just some of a wide variety of OEPs which must also be targeted to the chloroplast outer membrane to perform their functions, but most OEPs use targeting strategies which do not involve transit peptides. Novel strategies for outer envelope targeting and localization are still being added to our understanding of OEPs. In the Chuong and Smith labs, a C-terminal TP-like signal was recently added to the list of known pathways and was discovered in TOC159. In an effort to test other OEPs which may posses a similar signal all OEPs were input into an N-terminal TP prediction tool, ChloroP, in their reverse orientation. The protein annotated at At4G02482 at the time of this ChloroP analysis was OEP15-1 and was one of the proteins which scored a high enough likelihood of C-terminal TP presence to be considered a candidate for this novel pathway. The research outlined in this thesis aims to determine the localization pathway or targeting strategy of OEP15-1 and the more recently annotated gene product of the same accession number, OEP7.3. The majority of the thesis focusses on the expression patterns of transiently expressed recombinant fusion proteins in onion epidermal cells and A. thaliana protoplasts which were analyzed by fluorescence microscopy and Western blotting. The expression patterns confirm that OEP7.3 and OEP15-1 are both targeted to the outer envelope of chloroplasts and suggest that they may achieve this using β-strands in a pathway similar to both mitochondria-targeting β-barrel proteins and a few studied OEP β-barrel proteins. It is not currently known if both proteins annotated at At4G02482 are produced in A. thaliana, but the data in this thesis suggests that they are. The function(s) of OEP15-1 and OEP7.3 is also unknown, but bioinformatic analyses described in this thesis point towards OEP15-1 existing as a β-barrel protein in the outer envelope of chloroplasts which may be involved in cross-membrane transport.