Date of Award

8-2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Barry D. Bruce

Committee Members

Andreas Nebenfuehr, Francisco Barrera, Xiaolin Cheng, Todd Reynolds

Abstract

Eukaryotic organisms are defined by their compartmentalization and various organelles. The membranes that define these organelles require complex nanomachines (known as translocons) to selectively mediate the import of proteins from the cytosol where they are synthesized into the organelle. The plastid, (specifically the chloroplast) which is characteristic of plant cells, possibly represents the most complex system of protein sorting, requiring many different translocons located in the three membranes found in this organelle. Despite having a small genome, the vast majority of plastid-localized proteins are nuclear-encoded and must be post-translationally imported from the cytosol. These proteins are encoded as a larger molecular weight precursor that contains a special “zip code” at their N-terminus appropriately called a transit peptide (TP).

The mechanism of how the transit peptide mediates the selective targeting and translocation of precursor proteins into chloroplasts remains unclear. We aim to precisely understand how transit peptides are recognized and processed by the Translocon of the Outer Chloroplast membrane (abbreviated TOC). This is still a paradox, since several thousand different transit peptides exist in a given plant species, yet show very little similarity with each other – however, they seem to function in a common pathway that involves the recognition one or more receptor GTPases, which are proposed to function as “gatekeepers.” The current study extends our understanding of both TP physicochemical motifs as well as our understanding of the TOC34 GTP hydrolysis cycle. Furthermore, this study has advanced methodologies for transient expression and in vivo localization imaging using Pisum sativum. Together, our results have made a significant step forward in our understanding of the plastid protein import cycle.

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