Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Biology

Program Name/Specialization

Integrative Biology

Faculty/School

Faculty of Science

First Advisor

Dr. Matthew Smith

Advisor Role

Thesis Supervisor

Abstract

Chloroplast-destined preproteins are translated in the cytosol, and posttranslationally targeted to and translocated across the double envelope membrane of the chloroplast by the coordinated activities of two translocon complexes: the Translocons at the Outer and Inner envelope membrane of the Chloroplast (TOC and TIC, respectively). In the model organism Arabidopsis thaliana the core TOC components include two families of GTPase receptors: TOC159 (atToc159, -132, and -120) and TOC34 (atToc33 and -34). These receptor families are hypothesized to assemble into distinct complexes and recognize transit peptides present on the N-terminus of chloroplast-destined preproteins. The GTPase domains of the TOC159 and TOC34 family members are hypothesized to interact in such a way that structurally and functionally distinct TOC complexes are formed. These distinct complexes are thought to have specificity for different subsets of preproteins. Chloroplasts must differentiate between different subsets of proteins because they are needed in different amounts during various stages of chloroplast biogenesis. This investigation examines the propensity for atToc33 and atToc34 to associate with atToc159 or atToc132, how these interactions affect TOC complex formation, as well as what protein domains are conferring this preference. In vitro competitive chloroplast targeting assays, in which the GTPase domains of atToc33 or atToc34 are used as competitors for targeting of atToc159 or atToc132 to chloroplasts, and in vitro solidphase binding assays, in which the GTPase domains of atToc33 or atToc34 are used as "bait" to test interactions with "prey" atToc159 or atToc132 are used to characterize these interactions. In order to study the influence of the highly divergent A-domain, these

associations are also being investigated using A-domain deletion mutants, atToc159GM and atToc132GM as well as A-domain swapped mutants 159A132GM and 132A159GM. This investigation has revealed that the mechanisms governing TOC GTPase interactions in Arabidopsis may be dictated by the A-domain of atToc132 and the G-domain of atToc159, thereby giving insight into how key TOC components are assembled into distinct TOC complexes at the chloroplast surface. Distinct complexes are responsible for the critical identification and import of different subsets of preproteins, all of which are necessary for plant growth and development.

Convocation Year

2013

Convocation Season

Spring

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