Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Biology

Program Name/Specialization

Integrative Biology

Faculty/School

Faculty of Science

First Advisor

Matthew Smith

Advisor Role

Supervision, Collaboration on Experimental Design, Grant Funding, Thesis Feedback

Second Advisor

Simon Chuong

Advisor Role

Supervision, Collaboration on Experimental Design, Thesis Feedback

Abstract

Chloroplast biogenesis and function relies on proper import of nuclear-encoded proteins and their post-translational targeting to the chloroplast via a cleavable, N-terminal transit peptide (TP). Many of these proteins are destined for and function within the chloroplast outer membrane, having been identified to utilize a variety of targeting mechanisms, including traditional TPs, signal- and tail-anchored mechanisms, β-barrel signals, and reverse TP-like C-terminal sequences. Recent expansion of the known and predicted chloroplast outer membrane proteome has prompted the idea that some OEPs may not utilize or rely on previously established targeting signals or mechanisms. My research focuses on a novel OEP, OEP6, which has been recently identified in the chloroplast outer membrane of Arabidopsis thaliana. OEP6 contains a single ⍺-helical membrane spanning domain, with a poorly understood targeting mechanism and classification. This study aimed to examine molecular features believed to be important in OEP6 targeting, such as its ⍺-helical transmembrane domain and positively charged flanking regions, as well as investigate its previous identification as a tail-anchored protein. A series of OEP6 deletion and truncation mutants fused to EGFP (Enhanced Green Fluorescent Protein) were transformed into onion epidermal cells using biolistic bombardment and introduced into Arabidopsis mesophyll protoplasts using polyethylene glycol-mediated protocols, where their intracellular localization was determined using epifluorescence and confocal microscopy, respectively. The localization patterns were complemented by biochemical analyses, involving cellular fractionation and Western blotting, as well statistical analyses. These analyses determined N-terminal positive charges flanking the transmembrane ⍺-helix of OEP6 are key features in its chloroplast targeting signals. A reverse-mutation involving mitochondrial outer membrane protein, Tom7, revealed a C-terminal charged sequence responsible for organelle-specific targeting. A combination of these findings determined that OEP6 may utilize a novel targeting mechanism, one that is reliant on the presence of a bipartite charged signal, contradicting its previous tail-anchored classification and supporting the existence of novel targeting mechanisms for smaller outer membrane proteins. Overall, the findings generated from this study contribute to the broader understanding of intracellular protein trafficking; with specific advances to the understanding of chloroplast protein import and targeting having potential applications to both the agricultural and biotechnological industries.

Convocation Year

2026

Convocation Season

Spring

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Available for download on Tuesday, December 22, 2026

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