Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Biology

Program Name/Specialization

Integrative Biology

Faculty/School

Faculty of Science

First Advisor

Dr. Simon Chuong

Advisor Role

Supervisor

Abstract

Approximately 95% of chloroplast proteins are encoded by nuclear DNA and are post-translationally targeted to the chloroplast. Over half of the 138 known or predicted chloroplast outer membrane (OM) proteins are predicted to utilize four non-canonical targeting pathways. The first two pathways are characterized by containing a single α-helical transmembrane domain (TMD) within the first or last 50 amino acids (AAs), the third pathway is utilized by β-barrels, and the last requires an N-terminal cleavable transit peptide (TP). Recently, a fifth novel pathway called Toc159-like (TOC159L) has been characterized by the presence of a C-terminal reverse TP in Toc159. Subsequent analysis of all known chloroplast outer membrane proteins (OMPs) for this TOC159L feature identified 36 candidates including OEP18. However, recently developed structural prediction tools suggest that OEP18 is not a TOC159L candidate but it does contains a region of highly concentrated predicted secondary structures, referred to as the β-domain which is part of a conserved domain that is traced back over 1 billion years. In this thesis, the β-domain with and without the C-terminus were transiently expressed in onion epidermal cells and Arabidopsis protoplasts showing targeting to the plastids and the chloroplast exterior, respectively. This was also true for an OEP18 construct only containing the C-terminus. This suggests that both the β-domain and the C-terminus are sufficient for targeting to plastids. Western blotting showed targeting of a construct containing both features to the chloroplast, but this was not seen in other constructs. The mechanism is hypothesized as the C-terminus getting the protein to be in close proximity to the chloroplast OM, and the β-domain facilitating the interaction with the membrane. The presence of multiple targeting signals within a single protein led to the re-examination of a targeting motif previously identified in tail-anchored (TA) proteins. RK/ST residue motifs were previously shown to be an interchangeable and required targeting signals in some tail-anchored proteins. As such all chloroplast and mitochondrial OMPs, and endomembrane proteins were analyzed for a motif in close proximity to a TMD, since the RK/ST residue motif was found within 10 AAs of an α-helical TMD. This provides information that helps to close the gap of knowledge on how almost half of the chloroplast OMPs target the membrane.

Convocation Year

2024

Convocation Season

Fall

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Available for download on Saturday, May 30, 2026

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