Page 267 - PC2019 Program & Proceedings

PLANT CANADA 2019

P107. A novel method of producing the putative c-terminal transit peptide of attoc159 for
characterization of its targeting to the chloroplast outer membrane
2
1
*1
Fish, M. ; M. Jelokhani-Niaraki ; S. Chuong ; M. Smith
1
2
1 Wilfrid Laurier University; University of Waterloo
Chloroplast preproteins that are destined for interior chloroplast compartments and that are encoded in the
nucleus rely on N-terminal transit peptides (TPs) that are recognized and translocated by members of the
Toc (translocon at the outer membrane of chloroplasts) complex, including Toc159. Proteins that reside in
the chloroplast outer membrane (COM), instead, are typically directed to the chloroplast by intrinsic
transmembrane domains. Recently two COM proteins, Toc159 and OEP18, have been shown to be
targeted to the chloroplast outer membrane by a novel reverse TP-like C-terminal sequence. The objective
of my research is to characterize this novel targeting mechanism in order to understand the molecular
interactions involved and determine the role of membrane lipid composition in targeting specificity.
Bioinformatic analysis revealed three highly conserved pseudo-domains within the putative C-terminal
targeting sequence. One of these pseudo-domains is an amphipathic helix rich in hydrogen bonding
partners with the potential to make preferential interactions with galactolipids that are unique to the
chloroplast membrane. The Toc159 C-terminal peptide was overexpressed as a fusion protein in E.
coli and purified from inclusion bodies using immobilized metal affinity chromatography. Biophysical
techniques, including circular dichroism spectroscopy, will be used to investigate secondary structures
and conformational changes under different conditions, and surface plasmon resonance will be used to
investigate interactions with lipids and other proteins. Our most recent results will be presented.

Michael Fish (fish1960@mylaurier.ca)

P108. Investigating protein localization to the outer membrane of chloroplasts
2
*1
Nash, D. ; M. Smith ; S. Chuong 1
1 University of Waterloo
2 Wilfrid Laurier University

The chloroplast evolved through an endosymbiotic event wherein a photosynthetic bacterium was
engulfed by a heterotrophic eukaryote. Over time, genes have been transferred from the endosymbiotic
bacterial genome to the host nuclear genome. Consequently, the proteins encoded by these genes gained
chloroplast-targeting information; allowing them to be directed to the chloroplast post-
translationally. For chloroplast stromal-localized proteins this targeting information is typically a peptide
extension found on the N-terminus called a transit peptide (TP). With the exception of Toc75, the
majority of chloroplast outer envelope proteins (OEPs) do not possess an N-terminal transit peptide and
are targeted to the chloroplast by at least three different mechanisms. Recently, a novel chloroplast-
targeting pathway was identified in the OEP, Toc159, of Bienertia sinuspersici. It was shown that Toc159
uses a transit peptide-like sorting signal in its C-terminus to target to the chloroplast outer
membrane. The goal of my research is to investigate whether OEP16-2, one of eight OEPs predicted to
have a TP-like targeting signal in the C-terminus, uses this novel targeting pathway. Transient expression
assays of onion epidermal cells and Arabidopsis mesophyll protoplasts will be performed to examine the
subcellular localization of OEP16-2. The findings from this study will enhance our knowledge of protein
targeting to the chloroplast.

Delaney Nash (d2nash@uwaterloo.ca)

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