Biology

Permanent URI for this collectionhttps://uwspace.uwaterloo.ca/handle/10012/9938

This is the collection for the University of Waterloo's Department of Biology.

Research outputs are organized by type (eg. Master Thesis, Article, Conference Paper).

Waterloo faculty, students, and staff can contact us or visit the UWSpace guide to learn more about depositing their research.

Browse

Browsing Biology by Author "Chuong, Simon"

Filter results by typing the first few letters
Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Investigating Protein Targeting to the Outer Membrane of Plastids
    (University of Waterloo, 2021-02-03) Nash, Delaney; Chuong, Simon
    Plastids are plant organelles with specialized functions, such as photosynthesis. The specialized function of each plastid is informed by its distinct and dynamically regulated proteome. The vast majority of plastid proteins are synthesized in the cytosol and are imported into the plastid post-translationally. A variety of receptors and channels embedded within the plastid outer and inner envelope regulate the import of plastid proteins, thus, control the plastid proteome composition. Proteins embedded within the outer envelope membrane of plastids have been generally categorized into four groups which include, B-barrel proteins, tail-anchored proteins, signal-anchored proteins, and CT TP-like proteins. Each group is defined by distinct structural and plastid-targeting characteristics. B-barrel proteins are composed of B-sheets and their plastid-targeting signal and mechanism is not well understood. Tail-anchored and signal-anchored proteins are tethered to the plastid outer envelope membrane by a single transmembrane alpha-helix located at the proteins C-terminus or N-terminus, respectively, and use a variety of physiochemical features for plastid-targeting. Lastly, the only currently defined CT TP-like protein, Toc159, utilizes a C-terminal plastid-targeting signal with transit peptide-like features. In this study, the structure and plastid-targeting signal of the plastid protein Outer Envelope Protein 16-2 (OEP16-2) was investigated. Computational structural analysis showed that OEP16-2 is embedded within the outer envelope membrane by four alpha-helical transmembrane domains and does not share structural similarity with defined categories of outer envelope proteins. Furthermore, three internal transmembrane alpha helical domains were sufficient for plastid-targeting. These internal targeting domains cannot be characterized by currently defined outer envelope protein targeting strategies. Thus, OEP16-2 was classified in a fifth outer envelope protein category, defined by multiple transmembrane alpha helices and internal targeting domains. Future experiments will examine the structure and plastid-targeting signal of other outer envelope proteins with multiple transmembrane helices.
  • Thumbnail Image
    Item
    Investigating the Outer Envelope Localization of OEP 18 in A. thaliana Chloroplasts
    (University of Waterloo, 2021-10-26) Zhou, Tianlun; Chuong, Simon; Smith, Matthew
    Plastids are plant organelles with specialized functions including photosynthesis. The specialized function of each plastid is informed by its distinct and dynamically regulated proteome. The majority of plastid proteins are synthesized in the cytosol and are imported into the plastid post-translationally. A variety of receptors and channels embedded within the plastid outer and inner envelope membranes regulate the import of plastid proteins, and thus control the plastid proteome composition. There are 117 known Outer Envelope Proteins (OEPs) that reside in the chloroplast outer membrane. Of these 117 OEPs, only a few have been extensively studied regarding their targeting. With the exception of Toc75, which uses the general pathway involving an N-terminal cleavable transit peptide (TP) for targeting, the OEPs that have been studied so far either use the signal-anchored, tail-anchored, or β-barrel pathway for targeting. However, previous studies showed that a key component of the translocon at the outer envelope of chloroplasts (Toc complex), Toc159, possesses a novel reverse TP-like sorting signal and an unconventional membrane anchor within the C-terminus for its own targeting and insertion into the outer membrane. Moreover, recent studies using bioinformatics tools predicted that eight additional OEPs also contain transit peptide-like sequences at their C-termini. Transient expression assays in Arabidopsis mesophyll protoplasts demonstrated that one of the candidates, OEP18, appears to be targeted to the chloroplast outer membrane using such a signal. My research was able to determine the precise subcellular location of OEP18 using protoplast transient expression assays, chloroplast fractionation, and Western blot analysis. Secondary structure and membrane topology prediction analyses for all 117 OEPs were also used for grouping according to their targeting pathways. My research also determined that the predicted reverse TP region of OEP18 is responsible for specific targeting to the chloroplast outer membrane, but not its anchoring. These findings will allow for a better understanding of protein targeting to the outer membrane of chloroplasts.
  • Thumbnail Image
    Item
    OEP15-1 and OEP7.3 Localize to the Outer Envelope of Chloroplasts Using a Novel and Uncharacterized Targeting Pathway
    (University of Waterloo, 2021-09-27) Overton, Alyssa Kate; Chuong, Simon; Smith, Matthew
    Chloroplasts originated from an endosymbiotic event where a Gram-negative cyanobacterium was engulfed by an ancestral eukaryotic cell. The genome of the endosymbiont has since gone through extensive gene transfer to the host cell nucleus during the evolutionary transition to an organelle. The majority of the plastid proteome is now encoded in the nucleus of plant cells. This means that plastid targeted proteins are translated in the host cytosol and must be translocated across the two membranes surrounding the plastids before they can perform their function. Stroma-targeted proteins posses transit peptides that are recognized for translocation by the TOC and TIC complexes. The proteins which make up the TOC complex are just some of a wide variety of OEPs which must also be targeted to the chloroplast outer membrane to perform their functions, but most OEPs use targeting strategies which do not involve transit peptides. Novel strategies for outer envelope targeting and localization are still being added to our understanding of OEPs. In the Chuong and Smith labs, a C-terminal TP-like signal was recently added to the list of known pathways and was discovered in TOC159. In an effort to test other OEPs which may posses a similar signal all OEPs were input into an N-terminal TP prediction tool, ChloroP, in their reverse orientation. The protein annotated at At4G02482 at the time of this ChloroP analysis was OEP15-1 and was one of the proteins which scored a high enough likelihood of C-terminal TP presence to be considered a candidate for this novel pathway. The research outlined in this thesis aims to determine the localization pathway or targeting strategy of OEP15-1 and the more recently annotated gene product of the same accession number, OEP7.3. The majority of the thesis focusses on the expression patterns of transiently expressed recombinant fusion proteins in onion epidermal cells and A. thaliana protoplasts which were analyzed by fluorescence microscopy and Western blotting. The expression patterns confirm that OEP7.3 and OEP15-1 are both targeted to the outer envelope of chloroplasts and suggest that they may achieve this using β-strands in a pathway similar to both mitochondria-targeting β-barrel proteins and a few studied OEP β-barrel proteins. It is not currently known if both proteins annotated at At4G02482 are produced in A. thaliana, but the data in this thesis suggests that they are. The function(s) of OEP15-1 and OEP7.3 is also unknown, but bioinformatic analyses described in this thesis point towards OEP15-1 existing as a β-barrel protein in the outer envelope of chloroplasts which may be involved in cross-membrane transport.