Dissertation/Thesis Abstract

Interrogation of quality control mechanisms and protein trafficking in Saccharomyces cerevisiae
by Young, Carissa Leigh, Ph.D., University of Delaware, 2012, 381; 3543550
Abstract (Summary)

In eukaryotes, the endoplasmic reticulum (ER) is responsible for maintaining the fidelity of protein synthesis and maturation. As such, we have interrogated several quality controls mechanisms in the model eukaryote, S. cerevisiae . In this thesis, we investigated the intricate processes of the unfolded protein response (UPR), ER-associated degradation (ERAD), and autophagy to elucidate the cell's control of ER homeostasis; thus, focusing on the advancement of our fundamental understanding of cellular regulation.

Due to the intrinsic complexity of biological systems, the integration of experimental and computational approaches was utilized. To establish that the UPR is a global response to localized perturbations, we investigated transcriptional effects in response to UPR activation. Interestingly, microarray analysis and q-PCR validation confirmed the novel repression of 206 genes – highly enriched in protein synthesis and metabolic biological functions – following ER stress. A putative regulatory network has been established and mechanistic insight is provided by the analysis of yeast deletion strains.

To determine that protein redistribution, spatial effects, and organelle modifications are a consequence of the cell's response to ER stress, high-resolution imaging techniques were utilized in combination with DNA recombinant systems to label selective endogenous proteins. For these studies, multiple yeast expression cassettes were designed to test the effects of codon-optimized fluorescent variants, small epitope tags, polylinker length for C-terminal tags, and the inclusion of essential retrieval sequences for ER luminal chaperones and foldases. Using fluorescent protein variants as reporters, we monitored protein trafficking, analyzed the spatiotemporal effects of ERAD proteins, examined organelle dynamics under various environmental conditions, and confirmed the existence of cellular variability during UPR activation.

Using state-of-the-art techniques such as Structure Illumination Microscopy (SIM), Fluorescence Photoactivated Localization Microscopy (F-PALM), and Focused Ion Beam (FIB) microscopy to image cells, we developed entire three-dimensional organelle reconstructions of yeast cells. As a direct consequence of single-cell analyses, we established that endogenous proteins involved in quality control redistribute within the cell, specifically the ER, in order to perform essential functions that maintain cell homeostasis. Furthermore, high-resolution imaging techniques provided new perspectives regarding the expression, trafficking, and regulation of recombinant G protein-coupled receptors (GPCRs) in yeast. Chimeric receptors were rationally engineered to identify domains and motifs that affect production, subcellular localization, and activity of adenosine receptors hA1R, hA2aR, and hA3R.

Indexing (document details)
Advisor: Robinson, Anne Skaja
Commitee: Czymmek, Kirk J., Duncan, Randall L., Lenhoff, Abraham M., Ogunnaike, Babatunde A.
School: University of Delaware
Department: Department of Chemical Engineering
School Location: United States -- Delaware
Source: DAI-B 74/03(E), Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Molecular biology, Cellular biology, Chemical engineering, Nanotechnology
Keywords: Endoplasmic reticulum, Fluorescent proteins h/kdel, Gpcr, Protein trafficking, Quality control s. cerevisiae, Super-resolution imaging techniques, Upr
Publication Number: 3543550
ISBN: 9781267724830
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