Defense Date

2-4-2019

Graduation Date

Spring 5-10-2019

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Biological Sciences

Committee Chair

Jana Patton-Vogt

Committee Member

Joseph McCormick

Committee Member

Michael Jensen-Seaman

Committee Member

Michael Cascio

Keywords

Gpc1, Ale1, phosphatidylcholine, glycerophosphocholine, lipid remodeling, acyltransferase, membrane fluidity, lipid saturation

Abstract

Biomembranes are permeable barriers that enclose the cell and the intracellular organelles in a cell. The selective nature of these robust barriers acts as the first line of defense towards the harsh factors that can compromise cell survival. Biomembranes primarily consist of membrane lipids that are organized into layers to form a dynamic bilayer structure. The dynamic nature of the membrane requires the synchronized modulation of lipid composition through de novo synthesis, degradation, intracellular movement, and remodeling. Phospholipids are the major membrane lipid class, and phosphatidylcholine (PC) is the most abundant phospholipid in most eukaryotic biomembranes.

PC is primarily produced via two major pathways: the Kennedy and PE methylation pathways. Through a collaborative project, we have discovered a lipid-modifying enzyme, a glycerophosphocholine acyltransferase, Gpc1, involved in a new pathway for PC biosynthesis and PC remodeling. Here, I have examined this alternative route for PC biosynthesis and remodeling using Saccharomyces cerevisiae (S. cerevisiae) as a model organism. First, I performed in vivo metabolic labeling studies to delineate the role of Gpc1 in the alternative route for PC biosynthesis. I have also determined the apparent molecular weight of this novel Gpc1 protein by constructing a C-terminal epitope tag followed by Western blot analysis. These studies are described in more detail in Chapter two.

In the second half of my dissertation, I have examined the physiological function and regulatory aspects of Gpc1. Using LC-MS analysis, I have determined the PC species profile generated via this alternative pathway. Gpc1 was found to be involved in a PC deacylation/reacylation remodeling pathway (PC-DRP) that results in an increase in monounsaturated PC species at the expense of diunsaturated PC species. Here, I report inositol, a phospholipid precursor, as a down-regulator for GPC1 expression. Also, I have identified a phenotype associated with the loss of GPC1. Loss of GPC1 results in decreased cell viability for cells at the stationary phase. These studies are described in detail in Chapter three. Overall, in Chapters two and three, I have identified Gpc1 as a member of PC-DRP, a pathway that increases the saturation state of the PC. This increase may impact on the physiochemical properties of the membrane.

Language

English

Additional Citations

For chapter 2: Glab, B., et al. (2016). Cloning of Glycerophosphocholine Acyltransferase (GPCAT) from Fungi and Plants: A NOVEL ENZYME IN PHOSPHATIDYLCHOLINE SYNTHESIS. The Journal of Biological Chemistry. 291: 25066-25076.

For chapter 3: Anaokar, S, et. al. (2018). The glycerophosphocholine acyltransferase, Gpc1 is part of a defined phosphatidylcholine (PC)-remodeling pathway that alters PC species in yeast. The Journal of biological chemistry.

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