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Title:Characterization of the first steryl ester transporter in biology, Yol075c, and its pleiotropic effects
Author(s):Ellis, Ez rhodes Coriander
Director of Research:Fratti, Rutilio
Doctoral Committee Chair(s):Fratti, Rutilio
Doctoral Committee Member(s):Gerlt, John; Gennis, Robert; Zhang, Kai
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Yeast, ABC transporter, Sterol, Steryl Ester
Abstract:Sterol transport between the endoplasmic reticulum (ER) and the plasma membrane (PM) in the yeast Saccharomyces cerevisiae occurs through a transport pathway linking the lipid droplet (LD) stored steryl esters and mediated by the ABC transporter Yol075c. Sterols are synthesized in the ER and are bound for one of two fates, either free or esterified. Sterols being metabolically expensive to produce and toxic in high quantities may be esterified with a fatty acid and stored in an inert form inside the lipid droplet. When there is a need for free sterols the steryl esters can be hydrolyzed removing the fatty acid and free sterols are then incorporated into the cellular membranes. There are three steryl ester hydrolases in Saccharomyces cerevisiae Yeh1p, Tgl1p, and Yeh2p. The first two hydrolases are lipid droplet associated proteins. The final steryl ester hydrolase, Yeh2p, is PM associated with its catalytic domain on the extracellular surface of the membrane. We have demonstrated using terbinafine, a drug that inhibits sterol synthesis thereby forcing steryl ester hydrolysis, genetic knockouts of the steryl ester hydrolases, and thin layer chromatography (TLC) that Yol075c is essential for the transport and presentation of steryl esters to Yeh2p on the extracellular surface. This represents the first characterized case in biology of a steryl ester transporter. The effects of knocking out Yol075c are far reaching as cellular homeostasis of sterol content in many membranes is disturbed. We purified organelles including the lipid droplet, plasma membrane, nucleus, vacuole, mitochondria, microsome, peroxisome, and whole cells saponified the lipids and measured sterol amount using UV absorbance spectroscopy. The overall sterol content for whole cells in both wild type and Yol075c was identical. The organelles, in contrast, showed a very different sterol distribution. The content of sterols in the lipid droplet of wild type cells was dramatically decreased compared to the Yol075c. In the plasma membrane, nucleus, vacuole, and peroxisome the wild type organelles had a much higher sterol content relative to the Yol075c knockout. The mitochondria of the wild type had a small increase in sterol over the Yol075c and the microsomes appear unchanged between the two strains. A host of pleiotropic effects in the Yol075c strain have been observed in the lipid droplet, vacuole, mitochondria, and plasma membrane. Cells were stained with the lipid droplet specific fluorescent dye biodipy 493/503 and lipid droplets were counted before and after treatment of cells with terbinafine using fluorescence microscopy. Initially, the number of lipid droplets in the cells was less for the Yol075c cells, but after treatment with terbinafine the number stayed largely consistent in the wild type strain but increased dramatically in the knockout. Fluorescent microscopy and the vacuolar specific dye FM4-64 was used to determine vacuole number and susceptibility to salt induced vacuole fission showing a slightly larger vacuole in the Yol075c cells which can be attributed to the observed but slight fission defect in the knockout. Both cell lines were transformed with a Su9-GFP expressing plasmid which labels the mitochondria and the Yol075c show a dramatically increased size of mitochondria. Cells were then tested for resistance to oxidative stress induced by H2O2 and the Yol075c were found to be somewhat more resistant. The ability of cells to efflux rhodamine 6g, as a measure of drug efflux ability, was tested and the Yol075c cells had a greatly diminished ability for transport. With this work we have shown that Yol075c is responsible for the transport of steryl ester from the lipid droplet to the plasma membrane and by disrupting this process the sterol content of membranes throughout the cell are disturbed leading to many physiological differences.
Issue Date:2020-03-31
Type:Thesis
URI:http://hdl.handle.net/2142/108235
Rights Information:copyright 2020 Ez Ellis
Date Available in IDEALS:2020-08-27
Date Deposited:2020-05


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