Date Approved

2017

Degree Type

Open Access Senior Honors Thesis

Department

Chemistry

First Advisor

Steven Backues, Ph.D.

Second Advisor

Hedeel Evans, Ph.D.

Abstract

Autophagy is a cellular process conserved in eukaryotes that involves the trafficking of intracellular material from the cytosol to the vacuole/lysosome. This material is wrapped in a double membraned vesicle called an autophagosome, which ls constructed at the pre-autophagosomal structure. Many proteins are Involved In autophagy, one of them being Atgll. This protein is crucial in the selective autophagy pathway, and is responsible for proper formation of the pre-autophagosomal structure as well as recruiting the material intended for degradation to the autophagosome. Atgl 1 interacts with many other autophagy proteins, but it Is unknown whether it interacts with these binding partners spatially or temporally. To better understand the mechanism of Atg11' s interactions, it was tested against Yptl, an upstream regulator whose Interaction with Atgll is crucial for selective autophagy. Yptl with an N-terminal 6-histidine tag was expressed and purified using Rosetta cells, yielding a concentration of 0. 242 mg/ml seems low. This protein was then used in an in vitro binding test with pure GST ·Atgl1CC2·3 and imaged via Western blot using primary antibodies rabbit antihistidine {raHis) and rabbit anti-GST (raGST}, and secondary antibody goat anti-rabbit (Gar). The blot showed no Interaction between the proteins in vitro, so the interaction was tested in vivo using a yeast 2-hybrid screen with a multiple-knockout strain. BO-Yptl was tested against AD-Atg11CC2-3, pseudo-positive control AD-Atgll, and negative control AD-empty. Contrary to previous literature, i no interaction showed between the two proteins in any of the strains. This indicates that another type of selection must be used, or there might be another protein involved in the interaction. 3

Included in

Chemistry Commons

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