IDENTIFYING PROTEINS ASSOCIATED WITH PLASMA MEMBRANE TRANSPORTER AGP2 IN SACCHAROMYCE CEREVISIAE

Abstract

Influx transporters play vital roles in both eukaryotes and prokaryotes as they uptake essential substrates that are important for cellular activities. Agp2 is one of the members of amino acid-polyamine-organocation superfamily of proteins in Saccharomyces cerevisiae. Agp2 is a plasma membrane protein that has been implicated in the uptake of several substrates such as amino acids, L-carnitine, polyamines and bleomycin-A5. In the preliminary work carried out in the lab, it was also observed that Agp2 seems to play a role in the uptake of cycloheximide, a protein synthesis inhibitor. Recently, it had been proposed that Agp2 functions as a sensor rather than a transporter as Agp2 has been observed to regulate gene expression of several proteins. The signaling pathway of Agp2 has not yet been elucidated. Hence, we decided to identify signaling proteins associated with Agp2 on the plasma membrane by using cycloheximide as a substrate and determine the effect of cycloheximide on Agp2. Our results show that there are significant differences in proteins present on plasma membrane between the wild-type and agp2Δ strains under untreated condition, whereas a slight difference in proteins was observed when the strains were treated with 3.55 µM cycloheximide for 10 minutes and above. We also observed the disappearance of Agp2 protein from the crude plasma membrane upon treatment with cycloheximide for more than 7 minutes. Mass spectrometry was performed with triplicate samples of the plasma membrane derived from the wild-type and the agp2Δ mutant under normal and treated conditions. The data revealed that a significant number of mitochondrial proteins were weakly present in the agp2Δ mutant as compared to the parent. We interpret the data to suggest that the transport defects associated with the agp2Δ mutant is associated with its inability to produce energy and cycloheximide resistance might be dependent on Pdr5 overexpression in agp2Δ strains.

Date of Award	2022
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences