DEGRADATION OF THE YEAST TRANSCEPTOR AGP2 IS TRIGGERED BY THE PROTEIN SYNTHESIS INHIBITOR CYCLOHEXIMIDE AND REQUIRES THE SMALL PROTEIN BRP1

Abstract

The Saccharomyces cerevisiae Agp2 is a plasma membrane protein initially reported to be an uptake transporter for L-carnitine. Agp2 was later rediscovered, together with three additional proteins Sky1, Ptk2, and Brp1 to be involved in taking up the polyamine analogue bleomycin-A5, an anticancer drug. Mutants lacking either Agp2, Sky1, Ptk2, or Brp1 are resistant to polyamines and bleomycin-A5, suggesting they act in the same transport pathway. Pretreating cells with the protein synthesis inhibitor cycloheximide (CHX) blocked the uptake of bleomycin and initially suggested a component of this uptake pathway requires new protein synthesis to promote the drug entry. Subsequent findings revealed that Agp2 is responsible for regulating the expression of several genes including the uptake transporters Sam3 and Dur3. We propose that Agp2 may function as a sensor rather than a transporter and that its physiological state could be altered by the substrate it recognizes. In this dissertation, we monitor the fate of Agp2 as a GFP-tagged protein in response to CHX. We showed that CHX triggered the disappearance of Agp2 in a concentration- and time-dependent manner. Higher molecular weight forms of Agp2-GFP appeared before its complete disappearance within 15 to 30 mins of treatment with 10 µM CHX. Like the native form, these high molecular weight forms were also ubiquitylated, suggesting that Agp2 may undergo proteolysis in response to CHX. Interestingly, we found that CHX did not trigger the degradation of Agp2-GFP in the mutant strain lacking the small protein Brp1. We conclude that Agp2 is degraded upon sensing toxic compounds to prevent their uptake and that Brp1 plays a role in the degradation process.

Date of Award	2023
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences