Nutrient Regulated Control of Gene Expression
Abstract
Nutrient uptake is essential for life. Fungal and mammalian cells respond to the presence of extracellular amino acids by pleiotropically enhancing amino acid uptake, thus amino acids induce their own uptake. We study the mechanistic details of this process in fungi, specifically, in the yeast Saccharomyces cerevisiae and fungal pathogens of the Candida species complex that infect humans. We have a discovered the conserved SPS-sensing system that enables fungal cells to sense and respond to extracellular amino acids, and have defined the key signaling events required to transmit signals from the plasma membrane SPS-sensor to the promoters of responsive genes. Our studies have revealed several novel mechanisms of signal transduction, including receptor activated proteolysis (RAP) that controls the activity of two latently expressed cytoplasmic-localized transcription factors (Stp1 and Stp2), and transcriptional repression by integral components of the inner nuclear membrane (Asi E3-ubiquitin ligase complex). Additionally, we have discovered the existence of membrane-localized chaperones that function in the endoplasmic reticulum (ER) to prevent inappropriate molecular interactions between hydrophobic segments of polytopic membrane proteins as they co-translationally insert into the membrane. The primary sensing component of the SPS-sensor, Ssy1, and amino acid permeases exhibit a shared requirement for the ER membrane-localized chaperone Shr3 for correct folding.
We are now focused on filling the gaps in understanding regarding the SPS-sensing system. Specifically, we are probing protein-protein interactions that occur between the individual components during the resting “Off-state” and during the propagation of amino acid-induced signals, the “On-state. Initially, we are focused on investigating factors that contribute the latency of Stp1 and Stp2. Previous work has shown that the N-terminal regulatory domains of these transcription factors contain a small motif comprised of 16 amino acids that functions in a modular and transferable manner. This motif, designated RI, acts as a negative determinant with two intrinsic activities; RI acts as an effective cytoplasmic retention determinant that can be transferred and efficiently retain histone Htb2 in the cytoplasmic compartment, and as an Asi-dependent degron. The data show that both activities are required to prevent inappropriate transcription by Stp1 and Stp2 during the Off-state, and provided the first mechanistic insights into the function of the inner nuclear membrane proteins Asi1, Asi2 and Asi3 acting as an E3-ubiquitin ligase complex.
Here, we intend to use an AlphaPulldown-like strategy to identify potential protein-protein interactions that underlie the cytoplasmic retention of Stp1 and Stp2. We intend to screen the capacity of the RI to dock with putative binding proteins, probing theoretical interactions between RI and all ≈6 000 yeast proteins relying on high-throughput modelling of higher-order oligomers using AlphaFold-Multimer. Putative interactions can readily be tested using standard genetic approaches in yeast. The premise being that deletion of bona fide RI-binding proteins should activate Stp1/Stp2 gene expression in the absence of amino acid induction, effectively bypassing the need of the SPS-sensor.
