Substrate-Induced Ubiquitylation and Endocytosis of Yeast Amino Acid Permeases

Abstract

bMany plasma membrane transporters are downregulated by ubiquitylation, endocytosis, and delivery to the lysosome in response to various stimuli. We report here that two amino acid transporters of Saccharomyces cerevisiae, the general amino acid permease (Gap1) and the arginine-specific permease (Can1), undergo ubiquitin-dependent downregulation in response to their substrates and that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport catalysis itself. Following an approach based on permease structural modeling, mutagenesis, and kinetic parameter analysis, we obtained evidence that substrate-induced endocytosis requires transition of the permease to a conformational state preceding substrate release into the cell. Furthermore, this transient conformation must be stable enough, and thus sufficiently populated, for the permease to undergo efficient downregulation. Additional observations, including the constitutive downregulation of two active Gap1 mutants altered in cytosolic regions, support the model that the substrate-induced conformational transition inducing endocytosis involves remodeling of cytosolic regions of the permeases, thereby promoting their recognition by arrestin-like adaptors of the Rsp5 ubiquitin ligase. Similar mechanisms might control many other plasma membrane transporters according to the external concentrations of their substrates.

Dynamic internalization and recycling of a metal ion transporter: Cu homeostasis and CTR1, the human Cu+ uptake system

“…Similarly, endocytosis of the boron transporter in Arabidopsis thaliana, to avoid excess boron accumulation, occurs through transfer from the membranes to the vacuoles for degradation (Takano et al, 2005). The general amino acid permease, Gap1, of yeast also undergoes endocytosis in response to elevated levels of substrates via a ubiquitylation pathway (Ghaddar et al, 2014). It was concluded that the transporter engaged a conformation (caused by substrate binding) that would interact more efficiently with the endocytic machinery (Cain and Kaiser, 2011).…”

“…There is a need to regulate cellular composition to either protect against the toxic effects of certain substrates, as with elevation of P-glycoprotein and drug resistance (Bradley et al, 1989), or to ensure substrate is absorbed under low substrate conditions, as in the amino acid permease in yeast (Ghaddar et al, 2014). In these cases, transporter synthesis or degradation is regulated.…”

Identification of the endocytic sorting signal recognized by the Art1-Rsp5 ubiquitin ligase complex

“…, 2014). Several studies propose that substrate binding converts PM transporters into an activated conformational state, which acts as a signal for degradation (Cain and Kaiser, 2011; Keener and Babst, 2013; Ghaddar et al. , 2014).…”

Characterization of the Candida albicans Amino Acid Permease Family: Gap2 Is the Only General Amino Acid Permease and Gap4 Is an S -Adenosylmethionine (SAM) Transporter Required for SAM-Induced Morphogenesis

“…The induction of its expression as well as the activity of Sc Gap1 are tightly regulated according to both the quality and quantity of the nitrogen sources present in the medium. The presence of preferred nitrogen sources (ammonium, glutamate, and glutamine) or of high amino acid levels represses ScGAP1 expression and inactivates the transporter by ubiquitination and internalization for targeting to the vacuole and subsequent degradation (14–16). The transcription of ScGAP1 is regulated by the nitrogen catabolite repression (NCR) pathway (17).…”