The Bul1/2 Alpha-Arrestins Promote Ubiquitylation and Endocytosis of the Can1 Permease upon Cycloheximide-Induced TORC1-Hyperactivation

Abstract

Selective endocytosis followed by degradation is a major mechanism for downregulating plasma membrane transporters in response to specific environmental cues. In Saccharomyces cerevisiae, this endocytosis is promoted by ubiquitylation catalyzed by the Rsp5 ubiquitin-ligase, targeted to transporters via adaptors of the alpha-arrestin family. However, the molecular mechanisms of this targeting and their control according to conditions remain incompletely understood. In this work, we dissect the molecular mechanisms eliciting the endocytosis of Can1, the arginine permease, in response to cycloheximide-induced TORC1 hyperactivation. We show that cycloheximide promotes Rsp5-dependent Can1 ubiquitylation and endocytosis in a manner dependent on the Bul1/2 alpha-arrestins. Also crucial for this downregulation is a short acidic patch sequence in the N-terminus of Can1 likely acting as a binding site for Bul1/2. The previously reported inhibition by cycloheximide of transporter recycling, from the trans-Golgi network to the plasma membrane, seems to additionally contribute to efficient Can1 downregulation. Our results also indicate that, contrary to the previously described substrate-transport elicited Can1 endocytosis mediated by the Art1 alpha-arrestin, Bul1/2-mediated Can1 ubiquitylation occurs independently of the conformation of the transporter. This study provides further insights into how distinct alpha-arrestins control the ubiquitin-dependent downregulation of a specific amino acid transporter under different conditions.

2-deoxyglucose transiently inhibits yeast AMPK signaling and triggers glucose transporter endocytosis, potentiating the drug toxicity

“…These include a GPCR (Ste2), various classes of transporters, such as permeases, solute carriers such as multidrug transporters of the major facilitator superfamily (MFS) or ATP-binding cassette (ABC) types. This massive 2DG-induced endocytosis of membrane proteins is reminiscent of that observed in other situations of metabolic stresses such as acute glucose or nitrogen starvation [61][62][63], or upon treatment with other drugs, stressors or heat shock [26,[64][65][66][67][68][69]. Because 2DG is an artificial molecule, the observed endocytosis is unlikely to originate from an adaptation strategy but rather informs us that 2DGinduced metabolic blockade triggers endocytosis through mechanisms that we further explored.…”

2-deoxyglucose transiently inhibits yeast AMPK signaling and triggers glucose transporter endocytosis, potentiating the drug toxicity

“…These include a GPCR (Ste2), various classes of transporters, such as permeases, solute carriers such as multidrug transporters of the major facilitator superfamily (MFS) or ATP-binding cassette (ABC) types. This massive 2DG-induced endocytosis of membrane proteins is reminiscent of that observed in other situations of metabolic stresses such as acute glucose or nitrogen starvation [61][62][63], or upon treatment with other drugs, stressors or heat shock [26,[64][65][66][67][68][69]. Because 2DG is an artificial molecule, the observed endocytosis is unlikely to originate from an adaptation strategy but rather informs us that 2DG-induced metabolic blockade triggers endocytosis through mechanisms that we further explored.…”

Ferroptosis-Protective Membrane Domains in Quiescence

“…For quantifications, images were analyzed with custom-made FIJI macros, calculating the normalized mean fluorescence intensity, the MCC-to-total surface area or total-to-internal fluorescence intensity, as previously 23,79 . Briefly, for total-to-internal fluorescence intensity, two homocentric ellipses outlining the whole cell or the whole cells excluding the plasma membrane were manually drawn in middle-section images.…”

“…Western blotting. Total cell protein extracts were prepared and analyzed by SDS-PAGE as previously 79 . Proteins were transferred to a PVDF membrane (Immobilon, Macherey Nagel, Düren, NW, Germany) and probed with a mouse monoclonal anti-GFP (Roche, Basel, Switzerland), mouse monoclonal anti-mCherry (ab125096, Abcam PLC, Cambridge, UK) or anti-actin (sc47778, Santa Cruz Biotechnology, Dallas, TX, USA).…”