Specific analogues uncouple transport, signalling, oligo‐ubiquitination and endocytosis in the yeast Gap1 amino acid transceptor

Abstract

The Saccharomyces cerevisiae amino acid transceptor Gap1 functions as receptor for signalling to the PKA pathway and concomitantly undergoes substrate-induced oligo-ubiquitination and endocytosis. We have identified specific amino acids and analogues that uncouple to certain extent signalling, transport, oligo-ubiquitination and endocytosis. l-lysine, l-histidine and l-tryptophan are transported by Gap1 but do not trigger signalling. Unlike l-histidine, l-lysine triggers Gap1 oligo-ubiquitination without substantial induction of endocytosis. Two transported, non-metabolizable signalling agonists, β-alanine and d-histidine, are strong and weak inducers of Gap1 endocytosis, respectively, but both causing Gap1 oligo-ubiquitination. The non-signalling agonist, non-transported competitive inhibitor of Gap1 transport, l-Asp-γ-l-Phe, induces oligo-ubiquitination but no discernible endocytosis. The Km of l-citrulline transport is much lower than the threshold concentration for signalling and endocytosis. These results show that molecules can be transported without triggering signalling or substantial endocytosis, and that oligo-ubiquitination and endocytosis do not require signalling nor metabolism. Oligo-ubiquitination is required, but apparently not sufficient to trigger endocytosis. In addition, we demonstrate intracellular cross-induction of endocytosis of transport-defective Gap1Y395C by ubiquitination- and endocytosis-deficient Gap1K9R,K16R. Our results support the concept that different substrates bind to partially overlapping binding sites in the same general substrate-binding pocket of Gap1, triggering divergent conformations, resulting in different conformation-induced downstream processes.

mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids from Lysosomes and Use Protein as a Nutrient

“…This finding, along with previous data, supports the conclusion that SLC38A9 can serve as a direct sensor of lysosomal arginine levels for the mTORC1 pathway, and suggests that arginine transport is not required for arginine sensing. That a small molecule binds and regulates a transporter, but is not transported well, is an established concept in transporter biology, perhaps best exemplified by certain ligands of the GAP1 amino acid transporter (Hundal and Taylor, 2009; Popova et al, 2010; Van Zeebroeck et al, 2009; Van Zeebroeck et al, 2014). Future work, likely requiring high-resolution structures, will be needed to understand how arginine promotes the binding of SLC38A9 to Rag-Ragulator and how this interaction impacts Rag-Ragulator activity.…”

Role of the Transporter-Like Sensor Kinase CbrA in Histidine Uptake and Signal Transduction

“…Several lines of evidence suggest that the two are coupled, as found in the Saccharomyces cerevisiae Gap1 amino acid transceptor (47). The most compelling evidence comes from showing that two alleles of cbrA, one with a mutation in the N terminus (cbrA::189 nt) and the other with a mutation in the C terminus [cbrA(H766G)], complement one another.…”

Transceptors as a functional link of transporters and receptors

“…In the years to follow, Thevelein’s group has provided additional evidence supporting the concept of transceptors 101112131415. The new evidence concerned mostly, but not only, Gap1.…”