Specific analogues uncouple transport, signalling, oligo‐ubiquitination and endocytosis in the yeast G ap1 amino acid transceptor

Summary The S accharomyces cerevisiae amino acid transceptor G ap1 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 G ap1 but do not trigger signalling. Unlike l ‐histidine, l ‐lysine triggers G ap1 oligo‐ubiquitination without substantial induction of endocytosis. Two transported, non‐metabolizable signalling agonists, β‐alanine and d ‐histidine, are strong and weak inducers of G ap1 endocytosis, respectively, but both causing G ap1 oligo‐ubiquitination. The non‐signalling agonist, non‐transported competitive inhibitor of G ap1 transport, l ‐ A sp‐γ‐ l ‐ P he, induces oligo‐ubiquitination but no discernible endocytosis. The K m 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 Gap1 Y395C by ubiquitination‐ and endocytosis‐deficient Gap1 K9R,K16R . Our results support the concept that different substrates bind to partially overlapping binding sites in the same general substrate‐binding pocket of G ap1, triggering divergent conformations, resulting in different conformation‐induced downstream processes.