Specific analogues uncouple transport, signalling, oligo-ubiquitination and endocytosis in the yeast Gap1 amino acid transceptor.
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 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 Gap1, triggering divergent conformations, resulting in different conformation-induced downstream processes.
Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Belgium; Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, Leuven-Heverlee, Flanders, B-3001, Belgium.
Griet
G
Van Zeebroeck
Marta
M
Rubio-Texeira
Joep
J
Schothorst
Johan M
JM
Thevelein
D000595:Amino Acid Sequence; D026905:Amino Acid Transport Systems / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport; D002956:Citrulline / Q000378:metabolism; D004705:Endocytosis; D006639:Histidine / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014364:Tryptophan / Q000378:metabolism; D054875:Ubiquitination; D015091:beta-Alanine / Q000378:metabolism
D026905:Amino Acid Transport Systems; D000596:Amino Acids; C491145:GAP1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015091:beta-Alanine; D002956:Citrulline; D006639:Histidine; D014364:Tryptophan; D008239:Lysine