Specific analogues uncouple transport, signalling, oligo-ubiquitination and endocytosis in the yeast Gap1 amino acid transceptor.
Bottom Line: L-lysine, L-histidine and L-tryptophan are transported by Gap1 but do not trigger signalling.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.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.
Affiliation: 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.Show MeSH
Mentions: We previously showed that the Gap1Y395C protein, mutated in a residue located in TMDVIII has strongly reduced transport and signalling with regular amino acids (Van Zeebroeck et al., 2009). Transport of l-histidine and l-lysine was also strongly reduced in this mutant (Fig. 6A). When l-citrulline, l-histidine and l-lysine were added to nitrogen-starved cells we did not observe substantial disappearance of Gap1Y395C-GFP from the plasma membrane (Fig. 6B). A similar lack of enhanced internalization was also observed when Gap1Y395C-GFP was exposed to l-asparagine or to the non-metabolizable analogues β-alanine or d-histidine (Fig. S9). Even though the three non-signalling l-amino acids were unable to trigger endocytosis of this mutant form of Gap1, they still elicited oligo-ubiquitination of the mutated transceptor, as observed by detection of newly appearing di- and tri-ubiquitinated Gap1 (Fig. 6C). This further indicates that oligo-ubiquitination of Gap1 is not sufficient to elicit normal rates of endocytosis and that regular rates of transport are not required to trigger oligo-ubiquitination.
Affiliation: 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.