Regulation of GAP1p sorting in S. cerevisiae

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2003.Includes bibliographical references.The intracellular sorting of the general amino acid permease (Gaplp) of Saccharomyces cerevisiae is regulated by the nitrogen source in the growth medium. During growth on a non-preferred nitrogen source or during nitrogen starvation, Gaplp is sorted to the plasma membrane, where it is active and can transport amino acids into the cell. During growth on a preferred or intermediate nitrogen source, Gap p is sorted to the vacuole and degraded. To better understand the signals governing Gaplp sorting, we isolated mutants with defects in Gaplp sorting during growth on a non-preferred nitrogen source. We found that genes that regulate the glutamate and glutamine biosynthesis pathways have a pronounced effect on the intracellular sorting of Gaplp. Mutations in the genes encoding the principal enzymes for glutamate and glutamine synthesis, GDH1 and GLN1, decreased intracellular levels of glutamate and glutamine, respectively, and increased delivery of Gap Ip to the plasma membrane. In contrast, deletion of the MKS] gene, which encodes a negative regulator of glutamate synthesis, increased glutamate and glutamine levels and increased sorting of Gaplp to the vacuole. We also found that the LST8 gene, which was previously implicated in the regulation of Gap lp sorting, encodes a positively-acting component of the TOR signaling pathway. Because the TOR pathway inhibits glutamate and glutamine biosynthesis, lst8 mutants accumulated high levels of these amino acids and showed increased Gaplp sorting to the vacuole. Likewise, wild-type cells grown with a sublethal concentration of rapamycin showed increased glutamate and glutamine levels and increased Gaplp sorting to the vacuole.(cont.) Glutamate and glutamine were not unusual in their ability to affect Gaplp sorting, since all natural amino acids and many amino acid analogs, regardless of their utility as nitrogen sources, also triggered sorting of Gaplp to the vacuole. Finally, we found that many individual amino acids were toxic to mutants that sorted Gaplp constitutively to the plasma membrane. Together, these results indicate that amino acids are a signal for sorting Gaplp to the vacuole and suggest that the sensitivity of Gaplp sorting to amino acids represents a mechanism to protect yeast from an intracellular amino acid imbalance.by Esther J. Chen.Ph.D