Down-regulation of eukaryotic nitrate transporter by nitrogen-dependent ubiquitinylation.
In the yeast Hansenula polymorpha, the YNT1 gene encodes the high affinity nitrate transporter, which is repressed by reduced nitrogen sources such as ammonium or glutamine. Ynt1 protein is degraded in response to glutamine in the growth medium. Ynt1 disappears independently of YNT1 glutamine repression as shown in strains where YNT1 repression is abolished. Ynt1-green fluorescent protein chimera and a mutant defective in vacuolar proteinase A (deltapep4) showed that Ynt1 is degraded in the vacuole in response to glutamine. The central hydrophilic domain of Ynt1 contains PEST-like sequences whose deletion blocked Ynt1 down-regulation. Site-directed mutagenesis showed that Lys-253 and Lys-270, located in this sequence, were involved in internalization and subsequent vacuolar degradation of Ynt1. Ynt1-ubiquitin conjugates were induced by glutamine and not nitrate. We conclude that glutamine triggers Ynt1 down-regulation via ubiquitinylation of lysines in the central hydrophilic domain, and proteolysis in the vacuole.
Department of Biochemistry and Molecular Biology, Instituto Universitario de Enfermedades Tropicales, Universidad de La Laguna, E-38206 La Laguna, Tenerife, Canarias, Spain.
Francisco J
FJ
Navarro
Department of Biochemistry and Molecular Biology, Instituto Universitario de Enfermedades Tropicales, Universidad de La Laguna, E-38206 La Laguna, Tenerife, Canarias, Spain.
Félix
F
Machín
Department of Biochemistry and Molecular Biology, Instituto Universitario de Enfermedades Tropicales, Universidad de La Laguna, E-38206 La Laguna, Tenerife, Canarias, Spain.
Yusé
Y
Martín
Department of Biochemistry and Molecular Biology, Instituto Universitario de Enfermedades Tropicales, Universidad de La Laguna, E-38206 La Laguna, Tenerife, Canarias, Spain. Electronic address: jsiverio@ull.es.
José M
JM
Siverio
D027321:Anion Transport Proteins / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005973:Glutamine / Q000378:metabolism; D009154:Mutation; D000091285:Nitrate Transporters; D009584:Nitrogen / Q000378:metabolism*; D010843:Pichia / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D025801:Ubiquitin / Q000378:metabolism*
D027321:Anion Transport Proteins; D005656:Fungal Proteins; D000091285:Nitrate Transporters; D025801:Ubiquitin; D005973:Glutamine; D009584:Nitrogen