Down-regulation of Eukaryotic Nitrate Transporter by Nitrogen-dependent Ubiquitinylation

Abstract

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 (⌬pep4) 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.

Phosphorylation of the Yeast Nitrate Transporter Ynt1 Is Essential for Delivery to the Plasma Membrane during Nitrogen Limitation

“…Fluorescence Microscopy-To localize Ynt1 in the cell, the chromosomal copy of the YNT1 gene was substituted by a fusion of wild-type or mutant YNT1 to GFP (13). Images were obtained under an epifluorescence Nikon Eclipse 50i microscope equipped with a 1200F camera (Nikon).…”

“…Yeast Cell Extract Preparation, SDS-PAGE, and Immunoblotting-Extracts for immunoblot were prepared from 50 mg of cells as described elsewhere (13). Briefly, cells were homogenized in a FastPrep homogenizer device (Thermosavant LifeSciences, Hampshire, UK) for 20 s at 6.0 m/s with 300 l of Lysis Buffer I (50 mM Tris-HCl, pH 7.4, 15 mM EDTA, 15 mM EGTA, 10 mM NaN 3 , 10 mM Na 2 P 2 O 7 , 10 mM NaF plus Complete Mini protease inhibitor mixture, Roche Applied Science, Indianapolis, IN, and 2 mM phenylmethylsulfonyl fluoride) and 0.5-mm diameter glass beads.…”

“…Efficient endocytosis of Ynt1 requires the ubiquitinylation of lysine residues of the central intracellular loop, principally Lys-253 and Lys-270. Mutation of these residues, together with the nearby lysine residue Lys-243, produces a poor endocytosis of Ynt1 (13). To study if ubiquitinylation of these same residues was also involved in the sorting of Ynt1 from the Golgi apparatus to the vacuole of the non-phosphorylated transporter, we compared the degradation of mutant Ynt1 2SA (S244A and S246A) containing or lacking these lysine residues (S244K243R, K253R, and K270R) (Fig.…”

“…The role of the preferred nitrogen sources in regulating Ynt1 levels has been thoroughly documented by our group (10,11,13). However, the mechanism involved in preventing Ynt1 degradation when cells are exposed to nitrogen limitation is so far unknown.…”

“…Phosphorylation of AtNRT1;1, which increases affinity for nitrate, is stimulated when plants are exposed to a low concentration of nitrate, whereas the transporter is dephosphorylated at high nitrate concentration (12). Furthermore, we have reported that the nitrogen source controls the amount of Ynt1; glutamine or ammonium triggers its ubiquitinylation, endocytosis, and vacuolar degradation (13). Ynt1 endocytosis requires ubiquitinylation in lysine residues of the central intracellular loop of Ynt1 .…”