title: Phosphorylation of a conserved Thr357 in yeast Nedd4-like ubiquitin ligase Rsp5 is involved in down-regulation of the general amino acid permease Gap1.
abstract: Rsp5, an essential HECT-type ubiquitin ligase, is the only yeast Saccharomyces cerevisiae member of the Nedd4 family. Rsp5 triggers the ubiquitination-dependent endocytosis of the general amino acid permease Gap1 in response to a good nitrogen source. Previously, we showed that the Thr357Ala/Lys764Glu variant Rsp5 induces the constitutive inactivation of Gap1, which is mainly involved in uptake of the toxic proline analogue, l-azetidine-2-carboxylate (AZC). Here, our experimental results indicated that the Thr357Ala substitution in the substrate-recognizing WW2 domain of Rsp5 constitutively causes the down-regulation of four proline permeases (Gap1, Put4, Agp1 and Gnp1), leading to AZC tolerance to yeast cells. In RSP5(T357A) cells, Gap1 was highly ubiquitinated and constantly delivered to the vacuole from the Golgi without sorting to the plasma membrane. Analyses of RSP5 mutants using antiphosphopeptide antibody suggest that Thr phosphorylation occurred in all three WW domains and, interestingly, that Thr357 in the WW2 domain was phosphorylated, in agreement with the in vitro result for the mouse Rsp5 orthologue. Furthermore, the phosphorylation-mimic mutant (Thr357Asp) showed strong sensitivity to AZC. From these results, we propose a possible mechanism involved in the regulation of Rsp5 activity for Gap1 down-regulation via the phosphorylation of a conserved Thr357 in the Nedd4 family.
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
authors.forename: Toshiya
authors.initials: T
authors.lastname: Sasaki
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takagi
mesh_terms: D026905:Amino Acid Transport Systems / Q000378:metabolism*; D015536:Down-Regulation; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000235:genetics / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D026905:Amino Acid Transport Systems; D056827:Endosomal Sorting Complexes Required for Transport; C491145:GAP1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013912:Threonine; D043743:Ubiquitin-Protein Ligase Complexes; C469287:RSP5 protein, S cerevisiae