title: Functional analysis of the human orthologue of the RSP5-encoded ubiquitin protein ligase, hNedd4, in yeast.
abstract: hNedd4 and Rsp5p are orthologous ubiquitin ligases that contain a catalytic Hect domain, a C2 domain and multiple WW domains that mediate interactions with proteins. hNedd4 associates with the epithelial sodium channel and mutations disrupting this interaction lead to Liddle's syndrome, a heritable hypertension. Yeast Rsp5p ubiquitinates plasma membrane receptors and transporters and regulates their endocytosis. To determine whether the human enzyme has activity in yeast, hNEDD4 was expressed in yeast from the RSP5 or GAL1/10 promoters. Ectopic hNedd4 improved the growth and partially suppressed the endocytosis defect of rsp5 mutant cells, although it did not restore the viability of the rsp5-delta strain. Wild-type cells harboring hNedd4 grew better at elevated temperature and on media containing cycloheximide. In contrast, hNedd4 WW domain mutants inhibited the growth of yeast when expressed at high levels. Our results show that hNedd4 affects cell growth, endocytosis and cycloheximide tolerance of yeast cells.
authors.affiliation: Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland.
authors.forename: Beata
authors.initials: B
authors.lastname: Gajewska
authors.forename: Natalia
authors.initials: N
authors.lastname: Shcherbik
authors.forename: Danuta
authors.initials: D
authors.lastname: Oficjalska
authors.forename: Dale S
authors.initials: DS
authors.lastname: Haines
authors.forename: Teresa
authors.initials: T
authors.lastname: Zoładek
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D056827:Endosomal Sorting Complexes Required for Transport; D018014:Gene Transfer Techniques; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D000075702:Nedd4 Ubiquitin Protein Ligases; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000502:physiology; D043743:Ubiquitin-Protein Ligase Complexes / Q000235:genetics* / Q000502:physiology; D044767:Ubiquitin-Protein Ligases / Q000235:genetics* / Q000502:physiology*
chemical_list: D000935:Antifungal Agents; D056827:Endosomal Sorting Complexes Required for Transport; D029701:Saccharomyces cerevisiae Proteins; D003513:Cycloheximide; D043743:Ubiquitin-Protein Ligase Complexes; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; D044767:Ubiquitin-Protein Ligases; C469287:RSP5 protein, S cerevisiae