The yeast Npi1/Rsp5 ubiquitin ligase lacking its N-terminal C2 domain is competent for ubiquitination but not for subsequent endocytosis of the gap1 permease.
The yeast ubiquitin ligase Npi1/Rsp5 and its mammalian homologue Nedd4 are involved in ubiquitination of various cell surface proteins, these being subsequently internalized by endocytosis and degraded in the vacuole/lysosome. Both enzymes consist of an N-terminal C2 domain, three to four successive WW(P) domains, and a C-terminal catalytic domain (HECT) containing a highly conserved cysteine residue involved in ubiquitin thioester formation. In this study, we show that the conserved cysteine of the HECT domain is required for yeast cell viability and for ubiquitination and subsequent endocytosis of the Gap1 permease. In contrast, the C2 domain of Npi1/Rsp5 is not essential to cell viability. Its deletion impairs internalization of Gap1, without detectably affecting ubiquitination of the permease. This suggests that Npi1/Rsp5 participates, via its C2 domain, in endocytosis of ubiquitinated permeases.
Springael
J Y
JY
Laboratoire de Physiologie Cellulaire et de Génétique des Levures, Université Libre de Bruxelles-Campus Plaine CP244, Bd du triomphe, Bruxelles, B-1050, Belgium.
De Craene
J O
JO
André
B
B
D019943:Amino Acid Substitution; D026905:Amino Acid Transport Systems; D000818:Animals; D015153:Blotting, Western; D017124:Conserved Sequence / Q000235:genetics; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects; D056827:Endosomal Sorting Complexes Required for Transport; D004795:Enzyme Stability; D006801:Humans; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008970:Molecular Weight; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion / Q000235:genetics*; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases
D026905:Amino Acid Transport Systems; D056827:Endosomal Sorting Complexes Required for Transport; D026901:Membrane Transport Proteins; D000644:Quaternary Ammonium Compounds; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; C469287:RSP5 protein, S cerevisiae; D003545:Cysteine