Inhibition of ubiquitin/proteasome-dependent proteolysis in Saccharomyces cerevisiae by a Gly-Ala repeat.
The glycine-alanine (GA) repeat of the Epstein-Barr virus nuclear antigen-1 inhibits in cis ubiquitin-dependent proteolysis in mammalian cells through a yet unknown mechanism. In the present study we demonstrate that the GA repeat targets an evolutionarily conserved step in proteolysis since it can prevent the degradation of proteasomal substrates in the yeast Saccharomyces cerevisiae. Insertion of yeast codon-optimised recombinant GA (rGA) repeats of different length in green fluorescent protein reporters harbouring N-end rule or ubiquitin fusion degradation signals resulted in efficient stabilisation of these substrates. Protection was also achieved in rpn10delta yeast suggesting that this polyubiquitin binding protein is not required for the rGA effect. The conserved effect of the GA repeat in yeast opens the possibility for the use of genetic screens to unravel its mode of action.
Heessen
Stijn
S
Microbiology and Tumor Biology Center, Karolinska Institutet, Box 280, S-171 77 Stockholm, Sweden.
Dantuma
Nico P
NP
Tessarz
Peter
P
Jellne
Marianne
M
Masucci
Maria G
MG
D000409:Alanine; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins; D003062:Codon; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D005998:Glycine; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D025801:Ubiquitin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
C438144:ASI1 protein, S cerevisiae; D002352:Carrier Proteins; D003062:Codon; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009097:Multienzyme Complexes; C498506:PSMD4 protein, human; D016601:RNA-Binding Proteins; C122019:RPN10 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D008715:Methionine; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000409:Alanine; D005998:Glycine