Analysis of Nedd8-associated polypeptides: a model for deciphering the pathway for ubiquitin-like modifications.
Ubiquitin-like proteins modify target proteins, altering their activities or causing them to be slated for degradation. These modifications are used to efficiently regulate key events in the cell. To explore the set of proteins modified by a small ubiquitin-like protein, we have developed a proteomic approach. Affinity purification of an epitope-tagged Nedd8 allowed the identification of the majority of proteins known to be involved with the neddylation pathway. This purification not only isolated the known targets of neddylation but also the constellation of enzymes and complexes known to regulate neddylation and deneddylation, including the COP9 signalosome, Nub1, and enzymes in the neddylation cascade. This purification scheme can be applied to other small ubiquitin-like proteins, especially those with limited protein targets such as the SUMOs (1, 2, and 3), Isg15, or FAT10.
Norman
Jessica A
JA
Department of Cell and Molecular Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Shiekhattar
Ramin
R
D000075686:COP9 Signalosome Complex; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D044842:Cullin Proteins / Q000378:metabolism; D006801:Humans; D046912:Multiprotein Complexes / Q000378:metabolism; D000075687:NEDD8 Protein; D009842:Oligopeptides; D010447:Peptide Hydrolases / Q000378:metabolism; D010455:Peptides / Q000032:analysis* / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D013379:Substrate Specificity; D025801:Ubiquitin / Q000378:metabolism*; D014452:Ubiquitins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*
D044842:Cullin Proteins; D046912:Multiprotein Complexes; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D009842:Oligopeptides; D010455:Peptides; D025801:Ubiquitin; D014452:Ubiquitins; C085706:FLAG peptide; D010447:Peptide Hydrolases; D000075686:COP9 Signalosome Complex; D046988:Proteasome Endopeptidase Complex