Multiple functional categories of proteins identified in an in vitro cellular ubiquitin affinity extract using shotgun peptide sequencing.
To construct a high information content assay for examination of the function of the cellular ubiquitin system, we added his-tagged ubiquitin, ATP, and an ATP-regenerating system to endogenous human cellular ubiquitin system enzymes, and labeled cellular proteins with hexa-histidine tagged ubiquitin in vitro. Labeling depended on ATP, the ATP recycling system, the proteasome inhibitor MG132, and the ubiquitin protease inhibitor ubiquitin aldehyde, and was inhibited by iodoacetamide. Quadruplicate affinity extracted proteins were digested with trypsin, and the peptides were analyzed by 2D capillary LC-MS/MS, SEQUEST, MEDUSA, and support vector machine calculations. Identified proteins included 22 proteasome subunits or associated proteins, 18 E1, E2, or E3 ubiquitin system enzymes or related proteins, 4 ubiquitin domain proteins and 36 proteins in functional clusters associated with redox processes, endocytosis/vesicle trafficking, the cytoskeleton, DNA damage/repair, calcium binding, and mRNA splicing. This suggests a link between the ubiquitin system and these cellular processes. This map of cellular ubiquitin-associated proteins may be useful for further studies of ubiquitin system function.
Gururaja
Tarikere
T
Rigel Inc., South San Francisco, California 94080, USA.
Li
Weiqun
W
Noble
William Stafford
WS
Payan
Donald G
DG
Anderson
D C
DC
D000465:Algorithms; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D016000:Cluster Analysis; D019295:Computational Biology; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000502:physiology; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D009097:Multienzyme Complexes / Q000737:chemistry / Q000502:physiology; D010446:Peptide Fragments / Q000032:analysis; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000032:analysis* / Q000145:classification / Q000502:physiology; D040901:Proteomics / Q000379:methods*; D025801:Ubiquitin / Q000737:chemistry / Q000502:physiology*; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*
D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011506:Proteins; D025801:Ubiquitin; D043743:Ubiquitin-Protein Ligase Complexes; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex