Electrophoretic analysis of phosphorylation of the yeast 20S proteasome.
The 26S proteasome complex, consisting of two multisubunit complexes, a 20S proteasome and a pair of 19S regulatory particles, plays a major role in the nonlysosomal degradation of intracellular proteins. The 20S proteasome was purified from yeast and separated by two-dimensional gel electrophoresis (2-DE). A total of 18 spots separated by 2-DE were identified as the 20S proteasome subunits by peptide mass fingerprinting with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The alpha2-, alpha4- and alpha7-subunits gave multiple spots, which converged into one spot for each subunit when treated with alkaline phosphatase. The difference of pI between phosphorylated and dephosphorylated spots and their reaction against anti-phosphotyrosine antibody suggested that the alpha2- and alpha4-subunits are phosphorylated either at Ser or at Thr residue, and the alpha7-subunit is phosphorylated at Tyr residue(s). These phosphorylated subunits were analyzed by electrospray ionization-quadrupole time of flight-tandem MS (ESI-QTOF-MS/MS) to deduce the phosphorylation sites. The 20S proteasome has three different protease activities: chymotrypsin-like, trypsin-like and peptidylglutamyl peptide-hydrolyzing activities. The phosphatase treatment increased K(m) value for chymotrypsin-like activity of the 20S proteasome, indicating that phosphorylation may play an important role in regulating the proteasome activity.
Iwafune
Yuko
Y
Yokohama City University, Kihara Institute for Biological Research, Graduate School of Integrated Science, Yokohama, Japan.
Kawasaki
Hiroshi
H
Hirano
Hisashi
H
D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western / Q000379:methods; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D009097:Multienzyme Complexes; D010749:Phosphoprotein Phosphatases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex