Identification of three phosphorylation sites in the alpha7 subunit of the yeast 20S proteasome in vivo using mass spectrometry.
The 26S proteasome complex, which consists of a 20S proteasome and a pair of 19S regulatory particles, plays important roles in the degradation of ubiquitinated proteins in eukaryotic cells. The alpha7 subunit of the budding yeast 20S proteasome is a major phosphorylatable subunit; serine residue(s) in its C-terminal region are phosphorylated in vitro by CKII. However, the exact in vivo phosphorylation sites have not been identified. In this study, using electrospray ionization quadrupole time-of-flight mass spectrometry analysis, we detected a mixture of singly, doubly, and triply phosphorylated C-terminal peptides isolated from a His-tagged construct of the alpha7 subunit by nickel-immobilized metal affinity chromatography. In addition, we identified three phosphorylation sites in the C-terminal region using MS/MS analysis and site-directed mutagenesis: Ser258, Ser263, and Ser264 residues. The MS/MS analysis of singly phosphorylated peptides showed that phosphorylation at these sites did not occur successively.
Iwafune
Yuko
Y
Kihara Institute for Biological Research/Graduate School of Integrated Science, Yokohama City University, Maioka 641-12, Totsuka-ku, Yokohama 244-0813, Japan.
Kawasaki
Hiroshi
H
Hirano
Hisashi
H
D006639:Histidine / Q000378:metabolism; D013058:Mass Spectrometry; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013194:Staining and Labeling; D015003:Yeasts / Q000201:enzymology* / Q000378:metabolism
D012694:Serine; D006639:Histidine; D046988:Proteasome Endopeptidase Complex