Assembly of the 26S proteasome is regulated by phosphorylation of the p45/Rpt6 ATPase subunit

We investigated whether the assembly/disassembly of the 26S proteasome is r egulated by phosphorylation/dephosphorylation. The regulatory complex disas sembled from the 26S proteasome was capable of phosphorylating the p45Sug1/ Rpt6 subunit, suggesting that the protein kinase is activated upon dissocia tion of the 26S proteasome or that the phosphorylation site of p45 becomes susceptible to the protein kinase. In addition, the p45-phosphorylated regu latory complex was found to be incorporated into the 26S proteasome. When t he 26S proteasome was treated with alkaline phosphatase, it was dissociated into the 20S proteasome and the regulatory complex. Furthermore, the p45 s ubunit and the C3/alpha2 subunit were cross-linked with DTBP, whereas these subunits were not cross-linked by dephosphorylating the 26S proteasome. Th ese results indicate that the 26S proteasome is disassembled into the const ituent subcomplexes by dephosphorylation and that it is assembled by phosph orylation of p45 by a protein kinase, which is tightly associated with the regulatory complex. It was also revealed that the p45 subunit is directly a ssociated with the 20S proteasome alpha -subunit C3 in a phosphorylation-de pendent manner.