In vivo and in vitro phosphorylation of Candida albicans 20S proteasome

Abstract

In this paper we demonstrate that the Candida albicans 20S proteasome is in vivo phosphorylated and is a good in vitro substrate (S 0.5 14 nM) of homologous protein kinase CK2 (CK2). We identify α6/C2, α3/C9, and α5/Pup2 proteasome subunits as the main in vivo phosphorylated and in vitro CK2-phosphorylatable proteasome components. In vitro phosphorylation by homologous CK2 holoenzyme occurs only in the presence of polylysine, a characteristic that distinguishes the yeast proteasomes from mammalian proteasomes which are phosphorylated by CK2 in the absence of polycations. The major in vivo phosphate acceptor is the α3/C9 subunit, being phosphorylated in serine, both in vivo and in vitro. The phosphopeptides generated by endoproteinase Glu-C digestion from in vivo labeled α3/C9 subunit, from in vitro phosphorylation by homologous CK2 holoenzyme, and from the recombinant α3/C9 subunit phosphorylated by recombinant human CK2-α subunit are identical, suggesting that CK2 is likely responsible for in vivo phosphorylation of this subunit. Direct mutational analysis shows that serine 248 is the residue of the α3/C9 subunit phosphorylated by CK2. The in vitro stoichiometry of phosphorylation of the α6/C2 and α3/C9 proteasome subunits by CK2 can be estimated as 0.7–0.8 and 0.4–0.5 mol of phosphate per mole of subunit, respectively. These results are consistent with the relative abundance of the unphosphorylated and phosphorylated isoforms of these subunits present in the purified 20S proteasome preparation. Our demonstration of phosphorylation of C. albicans proteasome suggests that phosphorylation might be a general mechanism of regulation of proteasome activity.