Phosphorylation and Methylation of Proteasomal Proteins of the Haloarcheon Haloferax volcanii
Figure 1
Phosphatase-sensitive isoforms of α-type proteins of proteasomal CPs as a function of growth in H. volcanii cells. (a) An 1 isoform of pI 4.4 was present at all stages of growth with a more acidic isoform of pI 3.0 present in log- and early-stationary phase, but absent in late-stationary phase. Cell lyase was prepared from various stages of growth as indicated (1 OD600 unit 1 109 CFUmL-1), separated by 2-DE and analyzed by immunoblot using anti-α1 antibody (-1). (b) The two α1 isoforms of pI 3.0 and 4.4 are associated in proteasomal CPs. Proteasomal CPs were purified by Ni-NTA chromatography from early stationary-phase cells expressing -His6. Proteins were separated into two fractions: (i) flowed through the Ni-NTA column at 5 mM imidazole and (ii) bound and were eluted from the column at 500 mM imidazole. Protein fractions were separated by 2-DE and analyzed by immunoblot using anti-α1 antibody (-1). Fractions were also assayed for peptidase activity using Suc-LLVY-AMC. u.d., undetectable. (c) Both 1 and 2 isoforms are sensitive to phosphatase treatment. Proteasomal CPs (purified as above) were treated with (red) and without (green) phosphatase, separated by 2-DE, and probed by immunoblot with anti-α1 and anti-α2 antibodies as indicated (-1 and -2, resp.).
