Phosphorylation and methylation of proteasomal proteins of the haloarcheon Haloferax volcanii

Abstract

Proteasomes are composed of 20S core particles (CPs) of alpha- and beta-type subunits that associate with regulatory particle AAA ATPases such as the proteasome-activating nucleotidase (PAN) complexes of archaea. In this study, the roles and additional sites of post-translational modification of proteasomes were investigated using the archaeon Haloferax volcanii as a model. Indicative of phosphorylation, phosphatase-sensitive isoforms of alpha1 and alpha2 were detected by 2-DE immunoblot. To map these and other potential sites of post-translational modification, proteasomes were purified and analyzed by tandem mass spectrometry (MS/MS). Using this approach, several phosphosites were mapped including alpha1 Thr147, alpha2 Thr13/Ser14 and PAN-A Ser340. Multiple methylation sites were also mapped to alpha1, thus, revealing a new type of proteasomal modification. Probing the biological role of alpha1 and PAN-A phosphorylation by site-directed mutagenesis revealed dominant negative phenotypes for cell viability and/or pigmentation for alpha1 variants including Thr147Ala, Thr158Ala and Ser58Ala. An H. volcanii Rio1p Ser/Thr kinase homolog was purified and shown to catalyze autophosphorylation and phosphotransfer to alpha1. The alpha1 variants in Thr and Ser residues that displayed dominant negative phenotypes were significantly reduced in their ability to accept phosphoryl groups from Rio1p, thus, providing an important link between cell physiology and proteasomal phosphorylation.

Figure 1

Phosphatase-sensitive isoforms of α-type proteins of proteasomal CPs as a function of growth in H. volcanii cells. (a) An α1 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 CFU·mL⁻¹), separated by 2-DE and analyzed by immunoblot using anti-α1 antibody (α-α1). (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 β-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 (α-α1). Fractions were also assayed for peptidase activity using Suc-LLVY-AMC. u.d., undetectable. (c) Both α1 and α2 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 (α-α1 and α-α2, resp.).

Figure 2

Analysis of an H. volcanii psmA (α1) knockout expressing wild type and variant α-type proteasomal genes in trans. (a) Growth and pigmentation of an H. volcanii psmA mutant (GZ130) were influenced by expression of α1 variant proteins on ATCC novobiocin plates. Synthesis of α1 Thr147Ala and α1 Thr158Ala inhibited growth of the psmA mutant, while α1 Ser58Ala and α1 Thr147Ala altered pigmentation. (b) No significant difference in α1 protein levels were detected in the H. volcanii psmA mutant synthesizing the wild type and variant α1 proteins from genes provided in trans. The α-type proteins encoded by the genes expressed in trans are indicated with the following abbreviations: wt, wild type; S58A, Ser58Ala; Y28F, Tyr28Phe; T158A, Thr158Ala; T147A, Thr147Ala.

Figure 3

Rio-type protein kinases of H. volcanii. (a) Rio-type protein kinase homologs of H. volcanii, type-1 (HVO_0135) and type-2-like (HVO_0569), with conserved amino acid residues in bold and underlined. While HVO_0569 does not have a complete C-terminal domain, it does contain three of the five conserved residues of Rio type-2 kinases. (b) Purification of Rio1p (HVO_0135) from H. volcanii. Reducing SDS-PAGE gel of cell lysate (lane 1) and Rio1p purified by StrepTactin chromatography (lane 2) from in H. volcanii-pJAM2558. (c) Rio1p catalyzes its autophosphorylation. Autophosphotransfer was detected in vitro by autoradiography using [γ-³²P] ATP as a substrate with optimal activity detected at 10 to 50 mM MgCl₂. *, identity of band remains to be determined.

Figure 4

Rio1p phosphorylates α1 at Ser and Thr residues. (a) Phosphotransfer of Rio1p to α1 is catalyzed using α1-His₆ from recombinant E. coli as a substrate. Phosphotransfer was assayed using the protein substrates indicated as follows: α1-His₆ (boiled and unboiled) from recombinant E. coli, 20S CPs of α1-His₆ and β-StrepII subunits (boiled and unboiled) from H. volcanii and α-casein from bovine milk (boiled), as indicated. (b) Phosphotransfer of Rio1p to α1 is reduced by site-directed α1 protein variants Ser58Ala, Thr147Ala and Thr158Ala. The α1 wild type and protein variants were purified from recombinant E. coli and were not boiled prior to assay. Phosphotransfer was detected by autoradiography using Rio1p-StrepII purified by StrepTactin chromatography and γ-³²P P-ATP as a substrate.