Posttranslational modification of the 20S proteasomal proteins of the archaeon Haloferax volcanii

Abstract

20S proteasomes are large, multicatalytic proteases that play an important role in intracellular protein degradation. The barrel-like architecture of 20S proteasomes, formed by the stacking of four heptameric protein rings, is highly conserved from archaea to eukaryotes. The outer two rings are composed of alpha-type subunits, and the inner two rings are composed of beta-type subunits. The halophilic archaeon Haloferax volcanii synthesizes two different alpha-type proteins, alpha1 and alpha2, and one beta-type protein that assemble into at least two 20S proteasome subtypes. In this study, we demonstrate that all three of these 20S proteasomal proteins (alpha1, alpha2, and beta) are modified either post- or cotranslationally. Using electrospray ionization quadrupole time-of-flight mass spectrometry, a phosphorylation site of the beta subunit was identified at Ser129 of the deduced protein sequence. In addition, alpha1 and alpha2 contained N-terminal acetyl groups. These findings represent the first evidence of acetylation and phosphorylation of archaeal proteasomes and are one of the limited examples of post- and/or cotranslational modification of proteins in this unusual group of organisms.

FIG. 1.

Isoforms of 20S proteasomal proteins. 20S proteasomal proteins associated with α1-His were purified from recombinant H. volcanii and separated by 2-DE (45 and 25 μg total protein are shown in panels A and B, respectively). Isoforms of α1 and/or α1-His (a to h), β (i to m), and α2 (n and o) proteins were identified by MS analysis of protein spots. (A) Molecular mass standards (on the left) and a pI range of 3.9 to 5.1 (on top) are indicated. (B) Magnified region of a 2-DE gel.

FIG. 2.

MS/MS fragmentation of acet-M_oxQGQAQQQAYDR, an N-terminal fragment of the α1 protein of 20S proteasomes. The m/z of the doubly charged parental ion was 741.34 Da, which corresponds to the mass of the defined fragment plus oxidation (16.00 Da for methionine sulfoxide [M_ox]) and acetylation (42.01 Da for acet-) of the N terminus with a delta value of <0.1 Da. The individual Mascot ion score was 76 with an E value of 5.5e−7. Labeled peaks include b and y ions as well as dominate internal ion fragments. The loss of ammonia (*, 17.03 Da) is indicated.

FIG. 3.

MS/MS fragmentation of RGEDMSMQALpSTL, an internal phosphopeptide of the β subunit of 20S proteasomes. The fragment spans residues 119 to 131 of the deduced β protein and was generated by a sequential trypsin and chymotrypsin digest of the β subunit purified in complex with α1-His. The mass of the parent ion (1,518.66 Da) corresponds to the mass of the peptide (1,438.63 Da), deamination (1 Da) of glutamine [Q_(E)], and phosphorylation (79.97 Da) of serine (pS). The individual Mascot ion score was 62 with an E value of 6.8e−4. A predominant b ion series was detected due to the N-terminal arginine of the peptide. The characteristic collision-induced dissociation neutral loss of H₃PO₄ and subsequent formation of dehydroalanine at residue 11 verified the phosphorylation site at Ser129. b(11) − H₃PO₄ and b(12) − H₃PO₄ are b(11) and b(12) fragmentation ions with a neutral loss of H₃PO₄ and subsequent formation of dehydroalanine (loss of 97.99 Da total), respectively. The loss of ammonia (*, 17.03 Da) and H₂O (^o, 18.01 Da) is indicated. Noise filtering was used.

FIG. 4.

Multiple-sequence alignment of select α-type 20S proteasome proteins. Phosphorylation sites determined by biochemical studies are boxed and indicated by a closed circle below the alignment. These include Hs_a7 Ser249 (2, 10); Hs_a7 Tyr160, Hs_a2 Tyr23, and Hs_a2 Tyr97 (42); Rat_a2 Tyr120 (5); Hs_a5 Ser56 (2); Rat_a7 and monkey Ser243 and Ser250 (7, 9); and Cal_a3 Ser248 (34). Hs, Homo sapiens; Cal, Candida albicans; Rat, Rattus norvegicus; Hvo, Haloferax volcanii. Swiss-Prot or GenBank accession numbers are as follows: Hs_a1, P60900; Hs_a2, P25787; Rat_a2, P17220; Hs_a3, P25789; Cal_a3, 46441895; Hs_a4, O14818; Hs_a4-like, Q8TAA3; Hs_a5, P28066; Hs_a6, P25786; Hs_a7, P25788; Rat_a7, 203207; Hvo_a1, Q9V2V6; Hvo_a2, Q9V2V5. Conserved residues are highlighted in gray and black, with amino acid position numbers indicated on the right and left.

FIG. 5.

Multiple-sequence alignment of the central region of select β-type 20S proteasome proteins. Phosphorylation sites determined by biochemical studies are boxed and indicated by a closed circle below the alignment. These include Hs_b7 Tyr102 and Hs_b2 Tyr154 (42) and Hvo_b Ser129 (this study). Hs, Homo sapiens; Hvo, Haloferax volcanii. Swiss-Prot or GenBank accession numbers are as follows: Hs_b1, P28072; Hs_b1i, P28065; Hs_b2, Q99436; Hs_b2i, P40306; Hs_b3, P49720; Hs_b4, P49721; Hs_b5, P28074; Hs_b5i, P28062; Hs_b6, P20618; Hs_b7,P28070; and Hvo_b, Q9V2V4. Conserved residues are highlighted in gray and black, with amino acid position numbers indicated on the right and left.