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 ␣-type subunits, and the inner two rings are composed of ␤-type subunits. The halophilic archaeon Haloferax volcanii synthesizes two different ␣-type proteins, ␣1 and ␣2, and one ␤-type protein that assemble into at least two 20S proteasome subtypes. In this study, we demonstrate that all three of these 20S proteasomal proteins (␣1, ␣2, and ␤) are modified either post-or cotranslationally. Using electrospray ionization quadrupole time-of-flight mass spectrometry, a phosphorylation site of the ␤ subunit was identified at Ser129 of the deduced protein sequence. In addition, ␣1 and ␣2 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.26S proteasomes are central proteolytic enzymes of the ubiquitin-mediated degradation pathway in eukaryotes. The 20S core particle of 26S proteasomes is responsible for the hydrolysis of peptide bonds, and the overall architecture of this complex is conserved from archaea to eukaryotes (12). The 20S proteasomes are chambered proteases with the proteolytic N-terminal Thr active site sequestered within a cylindrical complex composed of 28 proteins associated as four heptameric rings of ␣-and ␤-type subunits (2). The ␣-type subunits form the outer two rings, and the ␤-type subunits form the inner two rings that comprise the central proteolytic chamber.In the halophilic archaeon Haloferax volcanii, there are two ␣-type (␣1 and ␣2) subunits and one ␤-type proteasomal subunit (54). The N-terminal protein sequence of the ␤ subunit purified from 20S proteasomes reveals an N-terminal Thr residue exposed via posttranslational cleavage which is likely to form the active site (54). In contrast, the ␣ subunits are not amenable to N-terminal sequencing, suggesting that these proteins are modified at their N termini (54).Two subtypes of 20S proteasome complexes have been identified in stationary-phase cells and are denoted ␣1␤-and ␣1␤␣2-20S, with the latter containing both ␣-type subunits in a single 20S proteasome (20). Interestingly, both ␣1 and ␣2 proteins form homo-oligomeric rings and ␣1-␣1 and ␣2-␣2 contacts (versus ␣1-␣2 contacts) are detected in 20S proteasome complexes (20). Thus, the ␣1␤␣2-20S core particle is proposed to be an asymmetric proteasome, with the stacked rings arranged in an ␣1:␤:␤:␣2 configuration. The ␣1␤-20S proteasome, in contrast, is in a symmetrical ␣1:␤:␤:␣1 configuration. The levels of ␣2 protein increase severalfold, while the levels of ␣1 and ␤ proteins remain relatively constant as cells enter stationary phase (41). This is likely to allow for the regul...

The N-Terminal Penultimate Residue of 20S Proteasome α1 Influences its N α Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of Haloferax volcanii

“…As previously reported, the proteasomal ␣1 protein of H. volcanii appears as a mixture of proteins that either are acetylated on the initiating methionine (Ac1) or are unacetylated with the methionine removed (N2) (16). In order to better understand the relative ratios of these forms, quantitative MS of ␣1-His 6 proteins purified by single-step Ni 2ϩ -affinity chromatography from H. volcanii DS70(pJAM204) cells was performed; thus, this initial analysis was of ␣1 proteins either unassembled or assembled into CPs.…”

“…Proteasomes purified from a nat1 mutant have twofold-higher chymotrypsin-like peptidase activity in the absence of SDS compared to the wild type, suggesting that N ␣ acetylation enhances closure of the ␣-gate (21). In H. volcanii, both ␣1 and ␣2 are N ␣ acetylated on their initiator methionine residue with a subset of ␣1 not acetylated and instead cleaved by an apparent methionine aminopeptidase (16). A large-scale proteomic survey reveals N ␣ acetylation is common to other proteasomal ␣-type proteins of the haloarchaea (10).…”

“…The ␣-type subunits of CPs are modified by N ␣ acetylation in several eukaryotes and haloarchaea, including Haloferax volcanii (10,16,20,21,44). In yeast, N-acetyltransferase 1 (NAT1), the catalytic component of NatA, is responsible for the N ␣ acetylation of five of the ␣-type subunits (␣1, ␣2, ␣3, ␣4, and ␣7).…”

Protein Ser/Thr/Tyr Phosphorylation in the Archaea

“…Analysis of the 20 S proteasome from H. volcanii by mass spectrometry revealed numerous covalent modifications, including phosphorylation of the ␣1, ␣2, and ␤ subunits on serine and/or threonine (58). Blocking phosphorylation of serine and threonine ␣1 subunits by substituting alanine produced variants that were unable to restore pigment production or salt tolerance to ␣1 knock-out strains (65).…”

Genetic and Proteomic Analyses of a Proteasome-Activating Nucleotidase A Mutant of the Haloarchaeon Haloferax volcanii

“…In both Crenarchaeota and Euryarchaeota, the initiator protein Cdc6 (Orc) is autophosphorylated by a DNA-regulated mechanism at a conserved Ser residue (10,16). Likewise, the ␤ subunit of 20S proteasomes from Haloferax volcanii is phosphorylated at a Ser residue (21). A zinc-dependent aminopeptidase, with a leucine zipper motif that associates with a CCT-TRiC family chaperonin, has also been shown to be phosphorylated in Sulfolobus solfataricus (8).…”