Archaeal ubiquitin-like SAMP3 is isopeptide-linked to proteins via a UbaA-dependent mechanism.

SAMP1 and SAMP2 are ubiquitin-like proteins that function as protein modifiers and are required for the production of sulfur-containing biomolecules in the archaeon Haloferax volcanii. Here we report a novel small archaeal modifier protein (named SAMP3) with a ß-grasp fold and C-terminal diglycine motif characteristic of ubiquitin that is functional in protein conjugation in Hfx. volcanii. SAMP3 conjugates were dependent on the ubiquitin-activating E1 enzyme homolog of archaea (UbaA) for synthesis and were cleaved by the JAMM/MPN+ domain metalloprotease HvJAMM1. Twenty-three proteins (28 lysine residues) were found to be isopeptide-linked to the C-terminal carboxylate of SAMP3, and 331 proteins were reproducibly found associated with SAMP3 in a UbaA-dependent manner based on tandem mass spectrometry (MS/MS) analysis. The molybdopterin (MPT) synthase large subunit homolog MoaE, found samp3ylated at conserved active site lysine residues in MS/MS analysis, was also shown to be covalently bound to SAMP3 by immunoprecipitation and tandem affinity purifications. HvJAMM1 was demonstrated to catalyze the cleavage of SAMP3 from MoaE, suggesting a mechanism of controlling MPT synthase activity. The levels of samp3ylated proteins and samp3 transcripts were found to be increased by the addition of dimethyl sulfoxide to aerobically growing cells. Thus, we propose a model in which samp3ylation is covalent and reversible and controls the activity of enzymes such as MPT synthase. Sampylation of MPT synthase may govern the levels of molybdenum cofactor available and thus facilitate the scavenging of oxygen prior to the transition to respiration with molybdenum-cofactor-containing terminal reductases that use alternative electron acceptors such as dimethyl sulfoxide. Overall, our study of SAMP3 provides new insight into the diversity of functional ubiquitin-like protein modifiers and the network of ubiquitin-like protein targets in Archaea.

The N-degradome of Escherichia coli: limited proteolysis in vivo generates a large pool of proteins bearing N-degrons.

The N-end rule is a conserved mechanism found in Gram-negative bacteria and eukaryotes for marking proteins to be degraded by ATP-dependent proteases. Specific N-terminal amino acids (N-degrons) are sufficient to target a protein to the degradation machinery. In Escherichia coli, the adaptor ClpS binds an N-degron and delivers the protein to ClpAP for degradation. As ClpS recognizes N-terminal Phe, Trp, Tyr, and Leu, which are not found at the N terminus of proteins translated and processed by the canonical pathway, proteins must be post-translationally modified to expose an N-degron. One modification is catalyzed by Aat, an enzyme that adds leucine or phenylalanine to proteins with N-terminal lysine or arginine; however, such proteins are also not generated by the canonical protein synthesis pathway. Thus, the mechanisms producing N-degrons in proteins and the frequency of their occurrence largely remain a mystery. To address these issues, we used a ClpS affinity column to isolate interacting proteins from E. coli cell lysates under non-denaturing conditions. We identified more than 100 proteins that differentially bound to a column charged with wild-type ClpS and eluted with a peptide bearing an N-degron. Thirty-two of 37 determined N-terminal peptides had N-degrons. Most of the proteins were N-terminally truncated by endoproteases or exopeptidases, and many were further modified by Aat. The identities of the proteins point to possible physiological roles for the N-end rule in cell division, translation, transcription, and DNA replication and reveal widespread proteolytic processing of cellular proteins to generate N-end rule substrates.

Prokaryotic proteasomes: nanocompartments of degradation.

Proteasomes are self-compartmentalized energy-dependent proteolytic machines found in Archaea, Actinobacteria species of bacteria and eukaryotes. Proteasomes consist of two separate protein complexes, the core particle that hydrolyzes peptide bonds and an AAA+ ATPase domain responsible for the binding, unfolding and translocation of protein substrates into the core particle for degradation. Similarly to eukaryotes, proteasomes play a central role in protein degradation and can be essential in Archaea. Core particles associate with and utilize a variety of ATPase complexes to carry out protein degradation in Archaea. In actinobacterial species, such as Mycobacterium tuberculosis, proteasome-mediated degradation is associated with pathogenesis and does not appear to be essential. Interestingly, both actinobacterial species and Archaea use small proteins to covalently modify proteins, prokaryotic ubiquitin-like proteins (Pup) in Actinobacteria and ubiquitin-like small archaeal modifier proteins (SAMP) in Archaea. These modifications may play a role in proteasome targeting similar to the ubiquitin-proteasome system in eukaryotes.

Extreme challenges and advances in archaeal proteomics.

Archaea display amazing physiological properties that are of interest to understand at the molecular level including the ability to thrive at extreme environmental conditions, the presence of novel metabolic pathways (e.g. methanogenesis, methylaspartate cycle) and the use of eukaryotic-like protein machineries for basic cellular functions. Coupling traditional genetic and biochemical approaches with advanced technologies, such as genomics and proteomics, provides an avenue for scientists to discover new aspects related to the molecular physiology of archaea. This review emphasizes the unusual properties of archaeal proteomes and how high-throughput and specialized mass spectrometry-based proteomic studies have provided insight into the molecular properties of archaeal cells.

Phosphorylation and methylation of proteasomal proteins of the haloarcheon Haloferax volcanii.

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.

Ubiquitin-like small archaeal modifier proteins (SAMPs) in Haloferax volcanii.

Archaea, one of three major evolutionary lineages of life, encode proteasomes highly related to those of eukaryotes. In contrast, archaeal ubiquitin-like proteins are less conserved and not known to function in protein conjugation. This has complicated our understanding of the origins of ubiquitination and its connection to proteasomes. Here we report two small archaeal modifier proteins, SAMP1 and SAMP2, with a beta-grasp fold and carboxy-terminal diglycine motif similar to ubiquitin, that form protein conjugates in the archaeon Haloferax volcanii. The levels of SAMP-conjugates were altered by nitrogen-limitation and proteasomal gene knockout and spanned various functions including components of the Urm1 pathway. LC-MS/MS-based collision-induced dissociation demonstrated isopeptide bonds between the C-terminal glycine of SAMP2 and the epsilon-amino group of lysines from a number of protein targets and Lys 58 of SAMP2 itself, revealing poly-SAMP chains. The widespread distribution and diversity of pathways modified by SAMPylation suggest that this type of protein conjugation is central to the archaeal lineage.

LccA, an archaeal laccase secreted as a highly stable glycoprotein into the extracellular medium by Haloferax volcanii.

Laccases couple the oxidation of phenolic compounds to the reduction of molecular oxygen and thus span a wide variety of applications. While laccases of eukaryotes and bacteria are well characterized, these enzymes have not been described in archaea. Here, we report the purification and characterization of a laccase (LccA) from the halophilic archaeon Haloferax volcanii. LccA was secreted at high levels into the culture supernatant of a recombinant H. volcanii strain, with peak activity (170 +/- 10 mU.ml(-)(1)) at stationary phase (72 to 80 h). LccA was purified 13-fold to an overall yield of 72% and a specific activity of 29.4 U.mg(-)(1) with an absorbance spectrum typical of blue multicopper oxidases. The mature LccA was processed to expose an N-terminal Ala after the removal of 31 amino acid residues and was glycosylated to 6.9% carbohydrate content. Purified LccA oxidized a variety of organic substrates, including bilirubin, syringaldazine (SGZ), 2,2,-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and dimethoxyphenol (DMP), with DMP oxidation requiring the addition of CuSO(4). Optimal oxidation of ABTS and SGZ was at 45 degrees C and pH 6 and pH 8.4, respectively. The apparent K(m) values for SGZ, bilirubin, and ABTS were 35, 236, and 670 muM, with corresponding k(cat) values of 22, 29, and 10 s(-)(1), respectively. The purified LccA was tolerant of high salt, mixed organosolvents, and high temperatures, with a half-life of inactivation at 50 degrees C of 31.5 h.

The N-terminal penultimate residue of 20S proteasome alpha1 influences its N(alpha) acetylation and protein levels as well as growth rate and stress responses of Haloferax volcanii.

Proteasomes are energy-dependent proteolytic machines. We elaborate here on the previously observed N(alpha) acetylation of the initiator methionine of the alpha1 protein of 20S core particles (CPs) of Haloferax volcanii proteasomes. Quantitative mass spectrometry revealed this was the dominant N-terminal form of alpha1 in H. volcanii cells. To further examine this, alpha1 proteins with substitutions in the N-terminal penultimate residue as well as deletion of the CP "gate" formed by the alpha1 N terminus were examined for their N(alpha) acetylation. Both the "gate" deletion and Q2A substitution completely altered the N(alpha)-acetylation pattern of alpha1, with the deletion rendering alpha1 unavailable for N(alpha) acetylation and the Q2A modification apparently enhancing cleavage of alpha1 by methionine aminopeptidase (MAP), resulting in acetylation of the N-terminal alanine. Cells expressing these two alpha1 variants were less tolerant of hypoosmotic stress than the wild type and produced CPs with enhanced peptidase activity. Although alpha1 proteins with Q2D, Q2P, and Q2T substitutions were N(alpha) acetylated in CPs similar to the wild type, cells expressing these variants accumulated unusually high levels of alpha1 as rings in N(alpha)-acetylated, unmodified, and/or MAP-cleaved forms. More detailed examination of this group revealed that while CP peptidase activity was not impaired, cells expressing these alpha1 variants displayed higher growth rates and were more tolerant of hypoosmotic and high-temperature stress than the wild type. Overall, these results suggest that N(alpha) acetylation of alpha1 is important in CP assembly and activity, high levels of alpha1 rings enhance cell proliferation and stress tolerance, and unregulated opening of the CP "gate" impairs the ability of cells to overcome salt stress.

Proteasomal components required for cell growth and stress responses in the haloarchaeon Haloferax volcanii.

Little is known regarding the biological roles of archaeal proteases. The haloarchaeon Haloferax volcanii is an ideal model for understanding these enzymes, as it is one of few archaea with an established genetic system. In this report, a series of H. volcanii mutant strains with markerless and/or conditional knockouts in each known proteasome gene was systematically generated and characterized. This included single and double knockouts of genes encoding the 20S core alpha1 (psmA), beta (psmB), and alpha2 (psmC) subunits as well as genes (panA and panB) encoding proteasome-activating nucleotidase (PAN) proteins closely related to the regulatory particle triple-A ATPases (Rpt) of eukaryotic 26S proteasomes. Our results demonstrate that 20S proteasomes are required for growth. Although synthesis of 20S proteasomes containing either alpha1 or alpha2 could be separately abolished via gene knockout with little to no impact on growth, conditional depletion of either beta alone or alpha1 and alpha2 together rendered the cells inviable. In contrast, the PAN proteins were not essential based on the robust growth of the panA panB double knockout strain. Deletion of genes encoding either alpha1 or PanA did, however, render cells more sensitive to growth on organic versus inorganic nitrogen sources and hypo-osmotic stress and limited growth in the presence of l-canavanine. Abolishment of alpha1 synthesis also had a severe impact on the ability of cells to withstand thermal stress. This contrasted with what was seen for panA knockouts, which displayed enhanced thermotolerance. Together, these results provide new and important insight into the biological role of proteasomes in archaea.

Shotgun proteomics of the haloarchaeon Haloferax volcanii.

Haloferax volcanii, an extreme halophile originally isolated from the Dead Sea, is used worldwide as a model organism for furthering our understanding of archaeal cell physiology. In this study, a combination of approaches was used to identify a total of 1296 proteins, representing 32% of the theoretical proteome of this haloarchaeon. This included separation of (phospho)proteins/peptides by 2-dimensional gel electrophoresis (2-D), immobilized metal affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC), and Multidimensional Protein Identification Technology (MudPIT) including strong cation exchange (SCX) chromatography coupled with reversed phase (RP) HPLC. Proteins were identified by tandem mass spectrometry (MS/MS) using nanoelectrospray ionization hybrid quadrupole time-of-flight (QSTAR XL Hybrid LC/MS/MS System) and quadrupole ion trap (Thermo LCQ Deca). Results indicate that a SCX RP HPLC fractionation coupled with MS/MS provides the best high-throughput workflow for overall protein identification.

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

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.

Proteasomes from structure to function: perspectives from Archaea.

Insight into the world of proteolysis has expanded considerably over the past decade. Energy-dependent proteases, such as the proteasome, are no longer viewed as nonspecific degradative enzymes associated solely with protein catabolism but are intimately involved in controlling biological processes that span life to death. The proteasome maintains this exquisite control by catalyzing the precisely timed and rapid turnover of key regulatory proteins. Proteasomes also interplay with chaperones to ensure protein quality and to readjust the composition of the proteome following stress. Archaea encode proteasomes that are highly related to those of eukaryotes in basic structure and function. Investigations of archaeal proteasomes coupled with those of eukaryotes has greatly facilitated our understanding of the molecular mechanisms that govern regulated protein degradation by this elaborate nanocompartmentalized machine.

Archaeal proteasomes and other regulatory proteases.

Numerous proteases have been shown to catalyze the precisely-timed and rapid turnover of key cellular proteins. Often these regulatory proteases are either energy-dependent or intramembrane-cleaving. In archaea, two different types of energy-dependent proteases have been characterized: 20S proteasomes associated with proteasome-activating nucleotidases and membrane-associated Lon proteases. Interestingly, homologs of all three mechanistic classes of intramembrane-cleaving proteases are widely distributed in archaea. Similar to their eucaryal and bacterial counterparts, members of these uncharacterized proteases might promote the controlled release of membrane-anchored regulatory proteins or liberate small peptide reporters and/or effectors that function in cell signaling.