Post-translation modification in Archaea: lessons from Haloferax volcanii and other haloarchaea

Abstract

As an ever-growing number of genome sequences appear, it is becoming increasingly clear that factors other than genome sequence impart complexity to the proteome. Of the various sources of proteomic variability, post-translational modifications (PTMs) most greatly serve to expand the variety of proteins found in the cell. Likewise, modulating the rates at which different proteins are degraded also results in a constantly changing cellular protein profile. While both strategies for generating proteomic diversity are adopted by organisms across evolution, the responsible pathways and enzymes in Archaea are often less well described than are their eukaryotic and bacterial counterparts. Studies on halophilic archaea, in particular Haloferax volcanii, originally isolated from the Dead Sea, are helping to fill the void. In this review, recent developments concerning PTMs and protein degradation in the haloarchaea are discussed.

Fig 1. N-glycosylation in Hfx. Volcanii

The Hfx. volcanii S-layer glycoprotein, a reporter of N-glycosylation in this species, is modified at Asn-13 and Asn-83 by a pentasaccharide comprising a hexose, two hexuronic acids, the methyl ester of a hexuronic acid and a mannose. The first four subunits of the pentasaccharide are sequentially assembled onto a DolP carrier via the activities of the glycosyltransferases, AglJ, AglG, AglI and AglE. At the same time, AglD adds the final pentasaccharide residue, mannose, onto a distinct DolP. Both charged DolP carriers are reoriented to face the cell exterior, with AglR thought to serve as the DolP-mannose flippase or to contribute to such activity. AglB acts to transfer the DolP-bound tetrasaccharide (and its precursors) to select Asn residues of target proteins, such as the S-layer glycoprotein. The final mannose subunit is then transferred to the protein-bound tetrasaccharide. AglF, AglM and AglP play various sugar-processing roles in the pathway. In the figure, DolP is presented as a vertical line, while hexoses are presented as red circles, hexuronic acids are presented as yellow squares and mannose is presented as a green circle.

Fig 2. The Hfx. volcanii S-layer glycoprotein undergoes differential N-glycosylation as a function of environmental salinity

Mass spectrometry was used to reveal that when Hfx. volcanii cells are grown in 3.4 M NaCl-containing medium, Asn-13 and Asn-83 are modified by the pentasaccharide portrayed in Fig 1. In the conditions, Asn-370 and Asn-498 are not modified. When however, the cells are grown at lower salt concentrations (i.e. in medium containing 1.75 M NaCl), S-layer glycoprotein Asn-498 is modified by a ‘low salt’ tetrasaccharide comprising a sulfated hexose, two hexoses and a rhamnose. At the same time, Asn-13 and Asn-83 are still modified by the pentasaccharide described above, albeit much less so. Asn-370 is still not modified. The N-glycosylation status of Asn-274, Asn-279 and Asn-732 was not considered. In the figure, hexoses are presented as red circles, hexuronic acids are presented as yellow squares, mannose is presented as a green circle and rhamnose is presented as a blue circle. Positions where no glycosylation is seen are indicated by ‘x’.

Fig 3. Protein modification in Hbt. salinarum taxis

Halobacterial transducers (Htrs) are soluble or membrane-bound complexes that associate with signal receptors (SRs). Htrs signal to a two-component regulatory system composed of an autophosphorylating histidine kinase CheA, which mediates phosphotransfer to CheY, the response regulator of the system. CheY targets the flagellar motor and regulates the switch for flagellar rotation (clockwise (CW) vs. counterclockwise (CCW)). Adaptation is promoted by the methylation status of conserved Glu and Gln residues of Htr, where CheB deamidates Htr Gln residues prior to O-methylesterification. Htr is methylated by CheR (+CH₃) and demethylated by CheB (−CH₃). CheA-mediated phosphorylation regulates the demethylation activity of CheB.

Fig 4. Phosphotransferase system of Hfx. Volcanii

A schematic diagram of the Hfx. volcanii phosphotransferase (PTS) system predicted to be responsible for responsible for the simultaneous transport and phosphorylation of sugar substrates (e.g., fructose and galacticol) and for the generation of dihydroyacetone phosphate (DHAP) from dihydroxyacetone (DHA) by DHA kinase. A series of enzyme intermediates, including EI, HPr, EIIA, EIIB, EIIC and DHA kinase (DhaM, L, K), are predicted to be phosphorylated.

Fig 5. Hfx. volcanii N-terminal proteome modified by Nα-acetylation (left) and/or cleavage by methionine aminopeptidase (right)

Pie charts based on the N-terminal proteome of Hfx. volcanii that was detected by MS/MS (Kirkland et al., 2008b). Based on this proteomic analysis, Hfx. volcanii proteins are often cleaved by methionine aminopeptidase (MAP) and/or Nα-acetylated. Penultimate residues exposed by MAP are often small and uncharged (Gly, Ala, Pro, Val, Ser or Thr). Of the N-termini that are acetylated, a relatively equal divide exists between proteins Nα-acetylated at their N-terminal Met versus a residue exposed after MAP cleavage (with Nα-acetylation of exposed Ser and Ala residues common). Among proteins with Nα-acetylated N-terminal Met residues, most (over 80%) have the Met residue followed by a small, uncharged residue (Gly, Ala, Pro, Val, Ser or Thr) typically cleaved by MAP.

Fig 6. Hypusine modification of eIF5A

Hypusine is a universal modification in archaea and eukaryotes on a single type of protein (eIF5A) by a sequential series of enzyme reactions. Deoxyhypusine synthase (DHS) transfers a 4-aminobutyl moiety from spermidine to the ε-amino group of a specific lysine residue on eIF5A. Deoxyhypusine hydroxylase (DOHH) hydroxylates the modified lysine to form hypusine.

Fig 7. Schematic depiction of haloarchaeal signal peptides and their processing

Type I and type II signal peptides that target proteins to the Sec or Tat translocation pathways, as well as type III signal peptides, have been reported in haloarchaea. In each signal peptide, the light blue N-terminal region contains positive charges (+) in the case of Sec pathway substrates or the twin arginine residues (RR) characteristic of Tat pathway substrates. Type I signal peptidases cleave the signal peptide after a C-terminal region (dark blue) often ending in alanine(s). Type II signal peptidases cleave the signal peptide upstream of the lipobox cysteine, found in the LAGC consensus sequence. The exposed cysteine that becomes the N-terminal residue of the mature protein may become lipid-modified. Type III signal peptidases act on a C-terminal domain upstream of a hydrophobic stretch (sky blue). The cleavage sites processed by the various signal peptidases are indicated by black triangles. For further details, see Pohlschroder et al. (2005).

Fig 8. Sampylation in Hfx. Volcanii

The Hfx. volcanii ubiquitin-like SAMP1/2 and UbaA (a ubiquitin-activating E1 enzyme homolog) function in protein conjugation (sampylation) and sulfur transfer (biosynthesis of MoCo and thiolated tRNA). In these pathways, UbaA is thought to adenylate the C-terminal glycine of the SAMP1/2. In protein conjugation, a thioester intermediate is thought to form between a conserved active site cysteine of UbaA (C188) and the C-terminal carboxyl group of the SAMP. SAMP1/2 are then transferred to lysine residues on protein targets to form an isopeptide bond. Archaeal proteins of the Jab1/Mov34/Mpr1 Pad1 N-terminal+ (MPN+) (JAMM) domain superfamily are proposed to cleave isopeptide bonds and, thus, render the pathway reversible.