posttranslation modification in archaea: lessons from haloferax

R EV I EW AR T I C L E

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

Jerry Eichler1 & Julie Maupin-Furlow2,3

1Department of Life Sciences, Ben Gurion University, Beersheva, Israel; 2Department of Microbiology and Cell Science, University of Florida,

Gainesville, FL, USA; and 3Genetics Institute, University of Florida, Gainesville, FL, USA

Correspondence: Jerry Eichler, Department

of Life Sciences, Ben Gurion University,

Beersheva 84105, Israel.

Tel.:+972 8646 1343; fax: +972 8647 9175;

e-mail: [email protected]

Received 12 July 2012; revised 13 November

2012; accepted 13 November 2012. Final

version published online 20 December 2012.

DOI: 10.1111/1574-6976.12012

Editor: Mecky Pohlschroder

Keywords

Archaea; haloarchaea; Haloferax volcanii;

post-translational modification; protein

degradation; proteome.

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 pro-

file. 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 counter-

parts. 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.

Introduction

The ability to address microorganisms and other life

forms at the level of the genome has revolutionized bio-

logical research. At the same time, it is becoming increas-

ingly clear that a major proprortion of the diversity that

exists within a cell is generated at the level of the prote-

ome. Numerous factors are responsible for the proteome

assuming additional levels of complexity not predicted at

the genome level. These include alternative RNA splicing,

differential expression of a given protein in response to

environmental cues or as a function of developmental

stage, and the plethora of possible protein–protein inter-

actions, as well as post-translational modifications

(PTMs) and regulated protein degradation.

Proteins can be modified post-translationally by the

permanent or temporary covalent attachment of one or

more of several classes of molecules, including sugars, lip-

ids, or other chemical groups. Flexibility in the spatial dis-

tribution of such linked moieties on a target protein and in

the timing of their addition, together with the ability to

introduce changes in the molecular composition of the

bound modifying groups, offers further sources of protein

variability. Intra-molecular disulphide linkages, formed via

the covalent bonding of Cys residues pairs, influence the

three-dimensional conformation of a protein. Disulphide

bonds formed between different protein subunits can yield

multimeric complexes (Fass, 2011). Proteolytic processing

likewise can allow for control of the folding and function

of a target protein. For instance, the post-translational

removal of targeting sequences and inteins respectively per-

mits the cell to control the site where a protein ultimately

resides, as well as the timing and manner in which a pro-

tein can act (Paulus, 2000; Gogarten et al., 2002; Jarvis &

Robinson, 2004; Hegde & Bernstein, 2006). Specifically,

any of these PTMs, either alone or in combination, can

affect protein function, interaction of the modified protein

with binding partners, protein localization, or the rate at

which a protein is degraded, among other traits.

At the same time, regulated protein degradation is crit-

ical for maintaining protein quality and controlling cell

functions. Proteases, which can discern and specifically

degrade proteins compromised by denaturation, misin-

corporation of amino acids, and other damaging events,

are important for regulating cellular homeostasis. Like-

wise, regulated protein degradation processes, in which

FEMS Microbiol Rev 37 (2013) 583–606 ª 2012 Federation of European Microbiological SocietiesPublished by John Wiley & Sons Ltd. All rights reserved

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the protease destroys key proteins that may be properly

folded but must be removed at specific times or moved

to locations to enable molecular mechanisms to occur,

are also important to cell function. Central to regulated

protein degradation are proteases that are coupled to

ATP hydrolysis (Gottesman, 2003). These energy-depen-

dent proteases have a self-compartmentalized structure, in

which the proteolytic active sites are housed within a pro-

tein nanoparticle that is chambered, gated, and linked to

an ATPase. The ATPase component of such proteases is

required for the unfolding of protein substrate, opening

of the gate, and facilitating the degradation process

(Lupas et al., 1997; Maupin-Furlow, 2012).

It is now clear that PTMs and regulated protein degra-

dation transpire in all three domains of life, namely Euk-

arya, Bacteria, and Archaea. Still, current understanding

of the archaeal versions of these processes lags behind

that of their eukaryal and bacterial counterparts. In the

following, we review what is known of PTM and regu-

lated protein degradation in the halophilic archaea, lar-

gely focusing on Haloferax volcanii. With the availability

of a complete genome sequence (Hartman et al., 2010),

simple growth requirements and advanced genetic, molec-

ular biology, proteomic and biochemical tools and tech-

niques (DasSarma & Fleishmann, 1995; Allers et al., 2004,

2010; Soppa, 2006; Kirkland et al., 2008b; Dyall-Smith,

2009), H. volcanii represents a strain of choice for molec-

ular studies in Archaea and has provided considerable

insight into the archaeal versions of these protein process-

ing events.

N-glycosylation

N-glycosylation, the covalent linkage of glycan moieties to

select Asn residues of a target protein, was among the

first haloarchaeal PTMs to be described. The Halobacteri-

um salinarum surface (S)-layer glycoprotein was the first

noneukaryotic protein shown to be N-glycosylated

(Mescher & Strominger, 1976). Two different Asn-linked

oligosaccharides modify the S-layer glycoprotein, namely

a repeating sulfated pentasaccharide linked via

N-acetylgalactosamine to Asn-2 and a sulfated glycan

linked by a glucose residue to ten other Asn residues

(Mescher & Strominger, 1978; Lechner et al., 1985a;

Lechner and Wieland, 1989; Wieland et al., 1980, 1983).

The H. salinarum flagellin [since renamed archaellin

(Jarrell & Albers, 2012)] was shown to bear the same gly-

can-linked sulfated polysaccharide (Wieland et al., 1985).

Although applying genetics to identify components of the

H. salinarum N-glycosylation pathway was not possible at

the time, biochemical approaches served to reveal various

aspect of the N-glycosylation pathway of this haloar-

chaeon. As such, it was shown that dolichol phosphate

(DolP) serves as the lipid carrier of the glucose-linked

sulfated glycan decorating 10 sites of N-glycosylation,

whereas dolichol pyrophosphate (DolPP) bears the

repeating sulfated pentasaccharide N-acetylgalactosamine-

linked to S-layer glycoprotein Asn-2 (Wieland et al.,

1980; Lechner and Wieland, 1989). A link between these

phosphodolichol-charged glycans to N-glycosylation was

supported by the observation that in H. salinarum, the

glycan moiety of the DolP-bound sulfated polysaccharide

is also detected on the S-layer glycoprotein and archael-

lins in this species (Lechner et al., 1985a; Wieland et al.,

1985). Moreover, the sulfated polysaccharide is methylat-

ed in the DolP-linked form but not when protein-bound

(Lechner et al., 1985b). The significance of this obser-

vation remains unclear. Finally, studies showing the

ability of H. salinarum cells to modify cell-impermeable,

sequon-bearing hexapeptides with sulfated oligosaccha-

rides localized the N-glycosylation event to the external

cell surface (Lechner et al., 1985a).

Despite these advances, the process of N-glycosylation

in the haloarchaea is currently best understood in H. vol-

canii. The S-layer glycoprotein, comprising the sole com-

ponent of the S-layer, contains seven putative N-

glycosylation sites, namely the motif Asn-X-Ser/Thr, where

X is any residue but Pro. Early studies reported modifica-

tion of the S-layer glycoprotein Asn-13 and Asn-498

positions by a linear string of glucose residues, whereas

Asn-274 and/or Asn-279 were supposedly decorated by a

glycan containing glucose, galactose, and idose (Sumper

et al., 1990; Mengele & Sumper, 1992). More recently, evi-

dence for N-glycosylation of the H. volcanii archaellins,

FlgA1 and FlgA2, was presented (Tripepi et al., 2010,

2012). Likewise, currently unidentified H. volcanii glyco-

proteins of 150, 105, 98, 58, 56, 54, and 52 kDa have been

detected (Zhu et al., 1995; Eichler, 2000), although some

of these species may correspond to the same polypeptide.

Moreover, it remains to be determined whether these

proteins indeed experience N-glycosylation rather than

O-glycosylation. The same is true of LccA, a glycosylated

laccase secreted by H. volcanii (Uthandi et al., 2010). The

H. volcanii S-layer glycoprotein, containing both N- and

O-linked glycans, thus remains the best-characterized

glycoprotein in this species (Sumper et al., 1990).

In the last few years, substantial progress in decipher-

ing the pathway of S-layer glycoprotein N-glycosylation

has been made, with the identification of a series of

archaeal glycosylation (agl) genes encoding proteins

involved in the assembly and attachment of a pentasac-

charide to select Asn residues of the S-layer glycoprotein.

Acting at the cytoplasmic face of the plasma membrane,

AglJ, AglG, AglI, and AglE sequentially add the first four

pentasaccharide residues (i.e. a hexose, two hexuronic

acids and the methyl ester of a hexuronic acid) onto

ª 2012 Federation of European Microbiological Societies FEMS Microbiol Rev 37 (2013) 583–606Published by John Wiley & Sons Ltd. All rights reserved

584 J. Eichler & J. Maupin-Furlow

a common DolP carrier, while AglD adds the final

pentasaccharide residue, mannose, to a distinct DolP

(Abu-Qarn et al., 2007, 2008b; Yurist-Doutsch et al.,

2008, 2010; Guan et al., 2010; Kaminski et al., 2010; Mag-

idovich et al., 2010). The use of DolP by Archaea as the

lipid carrier upon which the N-linked glycan is assembled

also holds true for eukaryal N-glycosylation (Burda &

Aebi, 1999; Hartley & Imperiali, 2012). In contrast, bacte-

rial N-linked glycans are first assembled on a different

isoprenoid, undecaprenol phosphate (Szymanski & Wren,

2005; Weerapana & Imperiali, 2006). N-glycosylation

roles have also been assigned to AglF, a glucose

-1-phosphate uridyltransferase (Yurist-Doutsch et al.,

2010), AglM, a UDP-glucose dehydrogenase (Yurist-Dou-

tsch et al., 2010) and AglP, a methyltransferase (Magido-

vich et al., 2010). Indeed, AglF and AglM were shown to

act in a sequential and coordinated manner in vitro,

transforming glucose-1-phophosphate into UDP-glucu-

ronic acid (Yurist-Doutsch et al., 2010). In a reaction

requiring the archaeal oligosaccharide transferase, AglB

(Abu-Qarn & Eichler, 2006; Chaban et al., 2006; Igura

et al., 2008), the lipid-linked tetrasaccharide and its pre-

cursors are delivered to select Asn residues of the S-layer

glycoprotein. The final mannose residue is subsequently

transferred from its DolP carrier to the protein-bound

tetrasaccharide (Guan et al., 2010) in a reaction requiring

AglR, a protein that either serves as the DolP-mannose

flippase or contributes to such activity (Kaminski et al.,

2012), and AglS, a DolP-mannose mannosyltransferase

(Cohen-Rosenzweig et al., 2012). Current understanding

of H. volcanii N-glycosylation is depicted in Fig. 1.

Insight gained from N-glycosylation in H. volcanii has

served to elucidate aspects of the parallel process in other

halophilic archaea. It was initially shown that H. volcanii

strains lacking either aglD or aglJ could be functionally

complemented by introduction of rrnAC1873 and

rrnAC0149, the respective homologues of these genes

from Haloarcula marismortui (Calo et al., 2010b, 2011).

Like H. volcanii, the haloarchaeon H. marismortui also

originates from the Dead Sea (Oren et al., 1990). Indeed,

subsequent efforts revealed the S-layer glycoprotein of

both species is decorated with N-linked pentasaccharides

comprising a hexose, two hexuronic acids, a methyl ester

of hexuronic acid, and a mannose (Calo et al., 2011).

Still, differences in the N-glycosylation pathways of these

two haloarchaea exist. While in H. volcanii the N-linked

pentasaccharide is derived from a tetrasaccharide sequen-

tially assembled on a single DolP and a final mannose

residue derived from a distinct DolP carrier (Guan et al.,

2010), a similar pentasaccharide N-linked to the

H. marismortui S-layer glycoprotein is first fully assem-

bled on a single DolP and only then transferred to the

protein target (Calo et al., 2011). The finding that H. vol-

canii N-glycosylation pathway components can be

replaced with homologues from other haloarchaea to

yield N-glycan variants has provided a proof-of-concept

for developing H. volcanii in a glyco-engineering platform

designed to produce tailored glycoproteins (Calo et al.,

2011). Such efforts will also exploit the proven ability of

H. volcanii to N-glycosylate introduced nonnative pro-

teins (Kandiba et al., 2012).

The H. volcanii N-glycosylation pathway, involving

multiple glycan-charged DolP carriers, recalls the parallel

eukaryal process. In higher Eukarya, the first seven

subunits of the 14-meric oligosaccharide assembled in the

endoplasmic reticulum are sequentially added to a com-

mon phosphodolichol carrier, whereas the second set of

seven sugar subunits are derived from single mannose- or

glucose-charged DolP (Burda & Aebi, 1999; Helenius &

Aebi, 2004; Hartley & Imperiali, 2012). The H. volcanii

N-glycosylation pathway further resembles its eukaryal

Fig. 1. N-glycosylation in Haloferax volcanii. The H. 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.


Protein modification in the haloarchaea 585

counterpart when one considers that even in cells lacking

the oligosaccharyltransferase, AglB, where pentasaccha-

ride-modified DolP would be expected to accumulate,

only tetrasaccharide-modified DolP could be detected

(Calo et al., 2011). This observation points to the final

mannose of the N-linked pentasaccharide as being added

to the tetrasaccharide already attached to the S-layer gly-

coprotein. This same general strategy is employed in Euk-

arya, where in the Golgi, additional sugar subunits are

attached to oligosaccharides already N-linked to the target

polypeptide. On the other hand, H. marismortui N-glyco-

sylation is similar to the parallel bacterial process in

which a heptasaccharide is assembled by the sequential

addition of seven soluble nucleotide-activated sugars onto

a common undecaprenol phosphate carrier (Szymanski &

Wren, 2005; Weerapana & Imperiali, 2006; Abu-Qarn

et al., 2008a). Yet, although delivered to the lipid-linked

rather than the protein-bound tetrasaccharide, the termi-

nal mannose subunit of the pentasaccharide N-linked to

the H. marismortui S-layer glycoprotein is derived from a

distinct DolP carrier, as in H. volcanii (Calo et al., 2011).

Although the absence or even the perturbation of

N-glycosylation compromises the ability of H. volcanii to

grow in high salt (Abu-Qarn et al., 2007) and modifies

S-layer stability and architecture (Abu-Qarn et al., 2007),

as well as S-layer resistance to added protease (Yurist-

Doutsch et al., 2008, 2010; Kaminski et al., 2010), cells

lacking AglB, and hence unable to perform N-glycosyla-

tion, are viable (Abu-Qarn et al., 2007). As such, it would

seem that this PTM is not essential for H. volcanii sur-

vival, yet nonetheless is advantageous to H. volcanii in

certain scenarios. Thus, one can hypothesize that H. vol-

canii modifies aspects of N-glycosylation in response to

changing growth conditions. This concept has gained

support from recent studies comparing N-glycosylation of

the S-layer glycoprotein in cells grown in 3.4 or 1.75 M

NaCl-containing medium (Guan et al., 2012). At the

higher salinity, S-layer glycoproteins Asn-13 and Asn-83

were shown to be modified by the pentasaccharide

described above, while DolP was shown to be modified

by the tetrasaccharide comprising the first four pentasac-

charide residues, again as discussed above. However, cells

grown at low salinity contain DolP modified by a distinct

tetrasaccharide comprising a sulfated hexose, two hexoses,

and a rhamnose not seen linked to DolP in cells grown at

high salinity. This is likely the same DolP-bound tetrasac-

charide observed by Kuntz et al. (1997) in H. volcanii

cells grown in 1.25 M NaCl-containing medium. The

same tetrasaccharide modified S-layer glycoprotein Asn-

498 in cells grown in low salt, whereas no glycan deco-

rated this residue in cells grown in the high-salt medium.

At the same time, Asn-13 and Asn-83 were modified by

substantially less pentasaccharide at the low-salt condi-

tions (Fig. 2). Hence, in response to environmental salin-

ity, H. volcanii not only modulates the N-linked glycans

decorating the S-layer glycoprotein but also residues sub-

jected to this PTM.

Finally, it should be noted that studies on the metha-

nogens, Methanococcus voltae and Methanococcus marip-

aludis, and the thermophiles, Sulfolobus acidocaldarius,

Pyrococcus furiosus, and Archaeoglobus fulgidus, have also

provided insight into archaeal N-glycosylation (Chaban

et al., 2006; Igura et al., 2008; VanDyke et al., 2009;

Meyer et al., 2011; Jones et al., 2012; Matsumoto et al.,

2012).

Phosphorylation

Phosphorylation is widely appreciated as a covalent form

of PTM that occurs at His, Asp, Ser, Thr, or Tyr residues.

Phosphorylation is rapid, reversible and generates confor-

mational changes in protein structure that mediate an

array of biological responses from signal transduction to

metabolism (Johnson & Barford, 1993). While early stud-

ies suggested that Archaea (and Bacteria) use mainly two-

component systems of His/Asp phosphorylation, it is now

appreciated that Archaea (and Bacteria) also perform Ser/

Thr/Tyr phosphorylation (once thought to be restricted to

eukaryotes) for creating highly sophisticated regulatory

Fig. 2. The Haloferax volcanii S-layer glycoprotein undergoes

differential N-glycosylation as a function of environmental salinity.

Mass spectrometry was used to reveal that when H. 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 ‘9’.



networks (Macek et al., 2008). A number of reviews and

genomic surveys are available that highlight the phosphor-

ylation of archaeal proteins and the enzymes (protein kin-

ases/phosphatases) likely to mediate and/or regulate this

PTM (Leonard et al., 1998; Kennelly & Potts, 1999; Ken-

nelly, 2003; Eichler & Adams, 2005; Tyagi et al., 2010). In

addition, a Phosphorylation Site Database is available

online that provides a guide to some of the Ser/Thr/Tyr-

phosphorylated proteins in Archaea (Wurgler-Murphy

et al., 2004). Thus, the discussion below highlights recent

studies on protein phosphorylation in halophilic archaea

and how this type of PTM may control cellular function.

Phospho-site mapping

Historically, halophilic archaea have provided a useful

model system to advance our understanding of how proteins

can be phosphorylated across domains of life. In fact, the

demonstration that proteins of H. salinarum were reversibly

phosphorylated by a light-regulated retinal-dependent

mechanism was the first report that proteins in Archaea

could be phosphorylated (Spudich & Stoeckenius, 1980).

Later use of molecular biology tools revealed a two-compo-

nent His/Asp phosphorelay system (analogous to the CheA/

CheY system of Bacteria) that was responsible for modulat-

ing the response of H. salinarum to chemotactic and photo-

tactic stimuli (Rudolph & Oesterhelt, 1995; Rudolph et al.,

1995) (Fig. 3). Early study of H. volcanii revealed a Mn2+-

stimulated protein phosphatase activity against synthetic

protein substrates phosphorylated at Ser/Thr residues

(Oxenrider & Kennelly, 1993). More recent studies using

high-throughput methods have facilitated the mapping of

phosphorylation sites on proteins of halophilic archaea (9

phospho-sites in H. volcanii and 81 phospho-sites in H. sali-

narum), thus increasing the number of phospho-sites (from

three total) previously detected in Archaea (Kirkland et al.,

2008a; Aivaliotis et al., 2009). In these high-throughput

approaches, wild-type and mutant strains with enhanced

levels of phosphoproteins [i.e. H. volcanii ΔpanA (protea-

some-activating nucleotidase A) and H. salinarum ΔserB(OE4405R phosphoserine phosphatase)] were used as input

material. Phosphopeptides were enriched from samples by

immobilized metal affinity chromatography and metal oxide

affinity chromatography (in parallel and sequentially), fol-

lowed by tandem mass spectrometry (MS/MS). While a

phospho-site consensus motif was not apparent, the major-

ity of sites mapped to Ser/Thr/Tyr residues.

Protein kinases/phosphatases

Halophilic archaea are predicted to encode histidine pro-

tein kinases and protein phosphatases of the ‘two-compo-

nent’ Asp/His phosphorelay system (Koretke et al., 2000;

Kim & Forst, 2001) (e.g. H. volcanii is predicted to

encode at least 30 histidine protein kinases). The best

studied example of an archaeal Asp/His phospho-relay

system is that of H. salinarum, in which a CheA histidine

protein kinase undergoes ATP-dependent autophosphory-

lation of a His residue and transfers the phosphoryl

group to an Asp residue on CheY, a response regulator

thought to be dephosphorylated by the protein phospha-

tase, CheC (and not CheZ) (Rudolph & Oesterhelt, 1995;

Rudolph et al., 1995; Muff & Ordal, 2007; Streif et al.,

2010). Ultimately, the phosphorylation status of CheY

impacts the ability of this protein to switch the flagellar

motor and regulate cellular movement toward favorable

light and nutrients (Nutsch et al., 2005). Interestingly,

variants of ‘two-component’ histidine protein kinases can

act as Ser/Thr/Tyr protein kinases in eukaryotes (Harris

et al., 1995) and Bacteria (Min et al., 1993; Yang et al.,

1996; Shi et al., 1999; Wu et al., 1999). Whether or not

this alternative type of phosphorylation also occurs in

Archaea is yet unknown.

Many halophilic archaea (including H. volcanii) as well

as the crenarchaeon Thermofilum pendens harbor homo-

logs of the phosphoenolpyruvate phosphotransferase sys-

tem (PTS) (Hartman et al., 2010) (Fig. 4). In analogy to

what is known for Bacteria (Barabote & Saier, 2005), the

H. volcanii PTS is predicted to mediate transfer of

the phosphoryl group on PEP to imported sugars (e.g.

fructose, galacticol) or endogenous dihydroxyacetone via

dihydroxyacetone kinase (Hartman et al., 2010). Halofe-

rax volcanii PTS homologs include a single enzyme I

(PtsI; His~P) and multiple copies of histidine protein

(HPr; His~P), enzyme IIA (EIIA; His~P), enzyme IIB (EIIB;

Cys~P), and enzyme IIC (EIIC; His~P), with the amino acid

PP

P

Htr CheW

CheR

CheB

Flagellarmotor

+ CH3CheY

CheA

–CH3

SAM

Flagella

cw

ccw

Signal

SR

Fig. 3. Protein modification in Halobacterium salinarum taxis. 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.



residue predicted to be phosphorylated during group

translocation provided in parenthesis. Recent work dem-

onstrates that the PTS gene cluster HVO_1495 to

HVO_1499, encoding PtsI, EIIB, HPr, EIIA, and EIIC

homologs, was highly upregulated as a cotranscript dur-

ing growth on fructose (Pickl et al., 2012). Deletion of

HVO_1499, encoding a homolog of the fructose-specific

membrane component EIIC of this cluster, resulted in

loss of growth on fructose compared to glucose (Pickl

et al., 2012). Thus, the PTS system has a functional

involvement in the metabolism of fructose in H. volcanii.

Atypical RIO-type Ser/Thr protein kinase homologs of

the type 1, type 2, and Bud32 (piD261) families are com-

mon among the Archaea (including the haloarchaea)

(Leonard et al., 1998; Shi et al., 1998; Ponting et al.,

1999; LaRonde-LeBlanc & Wlodawer, 2005a, b; Tyagi

et al., 2010). Protein kinases of the RIO-type 1 family are

distinguished by an STGKEA consensus sequence in their

N-terminal domain and a second region of homology

(IDXXQ, where X represents any amino acid residue) in

their C-terminal domain. Kinases of the RIO-type 2

family often have an N-terminal helix-turn-helix motif

followed by GXGKES and C-terminal IDFPQ sequences.

Members of the Bud32 family of Ser/Thr protein kinases

are associated with the KEOPS (ECK) complex, com-

posed of three additional subunits (Kae1, Pcc1, and

Cgi121), and shown to be required for formation of the

tRNA modification threonylcarbamoyladenosine (t6A) in

yeast (Srinivasan et al., 2011).

A RIO-type 1 homolog (Rio1p, HVO_0135) of H. vol-

canii has been characterized at the biochemical level.

Rio1p purifies as a monomer and can transfer the

c-phosphoryl group of ATP to a1, a protein that forms

the outer rings of 20S proteasomes in H. volcanii

(Humbard et al., 2010b). Rio1p-mediated phosphotrans-

fer is not observed and/or is diminished for a1 variants

T158A, S58A, and T147A. Thus, Rio1p can phosphorylate

a substrate protein (a1) at Ser/Thr residues based on

in vitro assay.

Homologs of the four subunits of the eukaryotic

KEOPS complex are conserved in Archaea, with

H. volcanii-encoding homologs of Pcc1 (HVO_0652) and

Cgi121 (HVO_0013) and a fusion of the Bud32 Ser/Thr

protein kinase to Kae1 (HVO_1895). The gene encoding

the Bud32-Kae1 homolog and its subdomains appear

essential in H. volcanii (Naor et al., 2012), suggesting

Bud32-mediated phosphorylation of Ser/Thr residues is

important for cell function. Homologs of KEOPS (Bud32,

Kae1, Cgi121 and Pcc1) from related Euryarchaeota

(Methanocaldococcus jannaschii and P. furiosus) have been

used for reconstitution of the complex, structural analysis

at the atomic level, heterologous complementation, and

in vitro phosphorylation assays (Hecker et al., 2008; Mao

et al., 2008). From this work, Bud32 is suggested to phos-

phorylate a Thr residue of an insert within the catalytic

cleft of Kae1 and, thus, regulate KEOPS function.

Whether or not Bud32 phosphorylates other proteins in

Archaea besides Kae1 is not clear. Both yeast Bud32 and

its human ortholog, p53-related protein kinase (PRPK),

can phosphorylate p53 (Abe et al., 2001; Facchin et al.,

2003). Likewise, Sulfolobus solfataricus SsoPK5 (a Bud32

homolog) catalyzes the phosphorylation of various

proteins in vitro (Haile & Kennelly, 2011).

In addition to the atypical RIO-type Ser/Thr/Tyr pro-

tein kinases, homologs of the Bacillus subtilis PrkA (Uni-

Prot P39134) are common among halophilic archaea

(e.g. H. volcanii HVO_2849 and HVO_2848). Members

of the PrkA family possess a Walker A-motif of nucleo-

tide-binding proteins and exhibit distant homology to

eukaryotic protein kinases (Fischer et al., 1996). In addi-

tion, amino acid residues within the active site of cyclic

adenosine 3′, 5′-monophosphate (cAMP)-dependent pro-

tein kinase are also conserved in PrkA (Fischer et al.,

1996). B. subtilis PrkA can phosphorylate a 60-kDa pro-

tein at a Ser residue (Fischer et al., 1996). However, fur-

ther analysis is needed to determine the identity of this

protein substrate and confirm that B. subtilis PrkA, and

its haloarchaeal relatives are Ser/Thr protein kinases.

Phosphorylation of proteasomes

Proteasomes are self-compartmentalized proteases,

which undergo a substantial number of post-/co-transla-

tional modifications, including phosphorylation. The

P~HPr3

outin

EIIC1,2EIIB1,2

Sugar

CM

DhaM(EIIA)

DhaL

DhaK

DHA DHAP

P~HPr1,2,3PEP EI

EI~Ppyruvate HPr1,2,3

EIIA1,2

~PEIIA1,2

Sugar -P

HPr3

P~

P~

P~

Fig. 4. PTS of Haloferax volcanii. A schematic diagram of the

H. 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.



phosphorylation of these complexes is of interest, because

proteasomes are important for cell function (e.g. growth

of H. volcanii) (Zhou et al., 2008). Proteasomes are com-

posed of a 20S catalytic core particle (of a- and b-typesubunits) and regulatory particles, including AAA+ ATP-

ases (homologs of Cdc48/VAT/p97 and Rpt subunits

termed proteasome-associated nucleotidases or PANs)

that mediate energy-dependent protein degradation (Bart-

helme & Sauer, 2012; Maupin-Furlow, 2012). In H. volca-

nii, proteasomes are modified by phosphorylation in

addition to Na-acetylation, methyl-esterification, and

cleavage of b subunit precursors that expose the

N-terminal threonine residue forming the active sites of

20S proteasomes (Table 1) (Wilson et al., 1999; Humbard

et al., 2006, 2010b). Eukaryotic 20S proteasomes and associ-

ated AAA+ ATPases (homologs of PAN termed Rpt1-6 and

Cdc48/VAT/p97) are also altered by phosphorylation and

other forms of covalent modification, such as the attach-

ment of O-linked N-acetylglucosamine, Ne- and Na-acety-lation, N-myristoylation, and cleavage of b subunit

precursors (Zhang et al., 2007; Ewens et al., 2010).

Haloferax volcanii 20S proteasomes and associated

PANs are phosphorylated at Ser and Thr residues. To

facilitate phospho-site mapping, 20S proteasomes and

PAN proteins were purified from H. volcanii by tandem

affinity chromatography using His6- and StrepII-tags

(Humbard et al., 2006, 2010b). Sites of phosphorylation

were identified by MS/MS (with precursor ion scanning)

and included b Ser129, a1 Thr147, and a2 Thr13/Ser14

(not distinguished) of 20S proteasomes and Ser340 of

PAN-A (Humbard et al., 2006, 2010b). A phospho-site

for PAN-B was not identified (Humbard et al., 2010b).

MS/MS analysis of phosphopeptides enriched from

H. volcanii proteomes suggests Cdc48/VAT/p97 homologs

are also phosphorylated; however, sites of modification

were not identified by this high-throughput method

(Kirkland et al., 2008a). In eukaryotes, phosphorylation

and acetylation regulate the function of Cdc48/VAT/p97

proteins.

Phosphorylation of the a-type subunits of H. volcanii

proteasomes has been investigated at various stages of cell

growth and assembly states. Humbard et al. (2010b) sepa-

rated the a-type subunits (of cell lysate and 20S proteo-

somes purified by affinity chromatography with bsubunits) by two-dimensional gel electrophoresis (2DE)

and detected a1 and a2 by immunoblot using polyclonal

antibodies specific to each protein. Phosphorylated iso-

forms (two specific for a1 and one specific for a2) were

determined by shifting the 2DE-protein spot to a more

basic pI after removal of the acidic phosphate groups by

phosphatase treatment. Of the two phosphorylated iso-

forms of a1 detected, the most acidic form was found

throughout growth, assembled in 20S proteasomes. In

contrast, the least phosphorylated isoform of a1 was pres-

ent as both unassembled and assembled subunits of 20S

proteasomes and was detected at reduced levels in later

stages of growth (Humbard et al., 2010b). The phosphor-

ylated isoform of a2 was also found associated with 20S

proteasomes. Thus, phosphorylation of the a-type pro-

teins is suggested to influence their assembly and/or be

important for 20S proteasome function.

Site-directed mutagenesis has been used to determine

the biological role of proteasome phosphorylation in

H. volcanii. Haloferax volcanii strains expressing a1 pro-

teins with Ala modifications in Ser/Thr residues likely to

be phosphorylated (based on MS analysis and an inability

to accept a phosphoryl group from Rio1p, in vitro) dis-

play dominant negative phenotypes for cell viability and

colony color (i.e. white vs. red) (Humbard et al., 2010b).

Thus, phosphorylation of the a1 subunit of 20S protea-

somes appears to be closely linked to cell growth and pig-

mentation (i.e. production of carotenoids) (Humbard

et al., 2010b). Interestingly, H. volcanii proteasomal

mutant strains deficient in the synthesis of PAN-A display

a striking increase in the number of cellular proteins that

are phosphorylated, suggesting an added link between

protein phosphorylation and proteasome function (e.g.

phosphorylation may target proteins for destruction by

energy-dependent proteases, in analogy to eukaryotes and

Bacteria) (Kirkland et al., 2008a).

Protein acetylation

Protein acetylation is the covalent attachment of an acetyl

group to a protein. In general, acetylation can impact

protein function, stability, and interactions with other

molecules. Two types of protein acetylation are known to

occur in living cells, Na-acetylation and Ne (or lysine)-

acetylation, with high-energy molecules, such as acetyl-

CoA, providing the acetyl group for these modifications.

Na-Acetylation is an irreversible mechanism in which an

acetyl group is covalently attached to the a-amino group

Table 1. PTMs of Haloferax volcanii proteasomes

Subunit Phosphorylated

Methyl-

esterified

Na-

acetylated

Exposed by

autocleavage

a1 Thr147 Asp20, Glu27,

Glu62, Glu112,

Glu161

Met1 n.d.

a2 Thr13/Ser14 n.d. Met1 n.d.

b Ser129 n.d. n.d. Thr50

PAN-A Ser340 n.d. n.d. n.d.

Residue number according to protein sequence in GenBank [GI:30

0669661 (a1), GI:12229945 (a2), GI:292655712 (b), GI:302425218

(PAN-A)].



of the N-terminal amino acid of a protein. In contrast,

Ne-acetylation occurs when the e-amino group of a lysine

residue is reversibly modified by the covalent attachment

of an acetyl group. Studies on H. volcanii have furthered

our understanding of both processes in Archaea.

Na-acetylation

Our current understanding of the prevalence of Na-acety-lation in Archaea is largely based on MS-based proteomic

surveys (Soppa, 2010; Maupin-Furlow et al., 2012). Like

Bacteria, Na-acetylation of ribosomal proteins appears

common among Archaea (Kimura et al., 1989; Hatakey-

ama & Hatakeyama, 1990; Klussmann et al., 1993; Mar-

quez et al., 2011). Interestingly, the number of proteins

reported to be Na-acetylated varies greatly among the dif-

ferent archaeal groups. In haloarchaea (apparently unlike

Bacteria), a relatively high proportion (14–29%) of the

proteome is modified by Na-acetylation (i.e. H. salina-

rum, Natronomonas pharaonis, and H. volcanii) (Falb

et al., 2006; Aivaliotis et al., 2007; Kirkland et al., 2008b).

Likewise, Na-acetylation appears to impact a large per-

centage of the S. solfataricus proteome, based on the find-

ing of 17 Na-acetylated N-termini of 26 total detected by

MS (Mackay et al., 2007). Interestingly, to date, only a

single protein (the a subunit of the 20S proteasome) is

reported to be Na-acetylated among the methanogens,

suggesting that this form of modification may be rare in

this group of Archaea (Forbes et al., 2004; Zhu et al.,

2004; Enoki et al., 2011).

In H. volcanii, like other Archaea, many of the

Na-acetylated proteins appear to be generated by a NatA-

type activity [i.e. acetylation of penultimate Ser or Ala

residues exposed after removal of N-terminal methionine

residues by methionine aminopeptidase (MAP)] (Fig. 5).

Na-Acetylated N-terminal methionine residues with pen-

ultimate Asp and Asn residues are also detected, consis-

tent with the NatB-like activity of eukaryotes (Kirkland

et al., 2008b). Proteins with a Na-acetylated N-terminal

methionine residue followed by a small penultimate resi-

due (Ser, Ala, Thr, Pro, and Gly) are also detected, sug-

gesting that Na-acetylation can restrict their cleavage by

MAP. Interestingly, many of the proteins of halophilic ar-

chaea (including those of H. volcanii) that are Na-acety-lated are also readily identified by semi-quantitative MS

spectral counting in unmodified and/or MAP-cleaved

forms (Falb et al., 2006; Aivaliotis et al., 2007; Kirkland

et al., 2008b). Liquid chromatography-multiple reaction

monitoring (LC-MRM) MS, a technique that provides a

more accurate perspective on the abundance of the

Na-acetylated state of a protein, reveals that the a1proteins that form 20S proteasomes are primarily in an

Na-acetylated Met form, as compared to the MAP

cleaved form (103 : 1 ratio), in H. volcanii (Humbard

et al., 2009). Whether or not Na-acetylation efficiency is

also near 100% for other H. volcanii protein substrates

and/or can be altered by growth conditions remains to be

determined.

While proteins with Na-acetylated Met-Gln and Met-

Asn sequences are not prevalent in Archaea, 20S protea-

some a-type subunits with these N-terminal sequences are

specifically Na-acetylated in the Euryarchaeota, H. salina-

rum, N. pharaonis, H. volcanii, and Methanothermobacter

thermoautotrophicus (Falb et al., 2006; Humbard et al.,

2006; Aivaliotis et al., 2007; Enoki et al., 2011). Site-direc-

ted mutagenesis of the N-terminal Met-Gln sequence of

the H. volcanii 20S proteasome a1 protein has been used

to further investigate this specific Na-acetylation (Hum-

bard et al., 2009). Variants of a1 were expressed in vivo

and analyzed by MS to detect alterations in N-terminal

modification (i.e. Na-acetylation, MAP cleavage). A Q2A

substitution rendered the a1 protein susceptible to cleav-

age by MAP followed by Na-acetylation of the penulti-

mate Ala by an apparent NatA-type activity similarly to

most Na-acetylated proteins in haloarchaea. However,

the N-termini of a1 proteins with the small penultimate

amino acid residues Ser and Val were detected

A

S

GV

P

T

D ENI W FL K Q

MAP cleaved (n = 143)

A

S

M

V TKD

F QLNα-acetylated (n = 72)

MS

MAMT

MPMGMV

MNMD MQ

Fig. 5. Haloferax volcanii N-terminal proteome modified by

Na-acetylation (left) and/or cleavage by MAP (right). Pie charts based

on the N-terminal proteome of H. volcanii that was detected by MS/

MS (Kirkland et al., 2008b). Based on this proteomic analysis,

H. volcanii proteins are often cleaved by MAP and/or Na-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 Na-acetylated at their

N-terminal Met vs. a residue exposed after MAP cleavage (with

Na-acetylation of exposed Ser and Ala residues common). Among

proteins with Na-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.



predominantly in the uncleaved forms, with

Na-acetylated methionine residues intact. Alteration of

penultimate amino acid residues to Asp, Pro, and Thr

resulted in a mixture of a1 protein in the Na-acetylatedmethionine, MAP cleaved and/or unmodified forms. Thus,

the enzyme(s) responsible for Na-acetylation of the a1N-terminal Met appears to have relaxed sequence specific-

ity with regard to the penultimate residue of the substrate.

Furthermore, only the Q2A substitution rendered the

N-terminus of a1 fully susceptible to MAP cleavage, sug-

gesting that structural elements of a1 or interacting part-

ners/chaperones may mask primary N-terminal sequences

that are otherwise optimal for MAP cleavage (e.g. Met-Ser

of the a1 Q2S variant). Interestingly, Na-acetylation of a1Met appears important in gating the 20S proteasome,

based on the enhanced peptidase activity of 20S protea-

somes with the MAP-cleaved a1 Q2A variant and the

inability of the gene encoding the a1 Q2A variant to com-

plement the a1 mutation for growth at ‘low’ salt

(� 1.3 M NaCl). Insight into archaeal enzyme(s) that

mediate Na-acetylation is provided through comparative

genomics, in vitro reconstitution of Na-acetyltransferaseactivity and crystallography. Members of the GNAT super-

family that use acetyl-CoAs to acylate their cognate

substrates, including the Na-acetylation of proteins, are

widely distributed among Archaea (Vetting et al., 2005).

Sulfolobus solfataricus ssArd1 (SSO0209) is an archaeal

GNAT related to Na-acetyltransferases that has been dem-

onstrated to acetylate the N-terminal residue (Ser) of the

DNA-binding protein, Alba (Mackay et al., 2007). Much

like the eukaryal Ard1 of NatA, SsArd1 preferentially

acetylates N-terminal Ser and Ala residues exposed after

methionine removal (Mackay et al., 2007). SsArd1 also

catalyzes appreciable Na-acetylation of N-terminal

Met-Glu and Met-Leu sequences, similar to Nat3 of NatB

in eukaryotes (Mackay et al., 2007). While ssArd1 can

Na-acetylate a variety of proteins in vitro, it shows prefer-

ence for proteins with N-termini that are disordered in

crystal structures (Mackay et al., 2007). Thus, it is unclear

whether ssArd1 functions post- or co-translationally in the

cell. The archaeal ssArd1 is thought to represent an ances-

tral form of some eukaryal Na-acetyltransferases based on

its relaxed sequence specificity. Recent crystallography of

SsArd1 now provides structural detail for analysis of this

ancestral function (Oke et al., 2010).

Na-acetylation and protein stability

Based on analogy to eukaryotes, Na-acetylation is

predicted to regulate protein turnover in H. volcanii and

other haloarchaea. The long-held argument that

Na-acetylation stabilizes a protein comes from several

lines of indirect evidence (Meinnel et al., 2006). For

example, proteins modified by Na-acetylation are often

over-represented in protein abundance profiles and

comprise a high proportion of the proteome (Falb et al.,

2006; Aivaliotis et al., 2007; Kirkland et al., 2008b;

Martinez et al., 2008). Furthermore, Na-acetylationblocks the Na-amino group of a protein from further

modification by destabilizing processes such as ‘linear’

ubiquitylation (Meinnel et al., 2005). However, recent

evidence suggests Na-acetylation can mark a protein for

destruction by the ubiquitin-proteasome system using a

mechanism named the Ac/N-end rule (Hwang et al.,

2010). The Ac/N-end rule is based on the finding that an

E3 ubiquitin ligase (named Doa10) can recognize proteins

with acetylated N-termini and facilitate their ubiquityla-

tion (at internal lysine residues) and degradation by pro-

teasomes in yeast (Hwang et al., 2010). To rationalize this

discrepancy between Na-acetylation in protein stability

and degradation, Hwang et al. (2010) provide a model

in which nascent proteins can ‘hide’ their acetylated

N-termini by rapid folding, interaction with chaperones,

and/or assembly into appropriate multi-subunit com-

plexes. Such sequestration of N-termini would render the

Na-acetylation-based degradation signals inaccessible for

recognition by the Doa10 E3 ubiquitin ligase, ultimately

stabilizing the protein. In contrast, delayed or defective

protein folding would expose acetylated N-termini and

allow for Doa10-dependent ubiquitylation and proteolysis

by proteasomes.

Insight into a potential archaeal Ac/N-end rule pathway

is provided by study of the a1 protein of 20S protea-

somes in H. volcanii (Humbard et al., 2009; Varshavsky,

2011). Here, the identity of the N-terminal penultimate

residue of a1 was found to dramatically alter the concen-

tration of a1 protein in the cell. In particular, the

levels of N-terminal a1 variants that were partially non-

Na-acetylated were remarkably higher than the levels of

Na-acetylated (wild type) a1 protein. Furthermore, most

of the a1 proteins associated in 20S proteasomes had

acetylated N-termini. Thus, proteasomal partners are

predicted to obstruct recognition of the acetylated

N-terminal domain of a1 and prevent its proteolytic

destruction by an Ac/N-end rule pathway.

Ne-acetylation

The reversible and differential Ne-acetylation of proteins

can have a major impact on transcription, translation,

stress response, detoxification, and carbohydrate and

energy metabolism (Hu et al., 2010; Jones & O’Connor,

2011; Thao & Escalante-Semerena, 2011). In eukaryotes,

histones are well known to be modified by Ne-acetyla-tion, in addition to Na-acetylation, methylation,

phosphorylation, ubiquitylation, ADP ribosylation,



glycosylation, and sumoylation (Shiio & Eisenman, 2003).

Numerous bacterial proteins are also differentially Ne-acetylated, with ‘K-acetylomes’ (subproteomes composed

of proteins with Ne-acetylated lysine residues) thought to

rival phosphoproteomes (Aka et al., 2011). In contrast,

Ne-acetylation of archaeal proteins is poorly understood.

In Archaea, only a few proteins are known to be

Ne-acetylated. A couple of early studies, focused on deter-

mining the amino acid sequence of 2Fe–2S ferredoxins

from the haloarchaea H. salinarum and H. marismortui,

revealed that a lysine residue near the C-terminus of these

proteins is conserved and Ne-acetylated (Hase et al.,

1978, 1980). Another archaeal protein that is Ne-acety-lated is Alba, a chromatin protein of S. solfataricus. Alba

is not only Na-acetylated on its N-terminal Ser (as dis-

cussed above) but is also Ne-acetylated on lysine 16 (Bell

et al., 2002). Acetylation of Alba Lys16 is mediated by the

protein acetyltransferase, Pat (Marsh et al., 2005). Pat is a

homolog of Salomonella Pat (YfiQ) and a member of the

family of NDP-forming acetyl-CoA synthetase enzymes

with GNAT domains (Starai & Escalante-Semerena,

2004). Alba Lys16 can be deacetylated by an NAD+-

dependent histone deacetylase (HDAC) class III homolog

of S. solfataricus (Sir2) (Bell et al., 2002). Ne-Acetylationof Alba reduces its binding affinity for DNA and RNA

and strongly prevents its ability to inhibit the DNA heli-

case activity of the mini-chromosome maintenance pro-

tein (Bell et al., 2002; Jelinska et al., 2005; Marsh et al.,

2006). Thus, Ne-acetylation of Alba is thought to have a

global impact on chromatin packaging and gene expres-

sion in Crenarchaeota, such as S. solfataricus (Wardle-

worth et al., 2002; Zhao et al., 2003).

Haloferax volcanii has served as a model for under-

standing the importance of histone acetyltransferase

(HAT) and HDAC gene homologs to archaeal cell func-

tion. In particular, gene homologs for three HATs (Pat1,

HVO_1756; Pat2, HVO_1821; Elp3, HVO_2888) and two

HDACs (Sir2, HVO_2194; HdaI, HVO_0522) were tar-

geted for deletion from the H. volcanii genome (Altman-

Price & Mevarech, 2009). Pat1 and Pat2 are related to the

S. solfataricus Pat, Elp3 is related to the yeast Elp3 sub-

unit of the elongator complex possessing acetyltransferase

activity, Sir2 is a class III HDAC homolog similar to

S. solfataricus Sir2, and HdaI is a class II HDAC homolog

related to yeast Hda1. Single deletion of sir2, pat1, pat2,

or elp3 genes or double deletion of pat1 with either pat2

or elp3 was found to have no detectable impact on the

viability of H. volcanii cells. In contrast, hdaI appeared

essential, based on the finding that the gene could only

be deleted when a wild-type copy of hdaI was provided in

trans. Attempts to create an elp3 deletion in any of the

pat2 null strains were unsuccessful, implying that these

two mutations are synthetically lethal and affect a single

function or pathway (Altman-Price & Mevarech, 2009).

Thus, Elp3- and Pat2-mediated acetylation and HdaI-

mediated deacetylation of lysine residues are predicted to

be important in H. volcanii.

Hypusine modification

Hypusine [Ne-(4-amino-2-hydroxybutyl)-L-lysine] is

formed upon PTM of a conserved lysine residue and

is found only in eukaryotic translation ‘initiation’ factor

5A (eIF5A) and the related archaeal aIF5A (Park et al.,

1981, 2010; Schumann & Klink, 1989; Bartig et al., 1992).

Hypusine is essential for the activity of eIF5A/aIF5A

(including that of H. salinarum), now considered impor-

tant in translation elongation (Wagner & Klug, 2007;

Saini et al., 2009; Park et al., 2010). In hypusine modifica-

tion of eIF5A, deoxyhypusine synthase (DHS) transfers a

4-aminobutyl moiety from spermidine to the e-amino

group of the conserved lysine residue to form a dexoxy-

hypusine intermediate, which is hydroxylated to a hypu-

sine residue by deoxyhypusine hydroxylase (DOHH)

(Fig. 6). In Archaea, DHS homologs are widespread,

suggesting the lysine residue of aIF5A is modified to a

deoxyhypusine residue by an enzyme similar to eukaryotic

DHS. In contrast, DOHH is an oxygen-dependent

enzyme, and its homologs are rare in (the often anaerobic)

Archaea. Thus, generation of the hypusine-modified form

of aIF5A from its deoxyhypusine precursor likely involves

a second enzyme distinct from DOHH in Archaea.

Spermidine

NH2

Lys

eIF5A

NH

Lys

eIF5A

H2N

NH

Lys

eIF5A

H2N

–OH

O2 H2O

1,3-diaminopropane

DHS DOHH

Deoxyhypusine Hypusine

donor-H2 acceptor

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. DHS transfers a 4-aminobutyl

moiety from spermidine to the e-amino group

of a specific lysine residue on eIF5A. DOHH

hydroxylates the modified lysine to form

hypusine.



Protein methylation

Protein methylation is a type of PTM that can mediate

many important biological processes, spanning from gene

regulation and signal transduction to protein stability

(Clarke, 1993; Lee et al., 2005; Paik et al., 2007). In this

PTM, nucleophilic oxygen, nitrogen, and sulfur atoms on

the polypeptide chain can be methylated, resulting in the

generation of methyl esters, methyl amines, methyl

amides, and other modifications (with only the hydroxyl

groups of Ser, Thr and Tyr not detected in methylated

forms). Methylation of the side chain hydroxyl group of

dicarboxylic amino acid residues (Asp, Glu) and the

hydroxyl group generated by deamidation of glutamine

residues (O-methylesterification) is typically reversible

and regulated. Recent evidence also supports the revers-

ible methylation of the amino groups in the side chains

of lysine (mono-, di-, and tri-methylated forms) and argi-

nine (mono- and di-methylated forms) residues of pro-

teins (Lee et al., 2005). S-adenosylmethionine (SAM)

commonly serves as the methyl group donor for the

methylation of proteins by methyltransferases that are

often specific for their protein substrate, while methyles-

terases and demethylases can remove the methyl groups.

In haloarchaea, O-methylesterification of proteins has

been detected and is the focus of the discussion below.

O-methylesterification

The methyl-accepting taxis proteins or transducers are a

large group of membrane-associated proteins that are typ-

ically methylated at glutamate residues (some of which

originate from glutamine residues via deamidation) (Por-

ter et al., 2011). Transducer homologs are relatively wide-

spread among Bacteria and Euryarchaeota (including

halophiles, methanogens, thermococci, and others). In

general, transducers act as (or interact with) sensory

receptors for a specific stimulus (e.g., light, attractant)

and are demethylated in response. The methylation state

of the transducer provides a ‘primitive memory’ or adap-

tation to a two-component regulatory system (CheA/

CheY) that switches the flagellar motor and alters cellular

movement. Among the archaeal transducers, the best

studied are the halobacterial transducers (Htrs) of H. sali-

narum, which comprise a group of 18 homologs (6 solu-

ble and 12 membrane-spanning) with 1–3 conserved

methylation sites per protein (Ng et al., 2000; Pfeiffer

et al., 2008) (Fig. 3). Early studies of H. salinarum dem-

onstrate methyl-[3H]-labeling of proteins and volatile

products of demethylation that are specific to taxis

and define the methylated proteins as Htrs (soluble and

membrane-spanning), based on the abolishment of

methyl-[3H]-labelling after gene deletion and site-directed

mutagenesis (Alam et al., 1989; Sundberg et al., 1990;

Nordmann et al., 1994; Lindbeck et al., 1995; Brooun

et al., 1997; Perazzona & Spudich, 1999; Storch et al.,

1999). Recently, MS-based proteomes have been used to

map three methylation sites in two of the six soluble Htrs

and 19 methylation sites in 10 of the 12 predicted mem-

brane-spanning Htrs (Koch et al., 2008). Sites include

singly methylated pairs of Glu and/or Gln, Asp-Glu, and

Ala-Glu residues (with Glu or deamidated Gln residues

methylated). Evidence supports the function of CheB in

deamidation and demethylation of the Htrs and CheR in

transfer of the methyl group from SAM co-factor to con-

served Glu or deamidated Gln residues of Htr (Perazzona

& Spudich, 1999; Koch & Oesterhelt, 2005; Koch et al.,

2008). Selective demethylation of Htr sites (e.g. one site is

demethylated upon stimulation by attractant, and another

site is demethylated upon simulation with repellent) is

proposed to control a diverse array of biological

responses, in analogy to the bacterium Bacillus subtilis

(Streif et al., 2010).

Like transducers, archaeal 20S proteasomes are

modified by O-methylesterification. In particular, five

unique acidic residues (Asp20, Glu27, Glu62, Glu112, and

Glu161) of the H. volcanii 20S proteasome a1 subunit are

methylated (Table 1) (Humbard et al., 2010b), as

detected by MS/MS with methods devoid of added meth-

anol (Chen et al., 2010). O-linked methylesterification is

typically a reversible form of PTM. Furthermore, both

methylated and unmethylated forms of a1 are readily

detected in the cell (Humbard et al., 2010b). Thus,

O-methylesterification of a1 is thought to be regulated

and to modulate proteasomal activity (e.g. stability in low

salt, high temperature, interaction with AAA+ ATPase

partners). While the enzyme responsible for a1 methyla-

tion is as yet unknown, a SAM-dependent methyltransfer-

ase homolog (HVO_1093) co-transcribed with the a1gene is a likely candidate (Gil et al., 2007).

N-terminal methionine removal

Methionine residues can be removed from the N-terminus

of proteins in all domains of life. Archaea and Eukarya use

methionine (Met) for the initiation of protein synthesis

(Ramesh & RajBhandary, 2001), and this N-terminal methi-

onine can be removed by a MAP as the protein emerges

from the ribosome (Lowther & Matthews, 2000). In contrast

to Met, Bacteria, and the related organelles of eukaryotes

(i.e. mitochondria and chloroplasts) use N-formylmethio-

nine (fMet) for initiation of protein synthesis. fMet is a

derivative of Met in which a formyl group has been added

to the amino group and is important for bacterial cell func-

tion (Guillon et al., 1992). With rare exceptions (Milligan &

Koshland, 1990) the N-formyl group is typically removed



from bacterial proteins by a peptide deformylase (Mazel

et al., 1994). After deformylation, the resulting N-terminal

methionine can be removed (Solbiati et al., 1999).

MAPs (EC 3.4.11.18) are metalloenzymes common to

all domains of life that remove N-terminal leading methi-

onine residues from nascent peptides during the early

stages of protein synthesis. MAPs generally cleave sub-

strates with penultimate residues that are one of the seven

small and uncharged amino acids (i.e. Gly, Ala, Ser, Thr,

Pro, Cys, and Val). MAPs belong to the MEROPS pepti-

dase family, M24 (clan MG), and are divided into two

types (types I and II), with the C-terminal domain of type

II distinguished by insertion of an extra � 60 amino acid

helical subdomain (Arfin et al., 1995). Bacteria typically

only contain the type I MAP (with some exceptions),

Archaea contain only type II MAP, while eukaryotes pos-

sess both type I and type II MAPs (Bazan et al., 1994;

Arfin et al., 1995; Lowther & Matthews, 2002).

Among the archaea, MAPs from the hyperthermophiles

P. furiosus (PfMAP) and Thermococcus sp. NA1 have been

purified in enzymatically active forms (Tsunasawa et al.,

1997; Tahirov et al., 1998; Lee et al., 2006). Of these two

enzymes, PfMAP is the most extensively studied. Similar

to the bacterial and eukaryal MAPs, PfMAP cleaves

N-terminal methionine from substrates whose penulti-

mate amino acid residues are Gly, Ala, Ser, Thr, Pro, and

Val, based on release of N-terminal methionine from the

synthetic peptide, Met-X-Ala-Ala-Ala (where X represents

the 19 common amino acids, excluding Cys) (Tsunasawa

et al., 1997). PfMAP also provided the first crystal struc-

ture of a type II MAP (Tahirov et al., 1998). Those

PfMAP residues (Asp82, Asp94, His153, Glu187, and

Glu280) that coordinate the two catalytic metal ions are

conserved in the Escherichia coli type I MAP crystal struc-

ture (with the identity of the metal ions used for catalysis

still under debate) (Roderick & Matthews, 1993). Like-

wise, the two positively charged residues (His62 and

His161) in the active site cleft of PfMAP that are thought

to shuffle protons from the nuclear center to the solvent

during catalysis are also conserved in E. coli MAP

(Roderick & Matthews, 1993; Tahirov et al., 1998).

While MAPs have not been purified from halophilic

archaea, type II MAP homologs are conserved and large-

scale proteomic studies provide evidence for MAP activity

in this group of microorganisms. In particular, nonredun-

dant N-terminal peptides identified by MS have been reli-

ably mapped to a large number of proteins from

H. salinarum (606 proteins), N. pharaonis (328 proteins),

and H. volcanii (236 proteins) (Falb et al., 2006; Aivaliotis

et al., 2007; Kirkland et al., 2008b). Of these proteins,

most (60–70%) were missing an N-terminal methionine

and presented N-termini suggesting that MAP cleavage

occurred predominantly when the penultimate residue

was small and uncharged (Gly, Ala, Pro, Val, Ser, or Thr)

(for H. volcanii, see Fig. 5). Interestingly, recent compari-

son of large data sets of nonredundant N-terminal pep-

tides detected by MS from diverse organisms suggests

that MAP cleavage efficiency is conserved across all

domains of life (Helbig et al., 2010).

Signal peptide cleavage

Proteins destined to reside beyond the confines of the

cytoplasm (i.e. secretory and membrane proteins)

are often synthesized as preproteins bearing cleavable

N-terminal targeting sequences termed signal peptides. In

H. volcanii, signal peptides direct preproteins to both the

Sec and the Tat translocation systems (Fig. 7) (Pohlsch-

roder et al., 2005; Calo & Eichler, 2011). Although the

major protein species of H. volcanii, the S-layer glycopro-

tein, relies on the Sec translocation system, bioinformat-

ics-based analysis predicts that the Tat translocation

system is preferentially used by exported H. volcanii

proteins (Dilks et al., 2003; Storf et al., 2010). In each sys-

tem, once the signal peptide has served its purpose,

namely targeting a preprotein to a proteinaceous translo-

cation complex, it is cleaved from the mature protein by

the actions of type I signal peptidase (SPase), although, as

considered below, the majority of Tat pathway lipoprotein

substrates are apparently processed by a type II SPase.

While general aspects of archaeal type I SPase biology have

been considered in several recent reviews (Ng et al., 2007;

Jarrell et al., 2010; Calo & Eichler, 2011), this enyzme has

only been biochemically addressed in a limited number of

Archaea to date, including H. volcanii (Fine et al., 2006;

Fink-Lavi & Eichler, 2008). In H. volcanii, genes encoding

two distinct type I SPases, Sec11a (HVO_2603) and

Sec11b (HVO_0002), are found (Fine et al., 2006). Of the

two, HVO_0002 is an essential gene. Observed differences

in the activities of the two purified enzymes toward a sig-

nal peptide-bearing reporter protein suggest that Sec11a

and Sec11b possess distinct substrate preferences.

At the molecular level, H. volcanii type I SPases present

a mosaic of eukaryal, bacterial, and archaeal properties

(Fine et al., 2006; Calo & Eichler, 2011). Like their eukar-

yal counterparts, the H. volcanii enzymes have replaced

the conserved lysine of the serine-lysine catalytic dyad

found in bacterial SPases with a histidine. Site-directed

mutagenesis studies revealed that both of these residues

are essential for Sec11b activity (Fink-Lavi & Eichler,

2008). Likewise, a third H. volcanii Sec11b residue, Asp-

280, was deemed as being essential for catalytic activity of

the enzyme. The equivalent residues were also shown to

be necessary for M. voltae SPase activity (Bardy et al.,

2005). As the yeast type I SPase also requires these resi-

dues for its functionality, it would appear that the



archaeal enzyme relies on a catalytic mechanism like that

employed by its eukaryal counterpart. On the other hand,

yeast SPase also requires the presence of the equivalent of

H. volcanii Sec11b Asp-273 (van Valkenburgh et al.,

1999), a residue not essential for the activity of the halo-

archaeal enzyme (Fink-Lavi & Eichler, 2008). At the same

time, the H. volcanii enzymes also share traits with their

bacterial counterparts. Tagged versions of Sec11a and

Sec11b were purified as single polypeptides, each able to

cleave the signal peptide from a reporter protein, suggest-

ing that the H. volcanii enzymes function independently

of other proteins, as do bacterial SPases. Eukaryal type I

SPases, by contrast, exist as part of a multimeric complex

in which additional proteins apart from Sec11 are essen-

tial for activity (Evans et al., 1986; YaDeau et al., 1991).

Still, the possibility remains that the H. volcanii SPases

operate optimally only when in complex with additional

components not captured under the conditions employed

nor identified in genome searches.

As discussed in more detail below, H. volcanii is

thought to contain a large number of lipoproteins. While

type II SPases remove characteristic lipoprotein signal

sequences, thereby unmasking the site of such lipid-based

PTM in target proteins, no such enzyme has been

detected in H. volcanii or any other archaea. It remains

unclear whether this reflects the absence of such an

archaeal enzyme or whether the archaeal enzyme, possibly

designed to act on the ether-based lipids of the archaeal

membrane, shares little sequence homology with its bac-

terial counterpart and, as such, has been overlooked.

Unlike type I SPases that cleave Sec or Tat transloca-

tion signal peptides, type III SPases process the unique

signal peptides found in archaellins, pili, and other cell-

surface structures (Albers et al., 2003). Analysis of the

H. volcanii genome reveals the presence of several prepro-

teins bearing a type III signal peptide, as well as a single

type III signal peptidase (Szabo et al., 2007), a homologue

of bacterial PilD, responsible for the processing of bacte-

rial type IV prepilin proteins (Strom et al., 1993). Indeed,

H. volcanii cells deleted of pibD (HVO_2993), encoding

the prepilin peptidase, fail to properly process proteins

bearing this class of signal peptide (Tripepi et al., 2010).

Lipid modification

Lipid modification of a protein involves the permanent

or temporary covalent attachment of lipid-based groups

at various positions within the polypeptide chain and

serves a variety of roles, with the most obvious being to

enhance the membrane affinity of the modified protein.

However, numerous other roles have been assigned to

such PTM, including modulation of protein–proteininteractions, signal transduction, embryogenesis, pattern

formation, protein trafficking through the secretory path-

way, evasion of the immune response by infectious para-

sites, and enzyme activation (for review, see Sinensky,

2000; Mann & Beachy, 2004; Pechlivanis & Kuhlmann,

2006; Nadolski & Linder, 2007; Charollais & Van Der

Goot, 2009; Greaves et al., 2009; Aicart-Ramos et al.,

2011). In efforts aimed at understanding the modes, path-

ways, and functions of lipid modification in archaea,

studies on H. volcanii have proven to be central.

In addressing the biogenesis of the H. volcanii S-layer

glycoprotein, it was shown that the protein is first

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).



synthesized as an immature precursor, possessing a lower

apparent molecular weight and a less hydrophobic char-

acter than the final version of the protein (Eichler,

2001). As the protein can be labeled with [3H] mevalon-

ic acid, an isoprene precursor, the post-translational

magnesium-dependent conversion to the mature form

apparently involves isoprenylation (Konrad & Eichler,

2002). Moreover, it was shown that such lipid modifica-

tion of the H. volcanii S-layer glycoprotein only occurs

once the protein has traversed the plasma membrane

(Eichler, 2001). Similarly, the H. salinarum S-layer glyco-

protein also undergoes such maturation and was shown

to be modified by a covalently linked diphytanylglyceryl

phosphate entity near an O-glycosylated Thr-rich stretch

found in the C-terminal region of the protein, upstream

of the predicted single transmembrane domain (Kikuchi

et al., 1999; Konrad & Eichler, 2002). At the same time,

several H. salinarum proteins are modified by a diphyta-

nylglycerol methyl moiety, linked to Cys residues of the

protein through a thioetheric bond (Sagami et al., 1994,

1995).

As the H. volcanii S-layer glycoprotein includes a pre-

dicted membrane-spanning domain (as does its H. salina-

rum counterpart) (Lechner & Sumper, 1987; Sumper

et al., 1990), it is unclear why an additional membrane

anchor in the form of a lipid would be required. The

recent identification of genes encoding archaeosortases

may explain this apparent paradox (Haft et al., 2012).

Archaeosortases are predicted by bioinformatics to corre-

spond to the archaeal homologues of bacterial exosortases,

enzymes involved in C-terminal anchoring of proteins to

membrane-embedded lipids. The archaeosortase, ArtA, is

predicted to cleave proteins bearing a C-terminal mem-

brane-spanning domain between this region and the Pro-

Gly-Phe motif found immediately upstream. The

processed protein is then delivered to the waiting lipid on

the external surface of the cell. Accordingly, the H. volca-

nii S-layer glycoprotein (and its H. salinarum counterpart;

Lechner & Sumper, 1987) present a Pro-Gly-Phe motif

found immediately upstream of the predicted transmem-

brane domain (Sumper et al., 1990). Indeed, the coordi-

nated transfer of the H. volcanii S-layer glycoprotein to a

lipid moiety following cleavage from the C-terminal mem-

brane-spanning domain could explain several apparent

paradoxes, such as the ability of EDTA to solubilize the

S-layer glycoprotein, an apparent integral membrane pro-

tein, without destroying membrane integrity (Charlebois

et al., 1987). In this scenario, EDTA treatment would

somehow interfere with the stabilization of newly lipid-

modified S-layer glycoprotein in the membrane or in the

S-layer, leading to the release of the protein from the cell

while leaving the plasma membrane intact. Most recently,

it has been reported that two H. volcanii S-layer glycopro-

tein populations exist, namely a lipid-modified, EDTA-

solublized pool and a second, detergent-solublized pool

likely retaining the transmembrane domain of the protein

(Kandiba et al., 2013).

In addition to such C-terminal post-translational lipid

modification, N-terminal lipid modification also occurs,

namely in processing of lipoproteins. Based on the pres-

ence the so-called lipobox sequence motif (composed of

the consensus Leu-Ala-Gly-Cys sequence) that includes

the cysteine residue that undergoes lipid modification

(Hayashi & Wu, 1990) near the start of predicted amino

acid sequence, halocyanin, a small blue copper protein of

the haloalkaliphile N. pharaonis is thought to undergo

such amino-terminal lipid modification (Mattar et al.,

1994). Although direct proof for such modification was

not provided, analysis of halocyanin by mass spectroscopy

was consistent with modification of the N-terminal Cys

residue by two C20 phytanyl (glycerol)diether lipids linked

via a thioether bond, as well as by acetylation. Indeed,

analysis of haloarchaeal genomes predicts numerous lipo-

box-containing proteins processed by the Tat protein

translocation system. In H. volcanii, 72 lipobox-contain-

ing proteins are predicted (Gim�enez et al., 2007). In the

specific cases of the H. volcanii iron-binding protein

(Ibp), DsbA-like thioredoxin domain protein (DsbA), and

maltose-binding protein (Mbp), replacement of the lipo-

box cysteine by serine in the precursor forms of these

predicted lipoproteins led to defective localization.

Mutant Ibp and DsbA were found in the growth medium

instead of being largely membrane associated, as are the

wild-type proteins; mutant Mpb is apparently unstable

and degraded (Gim�enez et al., 2007). On the other hand,

the predicted lipoproteins, HVO_B0139 and HVO_1242,

remained cell-associated in the lipobox cysteine-alanine

mutated forms (Storf et al., 2010).

The lipid-based modifications considered above essen-

tially serve to anchor the modified protein to the mem-

brane. The retinal proteins of haloarchaea (Oesterhelt &

Stoeckenius, 1971) provide another use for covalently

linked lipids. Bacteriorhodopsin, halorhodopsin, and the

two sensory rhodopsins [and related rhodopsins subse-

quently reported in Bacteria (Beja et al., 2000) and

eukaryotes (Bieszke et al., 1999)], comprise a family of

small, seven trans-membrane domain-containing proteins

involved in coupling light energy to the vectorial trans-

port of ions across a membrane. To achieve this feat,

these retinal proteins contain covalently linked retinal, an

isoprene-derived light-sensitive co-factor. Accordingly,

bacteriorhodopsin, the prototype rhodopsin, has served as

a useful reporter of the assembly of lipid modification of

proteins. For more information on this and related top-

ics, the reader is directed to reviews by Lanyi (2004) and

Grote & O’Malley (2011).



Sampylation

SAMPs are small archaeal ubiquitin-like modifier proteins

that form isopeptide bonds to lysine residues of archaeal

proteins (Fig. 8). While this system was first demon-

strated in H. volcanii (Humbard et al., 2010a), it is

predicted to exist in all Archaea and appears related to

other recently discovered protein conjugation systems,

including the TtuBC system of protein conjugation in the

hyperthermophilic bacterium, Thermus thermophilus

(Humbard et al., 2010a; Shigi, 2012), and the Urm1 sys-

tem of protein conjugation and sulfur transfer in eukary-

otes (Wang et al., 2011). Sampylation is thought to be

reversible, based on conservation of JAMM (JAB1/MPN/

Mov34 metalloenzyme) domain homologs in all Archaea

(Humbard et al., 2010a; Maupin-Furlow, 2012). In

eukaryotes, JAMM domain proteins include the Rpn11/

Poh1 subunit of 26S proteasomes, required for removal

of ubiquitin chains from proteins (Verma et al., 2002;

Yao & Cohen, 2002). Likewise, Csn5/Jab1 is a JAMM

domain protein subunit of the COP9 signalosome com-

plex that cleaves the ubiquitin-like Nedd8 from proteins

(Cope et al., 2002). While archaeal JAMM domain pro-

tein function has yet to be demonstrated, the solved crys-

tal structure of an A. fulgidus JAMM domain protein

(termed AfJAMM) was used to model the structures of

Rpn11/Poh1 and Csn5/Jab1 (Tran et al., 2003; Ambroggio

et al., 2004).

Much like the ubiquitin and ubiquitin-like protein

modifiers of eukaryotes, SAMPs have a b-grasp fold

structure and C-terminal diglycine motif that is impor-

tant to their function in protein conjugation (Humbard

et al., 2010a; Ranjan et al., 2010; Jeong et al., 2011). The

a-carboxyl of the C-terminal glycine of SAMPs can form

an isopeptide bond to the e-amino group of lysine resi-

dues of target proteins by a mechanism that requires

UbaA, an ubiquitin-activating E1 enzyme homolog of

Archaea (Humbard et al., 2010a; Miranda et al., 2011).

The physiological reason for the formation of these

ubiquitin-like protein conjugates in Archaea (and hyper-

thermophilic bacteria) remains to be determined. How-

ever, in analogy to ubiquitin, sampylation is thought to

alter the structure, enzymatic activity, and types of part-

ners that would associate with the modified protein,

including proteasomes, transcription factors, and others

(Maupin-Furlow, 2012). Interestingly, SAMPs not only

form isopeptide bonds with protein targets but also are

required for sulfur transfer to biomolecules, including

molybdenum cofactor (MoCo) and tRNA (similar to

Urm1 of eukaryotes and TtuB of the bacterium T. ther-

mophilus) (Miranda et al., 2011; Shigi, 2012). Sampyla-

tion differs from the system of protein conjugation

recently predicted for the archaeon Candidatus Caldiar-

chaeum subterraneum (based on metagenomics) (Nunoura

et al., 2011) and some bacteria (based on analogy) (Burr-

oughs et al., 2011). This latter system is rare in Archaea

(restricted to Candidatus C. subterraneum) and appears

to incorporate not only ubiquitin-activating E1 homologs

but also ubiquitin-conjugating E2 and ubiquitin ligase E3

homologs (Nunoura et al., 2011).

-C-OH

ATP PPi

C-terminalα-carboxyl group

(Gly residue)

Acyl-adenylate

-C-AMP

Isopeptide-linkedprotein conjugate

Thioester

SAMP JAMM

AMP

UbaA

SAMPSAMP

SAMP

Sulfur transferpathways

=O =

O

UbaA

SH

Cys188sulfhydryl

groupSC=O N-H

C=O

UbaA

Substrateprotein

Substrateprotein

+NH3Lysε-aminogroup

Protease

-C-S-XSAMP

=

O

Sulfur containing biomolecules(e.g. MoCo, thiolated tRNA)

Specificityfactors fortargeting?

Fig. 8. Sampylation in Haloferax volcanii. The H. 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.



Protein degradation

Proteases are important in maintaining cellular homeosta-

sis, regulating cellular signaling, and degrading exogenous

proteins for cellular metabolism. In these processes, pro-

teases are needed to catalyze the general turnover of

proteins, remove aberrant proteins (e.g. damaged or

viral-encoded proteins), and mediate the precisely timed

cleavage or turnover of regulatory proteins important to

cell functions ranging from transcription and cell division

to metabolism (Gottesman, 2003). Similarly to other

archaea, H. volcanii is predicted to encode a wide variety

of endo- and exo-proteases, including energy-dependent

and intramembrane-cleaving proteases, typically linked to

the early events of protein degradation and regulation

(Maupin-Furlow et al., 2005; De Castro et al., 2006). Of

the energy-dependent proteases, proteasomes and mem-

brane-associated Lon B-type proteases appear relatively

universal in the Archaea (including H. volcanii) (Cha

et al., 2010; Maupin-Furlow, 2012).

Among the various regulatory proteases (i.e. excluding

signal peptidases) predicted for H. volcanii, only the pro-

teasomes have been studied at the biochemical level (Mau-

pin-Furlow, 2012). 20S proteasomes are purified from

H. volcanii in at least three distinct subtypes, composed of

four stacked heptameric rings of different subunit compo-

sition (i.e. a17b7b7a17, a17b7b7a27, and a27b7b7a27,where subscript represents subunit stoichiometry) (Wilson

et al., 1999; Kaczowka & Maupin-Furlow, 2003; Karadzic

et al., 2012). The 20S proteasomes are catalytically active

in cleaving peptides and small denatured proteins (i.e. oxi-

dized bovine insulin B-chain protein) (Wilson et al., 1999;

Kaczowka & Maupin-Furlow, 2003; Karadzic et al., 2012).

Haloferax volcanii PANs are purified as PAN-A and

PAN-B subclasses but were not found to be active in stim-

ulating the energy-dependent degradation of proteins by

20S proteasomes (based on in vitro assay) (Reuter et al.,

2004; Humbard et al., 2010b). The levels of the a2 subunit

of 20S proteasomes and PAN-B are correlated with growth

phase (i.e. low levels detected in lag to log phase and high

levels detected in stationary phase) suggesting that these

two proteins are associated in a biologically related path-

way (Reuter et al., 2004).

Proteasomes appear important in cell division, protein

quality control, and other functions in H. volcanii. Like

eukaryotes, 20S proteasomes of H. volcanii are essential,

based on the conditional lethal phenotypes associated

with deletion of the b gene or double deletion of the a1and a2 genes (Zhou et al., 2008). The PANs are not

essential (panA panB double mutants are viable) (Zhou

et al., 2008), while Cdc48d (one of four Cdc48 homologs

in H. volcanii) appears essential, based on the inability to

delete this gene from the chromosome (Allers et al.,

2010). PAN, Cdc48/p97/VAT, and other members of the

AAA ATPase superfamily are thought to form networks

of ATPases that associate with and regulate the function

of 20S proteasomes in archaeal cells (Barthelme & Sauer,

2012; Forouzan et al., 2012; Maupin-Furlow, 2012). Thus,

Cdc48d and 20S proteasomes may regulate functions of

cell division. Consistent with a role in protein quality

control, proteasomes (PAN-A and a1) are needed for

H. volcanii to tolerate stressful conditions, including

exposure to L-canavanine (an amino acid analogue that

causes protein unfolding) and low salt (Zhou et al.,

2008). In addition, a1 is needed to overcome thermal

stress (Zhou et al., 2008). Whether sampylation is linked

to proteasome-mediated proteolysis, in analogy to the

ubiquitin-proteasome system of eukaryotic cells, remains

to be determined. Levels of SAMP1-protein conjugates

are, however, higher in proteasome mutants, as compared

to wild-type cells, providing indirect evidence for a con-

nection between these two systems (Humbard et al.,

2010a). Still, ubaA (encoding the single ubiquitin-activat-

ing E1 homolog) can be deleted without loss of cell

viability, suggesting that sampylation is not required for

cell division in H. volcanii (Miranda et al., 2011).

Conclusions

Across evolution, proteins can experience any of a long list

of PTMs that lead to numerous and varied effects on pro-

tein structure, function, localization, oligomerization sta-

tus, and stability. As is the case for much of archaeal

biology, the various PTMs performed by Archaea both

present traits unique to this life form and others shared by

eukaryotes and/or Bacteria. As presented in Table 2, post-

Table 2. List of some PTMs reported in Haloferax volcanii

PTM Experimentally-verified target(s)

N-glycosylation S-layer glycoprotein (HVO_2072),

FlgA1 (HVO_1210)

O-glycosylation S-layer glycoprotein (HVO_2072)

X-glycosylation* 150, 105, 98, 58, 56, 54 and

52 kDa proteins

Lipid modification S-layer glycoprotein (HVO_2072)

Signal peptide cleavage S-layer glycoprotein (HVO_2072),

FlgA1 (HVO_1210), FlgA2 (HVO_1211)

Sampylation SAMP2 (HVO_0202), UbaA, homologs

of ribose-1,5-bisphosphate isomerase

(HVO_0966), DNA gyrase B subunit

(HVO_1572), cysteine hydrolyase

(HVO_2328), NADH-quinone

oxidoreductase chain c/d (HVO_0980),

OsmC (HVO_1289), TBPe (HVO_1727), and

tandem rhodanese domain protein

(HVO_0025)

*X-glycosylation, form of glycosylation undetermined.



translationally modified proteins abound in H. volcanii,

justifying the use of this species for the study of the

responsible processes in archaea. With an ever-growing list

of tools available for the genetic, proteomic, biochemical,

and physiological examination of H. volcanii, it is likely

that future studies on this organism, and indeed, on other

haloarchaea, will continue to provide novel insight into

PTMs in general, and in archaea, in particular.

Acknowledgements

J.E. is supported by grants from the Israel Science Foun-

dation (8/11) and the US Army Research Office

(W911NF-11-1-520). J.M.-F. is supported by grants from

NIH (R01 GM057498) and DOE (DE-FG02-05ER15650).

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posttranslation modification in archaea: lessons from haloferax

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