Posttranslation modification in Archaea: lessons from Haloferax

24 Pages·2013·0.62 MB·English

REVIEW ARTICLE
Post-translation modification in Archaea: lessons from
Haloferax volcanii
and other haloarchaea
Jerry Eichler1 & Julie Maupin-Furlow2,3
1DepartmentofLifeSciences,BenGurionUniversity,Beersheva,Israel;2DepartmentofMicrobiologyandCellScience,UniversityofFlorida,
Gainesville,FL,USA;and3GeneticsInstitute,UniversityofFlorida,Gainesville,FL,USA
Correspondence:JerryEichler,Department Abstract
ofLifeSciences,BenGurionUniversity,
As an ever-growing number of genome sequences appear, it is becoming
Beersheva84105,Israel.
Tel.:[phone];fax:[phone]; increasingly clear that factors other than genome sequence impart complexity
e-mail:[email protected] to the proteome. Of the various sources of proteomic variability, post-
translational modifications (PTMs) most greatly serve to expand the variety of
Received12July2012;revised13November proteins found in the cell. Likewise, modulating the rates at which different
2012;accepted13November2012.Final
proteins are degraded also results in a constantly changing cellular protein pro-
versionpublishedonline20December2012.
file. While both strategies for generating proteomic diversity are adopted by
organisms across evolution, the responsible pathways and enzymes in Archaea
DOI:10.1111/1574-6976.12012
are often less well described than are their eukaryotic and bacterial counter-
Editor:MeckyPohlschroder 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
Keywords developments concerning PTMs and protein degradation in the haloarchaea
Archaea;haloarchaea;Haloferaxvolcanii; are discussed.
post-translationalmodification;protein
degradation;proteome.
S
W variability.Intra-moleculardisulphidelinkages,formedvia
Introduction
the covalent bonding of Cys residues pairs, influence the
E The ability to address microorganisms and other life three-dimensional conformation of a protein. Disulphide
I
V forms at the level of the genome has revolutionized bio- bondsformedbetweendifferentproteinsubunitscanyield
E logical research. At the same time, it is becoming increas- multimeric complexes (Fass, 2011). Proteolytic processing
R ingly clear that a major proprortion of the diversity that likewise can allow for control of the folding and function
exists within a cell is generated at the level of the prote- of a target protein. For instance, the post-translational
Y
ome. Numerous factors are responsible for the proteome removaloftargetingsequencesandinteinsrespectivelyper-
G
assuming additional levels of complexity not predicted at mits the cell to control the site where a protein ultimately
O the genome level. These include alternative RNA splicing, resides, as well as the timing and manner in which a pro-
differential expression of a given protein in response to tein can act (Paulus, 2000; Gogarten et al., 2002; Jarvis &
L
environmental cues or as a function of developmental Robinson, 2004; Hegde & Bernstein, 2006). Specifically,
O
stage, and the plethora of possible protein–protein inter- any of these PTMs, either alone or in combination, can
I
B actions, as well as post-translational modifications affectproteinfunction,interactionofthemodifiedprotein
O (PTMs) and regulated protein degradation. with binding partners, protein localization, or the rate at
Proteins can be modified post-translationally by the whichaproteinisdegraded,amongothertraits.
R
permanent or temporary covalent attachment of one or At the same time, regulated protein degradation is crit-
C
more of several classes of molecules, including sugars, lip- ical for maintaining protein quality and controlling cell
MI ids,orotherchemicalgroups.Flexibilityinthespatialdis- functions. Proteases, which can discern and specifically
tributionofsuchlinkedmoietiesonatargetproteinandin degrade proteins compromised by denaturation, misin-
the timing of their addition, together with the ability to corporation of amino acids, and other damaging events,
introduce changes in the molecular composition of the are important for regulating cellular homeostasis. Like-
bound modifying groups, offers further sources of protein wise, regulated protein degradation processes, in which
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584 J.Eichler&J.Maupin-Furlow
the protease destroys key proteins that may be properly (DolP) serves as the lipid carrier of the glucose-linked
folded but must be removed at specific times or moved sulfated glycan decorating 10 sites of N-glycosylation,
to locations to enable molecular mechanisms to occur, whereas dolichol pyrophosphate (DolPP) bears the
are also important to cell function. Central to regulated repeating sulfated pentasaccharide N-acetylgalactosamine-
protein degradation are proteases that are coupled to linked to S-layer glycoprotein Asn-2 (Wieland et al.,
ATP hydrolysis (Gottesman, 2003). These energy-depen- 1980; Lechner and Wieland, 1989). A link between these
dent proteases have a self-compartmentalized structure, in phosphodolichol-charged glycans to N-glycosylation was
which the proteolytic active sites are housed within a pro- supported by the observation that in H. salinarum, the
tein nanoparticle that is chambered, gated, and linked to glycan moiety of the DolP-bound sulfated polysaccharide
an ATPase. The ATPase component of such proteases is is also detected on the S-layer glycoprotein and archael-
required for the unfolding of protein substrate, opening lins in this species (Lechner et al., 1985a; Wieland et al.,
of the gate, and facilitating the degradation process 1985). Moreover, the sulfated polysaccharide is methylat-
(Lupas et al., 1997; Maupin-Furlow, 2012). ed in the DolP-linked form but not when protein-bound
It is now clear that PTMs and regulated protein degra- (Lechner et al., 1985b). The significance of this obser-
dation transpire in all three domains of life, namely Euk- vation remains unclear. Finally, studies showing the
arya, Bacteria, and Archaea. Still, current understanding ability of H. salinarum cells to modify cell-impermeable,
of the archaeal versions of these processes lags behind sequon-bearing hexapeptides with sulfated oligosaccha-
that of their eukaryal and bacterial counterparts. In the rides localized the N-glycosylation event to the external
following, we review what is known of PTM and regu- cell surface (Lechner et al., 1985a).
lated protein degradation in the halophilic archaea, lar- Despite these advances, the process of N-glycosylation
gely focusing on Haloferax volcanii. With the availability in the haloarchaea is currently best understood in H. vol-
of a complete genome sequence (Hartman et al., 2010), canii. The S-layer glycoprotein, comprising the sole com-
simple growth requirements and advanced genetic, molec- ponent of the S-layer, contains seven putative N-
ular biology, proteomic and biochemical tools and tech- glycosylationsites,namelythemotifAsn-X-Ser/Thr,where
niques (DasSarma & Fleishmann, 1995; Allers et al., 2004, X is any residue but Pro. Early studies reported modifica-
2010; Soppa, 2006; Kirkland et al., 2008b; Dyall-Smith, tion of the S-layer glycoprotein Asn-13 and Asn-498
2009), H. volcanii represents a strain of choice for molec- positions by a linear string of glucose residues, whereas
ular studies in Archaea and has provided considerable Asn-274 and/or Asn-279 were supposedly decorated by a
insight into the archaeal versions of these protein process- glycan containing glucose, galactose, and idose (Sumper
ing events. et al.,1990;Mengele&Sumper,1992).Morerecently,evi-
dence for N-glycosylation of the H. volcanii archaellins,
FlgA1 and FlgA2, was presented (Tripepi et al., 2010,
N-glycosylation
2012). Likewise, currently unidentified H. volcanii glyco-
N-glycosylation, the covalent linkage of glycan moieties to proteinsof 150,105, 98, 58,56, 54, and 52 kDahavebeen
select Asn residues of a target protein, was among the detected (Zhu et al., 1995; Eichler, 2000), although some
first haloarchaeal PTMs to be described. The Halobacteri- of these species may correspond to the same polypeptide.
um salinarum surface (S)-layer glycoprotein was the first Moreover, it remains to be determined whether these
noneukaryotic protein shown to be N-glycosylated proteins indeed experience N-glycosylation rather than
(Mescher & Strominger, 1976). Two different Asn-linked O-glycosylation. The same is true of LccA, a glycosylated
oligosaccharides modify the S-layer glycoprotein, namely laccase secreted by H. volcanii (Uthandi et al., 2010). The
a repeating sulfated pentasaccharide linked via H. volcanii S-layer glycoprotein, containing both N- and
N-acetylgalactosamine to Asn-2 and a sulfated glycan O-linked glycans, thus remains the best-characterized
linked by a glucose residue to ten other Asn residues glycoproteininthisspecies(Sumperet al.,1990).
(Mescher & Strominger, 1978; Lechner et al., 1985a; In the last few years, substantial progress in decipher-
Lechner and Wieland, 1989; Wieland et al., 1980, 1983). ing the pathway of S-layer glycoprotein N-glycosylation
The H. salinarum flagellin [since renamed archaellin has been made, with the identification of a series of
(Jarrell & Albers, 2012)] was shown to bear the same gly- archaeal glycosylation (agl) genes encoding proteins
can-linked sulfated polysaccharide (Wieland et al., 1985). involved in the assembly and attachment of a pentasac-
Although applying genetics to identify components of the charide to select Asn residues of the S-layer glycoprotein.
H. salinarum N-glycosylation pathway was not possible at Acting at the cytoplasmic face of the plasma membrane,
the time, biochemical approaches served to reveal various AglJ, AglG, AglI, and AglE sequentially add the first four
aspect of the N-glycosylation pathway of this haloar- pentasaccharide residues (i.e. a hexose, two hexuronic
chaeon. As such, it was shown that dolichol phosphate acids and the methyl ester of a hexuronic acid) onto
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Proteinmodificationinthehaloarchaea 585
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 Fig.1. N-glycosylation in Haloferax volcanii. The H.volcanii S-layer
requiring the archaeal oligosaccharide transferase, AglB glycoprotein, a reporter of N-glycosylation in this species, is modified
atAsn-13andAsn-83byapentasaccharidecomprisingahexose,two
(Abu-Qarn & Eichler, 2006; Chaban et al., 2006; Igura
hexuronic acids, the methyl ester of a hexuronic acid, and a
et al., 2008), the lipid-linked tetrasaccharide and its pre-
mannose. The first four subunits of the pentasaccharide are
cursors are delivered to select Asn residues of the S-layer
sequentially assembled onto a DolP carrier via the activities of the
glycoprotein. The final mannose residue is subsequently glycosyltransferases, AglJ, AglG, AglI, and AglE. At the same time,
transferred from its DolP carrier to the protein-bound AglDaddsthefinalpentasaccharideresidue,mannose,ontoadistinct
tetrasaccharide (Guan et al., 2010) in a reaction requiring DolP. Both charged DolP carriers are reoriented to face the cell
AglR, a protein that either serves as the DolP-mannose exterior,withAglRthoughttoserveastheDolP-mannoseflippaseor
to contribute to such activity. AglB acts to transfer the DolP-bound
flippase or contributes to such activity (Kaminski et al.,
tetrasaccharide (and its precursors) to select Asn residues of target
2012), and AglS, a DolP-mannose mannosyltransferase
proteins, such as the S-layer glycoprotein. The finalmannose subunit
(Cohen-Rosenzweig et al., 2012). Current understanding
is then transferred to the protein-bound tetrasaccharide. AglF, AglM,
of H. volcanii N-glycosylation is depicted in Fig. 1. and AglP play various sugar-processing roles in the pathway. In the
Insight gained from N-glycosylation in H. volcanii has figure, DolP is presented as a vertical line, while hexoses are
served to elucidate aspects of the parallel process in other presented as red circles, hexuronic acids are presented as yellow
halophilic archaea. It was initially shown that H. volcanii squaresandmannoseispresentedasagreencircle.
strains lacking either aglD or aglJ could be functionally
complemented by introduction of rrnAC1873 and canii N-glycosylation pathway components can be
rrnAC0149, the respective homologues of these genes replaced with homologues from other haloarchaea to
from Haloarcula marismortui (Calo et al., 2010b, 2011). yield N-glycan variants has provided a proof-of-concept
Like H. volcanii, the haloarchaeon H. marismortui also for developing H. volcanii in a glyco-engineering platform
originates from the Dead Sea (Oren et al., 1990). Indeed, designed to produce tailored glycoproteins (Calo et al.,
subsequent efforts revealed the S-layer glycoprotein of 2011). Such efforts will also exploit the proven ability of
both species is decorated with N-linked pentasaccharides H. volcanii to N-glycosylate introduced nonnative pro-
comprising a hexose, two hexuronic acids, a methyl ester teins (Kandiba et al., 2012).
of hexuronic acid, and a mannose (Calo et al., 2011). The H. volcanii N-glycosylation pathway, involving
Still, differences in the N-glycosylation pathways of these multiple glycan-charged DolP carriers, recalls the parallel
two haloarchaea exist. While in H. volcanii the N-linked eukaryal process. In higher Eukarya, the first seven
pentasaccharide is derived from a tetrasaccharide sequen- subunits of the 14-meric oligosaccharide assembled in the
tially assembled on a single DolP and a final mannose endoplasmic reticulum are sequentially added to a com-
residue derived from a distinct DolP carrier (Guan et al., mon phosphodolichol carrier, whereas the second set of
2010), a similar pentasaccharide N-linked to the seven sugar subunits are derived from single mannose- or
H. marismortui S-layer glycoprotein is first fully assem- glucose-charged DolP (Burda & Aebi, 1999; Helenius &
bled on a single DolP and only then transferred to the Aebi, 2004; Hartley & Imperiali, 2012). The H. volcanii
protein target (Calo et al., 2011). The finding that H. vol- N-glycosylation pathway further resembles its eukaryal
FEMSMicrobiolRev37(2013)583–606 ª2012FederationofEuropeanMicrobiologicalSocieties
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586 J.Eichler&J.Maupin-Furlow
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 Fig.2. The Haloferax volcanii S-layer glycoprotein undergoes
which a heptasaccharide is assembled by the sequential differential N-glycosylation as a function of environmental salinity.
MassspectrometrywasusedtorevealthatwhenH.volcaniicellsare
addition of seven soluble nucleotide-activated sugars onto
grown in 3.4M NaCl-containing medium, Asn-13 and Asn-83 are
a common undecaprenol phosphate carrier (Szymanski &
modified by the pentasaccharide portrayed in Fig.1. In the
Wren, 2005; Weerapana & Imperiali, 2006; Abu-Qarn
conditions, Asn-370 and Asn-498 are not modified. When, however,
et al., 2008a). Yet, although delivered to the lipid-linked the cells are grown at lower salt concentrations (i.e. in medium
rather than the protein-bound tetrasaccharide, the termi- containing1.75MNaCl),S-layerglycoproteinAsn-498ismodifiedby
nal mannose subunit of the pentasaccharide N-linked to a ‘low-salt’ tetrasaccharide comprising a sulfated hexose, two
the H. marismortui S-layer glycoprotein is derived from a hexoses,andarhamnose.Atthesametime,Asn-13andAsn-83are
still modified by the pentasaccharide described above, albeit much
distinct DolP carrier, as in H. volcanii (Calo et al., 2011).
less so. Asn-370 is still not modified. The N-glycosylation status of
Although the absence or even the perturbation of
Asn-274, Asn-279, and Asn-732 was not considered. In the figure,
N-glycosylation compromises the ability of H. volcanii to
hexosesarepresentedasredcircles,hexuronicacidsarepresentedas
grow in high salt (Abu-Qarn et al., 2007) and modifies yellowsquares,mannoseispresentedasagreencircle,andrhamnose
S-layer stability and architecture (Abu-Qarn et al., 2007), is presented asa blue circle. Positions where no glycosylation is seen
as well as S-layer resistance to added protease (Yurist- areindicatedby‘9’.
Doutsch et al., 2008, 2010; Kaminski et al., 2010), cells
lacking AglB, and hence unable to perform N-glycosyla- tions (Fig. 2). Hence, in response to environmental salin-
tion, are viable (Abu-Qarn et al., 2007). As such, it would ity, H. volcanii not only modulates the N-linked glycans
seem that this PTM is not essential for H. volcanii sur- decorating the S-layer glycoprotein but also residues sub-
vival, yet nonetheless is advantageous to H. volcanii in jected to this PTM.
certain scenarios. Thus, one can hypothesize that H. vol- Finally, it should be noted that studies on the metha-
canii modifies aspects of N-glycosylation in response to nogens, Methanococcus voltae and Methanococcus marip-
changing growth conditions. This concept has gained aludis, and the thermophiles, Sulfolobus acidocaldarius,
support from recent studies comparing N-glycosylation of Pyrococcus furiosus, and Archaeoglobus fulgidus, have also
the S-layer glycoprotein in cells grown in 3.4 or 1.75 M provided insight into archaeal N-glycosylation (Chaban
NaCl-containing medium (Guan et al., 2012). At the et al., 2006; Igura et al., 2008; VanDyke et al., 2009;
higher salinity, S-layer glycoproteins Asn-13 and Asn-83 Meyer et al., 2011; Jones et al., 2012; Matsumoto et al.,
were shown to be modified by the pentasaccharide 2012).
described above, while DolP was shown to be modified
by the tetrasaccharide comprising the first four pentasac-
Phosphorylation
charide residues, again as discussed above. However, cells
grown at low salinity contain DolP modified by a distinct Phosphorylation is widely appreciated as a covalent form
tetrasaccharide comprising a sulfated hexose, two hexoses, of PTM that occurs at His, Asp, Ser, Thr, or Tyr residues.
and a rhamnose not seen linked to DolP in cells grown at Phosphorylation is rapid, reversible and generates confor-
high salinity. This is likely the same DolP-bound tetrasac- mational changes in protein structure that mediate an
charide observed by Kuntz et al. (1997) in H. volcanii array of biological responses from signal transduction to
cells grown in 1.25 M NaCl-containing medium. The metabolism (Johnson & Barford, 1993). While early stud-
same tetrasaccharide modified S-layer glycoprotein Asn- ies suggested that Archaea (and Bacteria) use mainly two-
498 in cells grown in low salt, whereas no glycan deco- component systems of His/Asp phosphorylation, it is now
rated this residue in cells grown in the high-salt medium. appreciated that Archaea (and Bacteria) also perform Ser/
At the same time, Asn-13 and Asn-83 were modified by Thr/Tyr phosphorylation (once thought to be restricted to
substantially less pentasaccharide at the low-salt condi- eukaryotes) for creating highly sophisticated regulatory
ª2012FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolRev37(2013)583–606
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Proteinmodificationinthehaloarchaea 587
networks (Macek et al., 2008). A number of reviews and phospho-sitesinH. volcaniiand81phospho-sitesinH. sali-
genomicsurveysareavailablethathighlightthephosphor- narum),thusincreasingthenumberofphospho-sites(from
ylation of archaeal proteins and the enzymes (protein kin- three total) previously detected in Archaea (Kirkland et al.,
ases/phosphatases) likely to mediate and/or regulate this 2008a; Aivaliotis et al., 2009). In these high-throughput
PTM (Leonard et al., 1998; Kennelly & Potts, 1999; Ken- approaches, wild-type and mutant strains with enhanced
nelly, 2003; Eichler & Adams, 2005; Tyagi et al., 2010). In levels of phosphoproteins [i.e. H. volcanii ΔpanA (protea-
addition, a Phosphorylation Site Database is available some-activating nucleotidase A) and H. salinarum ΔserB
online that provides a guide to some of the Ser/Thr/Tyr- (OE4405Rphosphoserinephosphatase)]wereusedasinput
phosphorylated proteins in Archaea (Wurgler-Murphy material. Phosphopeptides were enriched from samples by
et al., 2004). Thus, the discussion below highlights recent immobilizedmetalaffinitychromatographyandmetaloxide
studies on protein phosphorylation in halophilic archaea affinity chromatography (in parallel and sequentially), fol-
andhowthistypeofPTMmaycontrolcellularfunction. lowed by tandem mass spectrometry (MS/MS). While a
phospho-site consensusmotif wasnot apparent, themajor-
ityofsitesmappedtoSer/Thr/Tyrresidues.
Phospho-site mapping
Historically, halophilic archaea have provided a useful
Protein kinases/phosphatases
modelsystemtoadvanceourunderstandingofhowproteins
can be phosphorylated across domains of life. In fact, the Halophilic archaea are predicted to encode histidine pro-
demonstrationthatproteinsofH. salinarumwerereversibly tein kinases and protein phosphatases of the ‘two-compo-
phosphorylated by a light-regulated retinal-dependent nent’ Asp/His phosphorelay system (Koretke et al., 2000;
mechanism was the first report that proteins in Archaea Kim & Forst, 2001) (e.g. H. volcanii is predicted to
could be phosphorylated (Spudich & Stoeckenius, 1980). encode at least 30 histidine protein kinases). The best
Lateruseofmolecularbiologytoolsrevealedatwo-compo- studied example of an archaeal Asp/His phospho-relay
nentHis/Aspphosphorelaysystem(analogoustotheCheA/ system is that of H. salinarum, in which a CheA histidine
CheYsystemofBacteria)thatwasresponsibleformodulat- protein kinase undergoes ATP-dependent autophosphory-
ingtheresponseofH. salinarumtochemotacticandphoto- lation of a His residue and transfers the phosphoryl
tactic stimuli (Rudolph & Oesterhelt, 1995; Rudolph et al., group to an Asp residue on CheY, a response regulator
1995) (Fig. 3). Early study of H. volcanii revealed a Mn2+- thought to be dephosphorylated by the protein phospha-
stimulated protein phosphatase activity against synthetic tase, CheC (and not CheZ) (Rudolph & Oesterhelt, 1995;
protein substrates phosphorylated at Ser/Thr residues Rudolph et al., 1995; Muff & Ordal, 2007; Streif et al.,
(Oxenrider & Kennelly, 1993). More recent studies using 2010). Ultimately, the phosphorylation status of CheY
high-throughput methods have facilitated the mapping of impacts the ability of this protein to switch the flagellar
phosphorylation sites on proteins of halophilic archaea (9 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
SR Htr CheWCheA et al., 1995) and Bacteria (Min et al., 1993; Yang et al.,
P
ccw 1996; Shi et al., 1999; Wu et al., 1999). Whether or not
+ CH3 –CH3 CheB P CheY P this alternative type of phosphorylation also occurs in
Signal CheR SAM Flagellar cw Archaea is yet unknown.
motor
Flagella Many halophilic archaea (including H. volcanii) as well
as the crenarchaeon Thermofilum pendens harbor homo-
logs of the phosphoenolpyruvate phosphotransferase sys-
Fig.3. Protein modification in Halobacterium salinarum taxis. Htrs
tem (PTS) (Hartman et al., 2010) (Fig. 4). In analogy to
aresolubleormembrane-boundcomplexesthatassociatewithsignal
receptors (SRs). Htrs signal to a two-component regulatory system what is known for Bacteria (Barabote & Saier, 2005), the
composed of an autophosphorylating histidine kinase CheA, which H. volcanii PTS is predicted to mediate transfer of
mediates phosphotransfer to CheY, the response regulator of the the phosphoryl group on PEP to imported sugars (e.g.
system.CheYtargetstheflagellarmotorandregulatestheswitchfor fructose, galacticol) or endogenous dihydroxyacetone via
flagellar rotation [clockwise (CW) vs. counterclockwise (CCW)].
dihydroxyacetone kinase (Hartman et al., 2010). Halofe-
Adaptation is promoted by the methylation status of conserved Glu
rax volcanii PTS homologs include a single enzyme I
and Gln residues of Htr, where CheB deamidates Htr Gln residues
priortoO-methylesterification.HtrismethylatedbyCheR(+CH )and (PtsI; His~P) and multiple copies of histidine protein
demethylated by CheB ((cid:1)CH ). CheA-mediated phosphory3lation (HPr; His~P), enzyme IIA (EIIA; His~P), enzyme IIB (EIIB;
3
regulatesthedemethylationactivityofCheB. Cys~P), and enzyme IIC (EIIC; His~P), with the amino acid
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588 J.Eichler&J.Maupin-Furlow
Rio1p purifies as a monomer and can transfer the
c-phosphoryl group of ATP to a1, a protein that forms
Sugar
the outer rings of 20S proteasomes in H. volcanii
Sugar -P
(Humbard et al., 2010b). Rio1p-mediated phosphotrans-
pyruvate EI~P HPr1,2,3 EIIA1,2 ~P fer is not observed and/or is diminished for a1 variants
PEP EI P~HPr1,2,3 EIIA1,2 EIIB1,2 EIIC1,2 Ta1s5u8bAs,trSa5te8Ap,raontdeinT1(4a71A). TathuSse,rR/Tioh1rprceasnidupehsosbpahsoerdylaotne
P~HPr3 HPr3 in vitro assay.
P~ DhaM(EIIA) Homologs of the four subunits of the eukaryotic
KEOPS complex are conserved in Archaea, with
P~ DhaL H. volcanii-encoding homologs of Pcc1 (HVO_0652) and
P~ DhaK Cgi121 (HVO_0013) and a fusion of the Bud32 Ser/Thr
in CM out
protein kinase to Kae1 (HVO_1895). The gene encoding
DHA DHAP
the Bud32-Kae1 homolog and its subdomains appear
Fig.4. PTS of Haloferax volcanii. A schematic diagram of the essential in H. volcanii (Naor et al., 2012), suggesting
H.volcanii phosphotransferase (PTS) system predicted to be Bud32-mediated phosphorylation of Ser/Thr residues is
responsible for responsible for the simultaneous transport and important for cell function. Homologs of KEOPS (Bud32,
phosphorylationofsugar substrates (e.g.fructose andgalacticol)and Kae1, Cgi121 and Pcc1) from related Euryarchaeota
for the generation of dihydroyacetone phosphate (DHAP) from
(Methanocaldococcus jannaschii and P. furiosus) have been
dihydroxyacetone (DHA) by DHA kinase. A series of enzyme
used for reconstitution of the complex, structural analysis
intermediates, including EI, HPr, EIIA, EIIB, EIIC, and DHA kinase
at the atomic level, heterologous complementation, and
(DhaM,L,K),arepredictedtobephosphorylated.
in vitro phosphorylation assays (Hecker et al., 2008; Mao
residue predicted to be phosphorylated during group et al., 2008). From this work, Bud32 is suggested to phos-
translocation provided in parenthesis. Recent work dem- phorylate a Thr residue of an insert within the catalytic
onstrates that the PTS gene cluster HVO_1495 to cleft of Kae1 and, thus, regulate KEOPS function.
HVO_1499, encoding PtsI, EIIB, HPr, EIIA, and EIIC Whether or not Bud32 phosphorylates other proteins in
homologs, was highly upregulated as a cotranscript dur- Archaea besides Kae1 is not clear. Both yeast Bud32 and
ing growth on fructose (Pickl et al., 2012). Deletion of its human ortholog, p53-related protein kinase (PRPK),
HVO_1499, encoding a homolog of the fructose-specific can phosphorylate p53 (Abe et al., 2001; Facchin et al.,
membrane component EIIC of this cluster, resulted in 2003). Likewise, Sulfolobus solfataricus SsoPK5 (a Bud32
loss of growth on fructose compared to glucose (Pickl homolog) catalyzes the phosphorylation of various
et al., 2012). Thus, the PTS system has a functional proteins in vitro (Haile & Kennelly, 2011).
involvement in the metabolism of fructose in H. volcanii. In addition to the atypical RIO-type Ser/Thr/Tyr pro-
Atypical RIO-type Ser/Thr protein kinase homologs of tein kinases, homologs of the Bacillus subtilis PrkA (Uni-
the type 1, type 2, and Bud32 (piD261) families are com- Prot P39134) are common among halophilic archaea
mon among the Archaea (including the haloarchaea) (e.g. H. volcanii HVO_2849 and HVO_2848). Members
(Leonard et al., 1998; Shi et al., 1998; Ponting et al., of the PrkA family possess a Walker A-motif of nucleo-
1999; LaRonde-LeBlanc & Wlodawer, 2005a, b; Tyagi tide-binding proteins and exhibit distant homology to
et al., 2010). Protein kinases of the RIO-type 1 family are eukaryotic protein kinases (Fischer et al., 1996). In addi-
distinguished by an STGKEA consensus sequence in their tion, amino acid residues within the active site of cyclic
N-terminal domain and a second region of homology adenosine 3′, 5′-monophosphate (cAMP)-dependent pro-
(IDXXQ, where X represents any amino acid residue) in tein kinase are also conserved in PrkA (Fischer et al.,
their C-terminal domain. Kinases of the RIO-type 2 1996). B. subtilis PrkA can phosphorylate a 60-kDa pro-
family often have an N-terminal helix-turn-helix motif tein at a Ser residue (Fischer et al., 1996). However, fur-
followed by GXGKES and C-terminal IDFPQ sequences. ther analysis is needed to determine the identity of this
Members of the Bud32 family of Ser/Thr protein kinases protein substrate and confirm that B. subtilis PrkA, and
are associated with the KEOPS (ECK) complex, com- its haloarchaeal relatives are Ser/Thr protein kinases.
posed of three additional subunits (Kae1, Pcc1, and
Cgi121), and shown to be required for formation of the
Phosphorylation of proteasomes
tRNA modification threonylcarbamoyladenosine (t6A) in
yeast (Srinivasan et al., 2011). Proteasomes are self-compartmentalized proteases,
A RIO-type 1 homolog (Rio1p, HVO_0135) of H. vol- which undergo a substantial number of post-/co-transla-
canii has been characterized at the biochemical level. tional modifications, including phosphorylation. The
ª2012FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolRev37(2013)583–606
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Proteinmodificationinthehaloarchaea 589
phosphorylation of these complexes is of interest, because Phosphorylation of the a-type subunits of H. volcanii
proteasomes are important for cell function (e.g. growth proteasomes has been investigated at various stages of cell
of H. volcanii) (Zhou et al., 2008). Proteasomes are com- growth and assembly states. Humbard et al. (2010b) sepa-
posed of a 20S catalytic core particle (of a- and b-type rated the a-type subunits (of cell lysate and 20S proteo-
subunits) and regulatory particles, including AAA+ ATP- somes purified by affinity chromatography with b
ases (homologs of Cdc48/VAT/p97 and Rpt subunits subunits) by two-dimensional gel electrophoresis (2DE)
termed proteasome-associated nucleotidases or PANs) and detected a1 and a2 by immunoblot using polyclonal
that mediate energy-dependent protein degradation (Bart- antibodies specific to each protein. Phosphorylated iso-
helme & Sauer, 2012; Maupin-Furlow, 2012). In H. volca- forms (two specific for a1 and one specific for a2) were
nii, proteasomes are modified by phosphorylation in determined by shifting the 2DE-protein spot to a more
addition to Na-acetylation, methyl-esterification, and basic pI after removal of the acidic phosphate groups by
cleavage of b subunit precursors that expose the phosphatase treatment. Of the two phosphorylated iso-
N-terminal threonine residue forming the active sites of forms of a1 detected, the most acidic form was found
20S proteasomes(Table 1)(Wilson et al., 1999;Humbard throughout growth, assembled in 20S proteasomes. In
et al.,2006,2010b).Eukaryotic20Sproteasomesandassoci- contrast, the least phosphorylated isoform of a1 was pres-
atedAAA+ATPases(homologsofPANtermedRpt1-6and ent as both unassembled and assembled subunits of 20S
Cdc48/VAT/p97) are also altered by phosphorylation and proteasomes and was detected at reduced levels in later
other forms of covalent modification, such as the attach- stages of growth (Humbard et al., 2010b). The phosphor-
mentofO-linkedN-acetylglucosamine,Ne-andNa-acety- ylated isoform of a2 was also found associated with 20S
lation, N-myristoylation, and cleavage of b subunit proteasomes. Thus, phosphorylation of the a-type pro-
precursors(Zhanget al.,2007;Ewenset al.,2010). teins is suggested to influence their assembly and/or be
Haloferax volcanii 20S proteasomes and associated important for 20S proteasome function.
PANs are phosphorylated at Ser and Thr residues. To Site-directed mutagenesis has been used to determine
facilitate phospho-site mapping, 20S proteasomes and the biological role of proteasome phosphorylation in
PAN proteins were purified from H. volcanii by tandem H. volcanii. Haloferax volcanii strains expressing a1 pro-
affinity chromatography using His6- and StrepII-tags teins with Ala modifications in Ser/Thr residues likely to
(Humbard et al., 2006, 2010b). Sites of phosphorylation be phosphorylated (based on MS analysis and an inability
were identified by MS/MS (with precursor ion scanning) to accept a phosphoryl group from Rio1p, in vitro) dis-
and included b Ser129, a1 Thr147, and a2 Thr13/Ser14 play dominant negative phenotypes for cell viability and
(not distinguished) of 20S proteasomes and Ser340 of colony color (i.e. white vs. red) (Humbard et al., 2010b).
PAN-A (Humbard et al., 2006, 2010b). A phospho-site Thus, phosphorylation of the a1 subunit of 20S protea-
for PAN-B was not identified (Humbard et al., 2010b). somes appears to be closely linked to cell growth and pig-
MS/MS analysis of phosphopeptides enriched from mentation (i.e. production of carotenoids) (Humbard
H. volcanii proteomes suggests Cdc48/VAT/p97 homologs et al., 2010b). Interestingly, H. volcanii proteasomal
are also phosphorylated; however, sites of modification mutant strains deficient in the synthesis of PAN-A display
were not identified by this high-throughput method a striking increase in the number of cellular proteins that
(Kirkland et al., 2008a). In eukaryotes, phosphorylation are phosphorylated, suggesting an added link between
and acetylation regulate the function of Cdc48/VAT/p97 protein phosphorylation and proteasome function (e.g.
proteins. phosphorylation may target proteins for destruction by
energy-dependent proteases, in analogy to eukaryotes and
Bacteria) (Kirkland et al., 2008a).
Table1. PTMsofHaloferaxvolcaniiproteasomes
Methyl- Na- Exposedby Protein acetylation
Subunit Phosphorylated esterified acetylated autocleavage
Protein acetylation is the covalent attachment of an acetyl
a1 Thr147 Asp20,Glu27, Met1 n.d.
group to a protein. In general, acetylation can impact
Glu62,Glu112,
protein function, stability, and interactions with other
Glu161
a2 Thr13/Ser14 n.d. Met1 n.d. molecules. Two types of protein acetylation are known to
b Ser129 n.d. n.d. Thr50 occur in living cells, Na-acetylation and Ne (or lysine)-
PAN-A Ser340 n.d. n.d. n.d. acetylation, with high-energy molecules, such as acetyl-
CoA, providing the acetyl group for these modifications.
Residue number according to protein sequence in GenBank [GI:30
0669661 (a1), GI:12229945 (a2), GI:292655712 (b), GI:302425218 Na-Acetylation is an irreversible mechanism in which an
(PAN-A)]. acetyl group is covalently attached to the a-amino group
FEMSMicrobiolRev37(2013)583–606 ª2012FederationofEuropeanMicrobiologicalSocieties
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590 J.Eichler&J.Maupin-Furlow
of the N-terminal amino acid of a protein. In contrast, MAP cleaved (n = 143)
Nα-acetylated (n = 72)
Nreesi-dauceetyilsatrieovnerosicbcluyrsmwohdeifinetdhebye-tahmeincoovgarleonutpaottfaachlmyseinnet TK FQL NIWEFL K Q
V D A D
of an acetyl group. Studies on H. volcanii have furthered A
our understanding of both processes in Archaea. T
S
P
Na-acetylation V S
Our current understanding of the prevalence of Na-acety- M G
lation in Archaea is largely based on MS-based proteomic
surveys (Soppa, 2010; Maupin-Furlow et al., 2012). Like MD MQ
Bacteria, Na-acetylation of ribosomal proteins appears MN
common among Archaea (Kimura et al., 1989; Hatakey- MV
ama & Hatakeyama, 1990; Klussmann et al., 1993; Mar- MG MS
MP
quez et al., 2011). Interestingly, the number of proteins MT
reported to be Na-acetylated varies greatly among the dif- MA
ferent archaeal groups. In haloarchaea (apparently unlike
Bacteria), a relatively high proportion (14–29%) of the Fig.5. Haloferax volcanii N-terminal proteome modified by
proteome is modified by Na-acetylation (i.e. H. salina- 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/
rum, Natronomonas pharaonis, and H. volcanii) (Falb
MS (Kirkland etal., 2008b). Based on this proteomic analysis,
et al., 2006; Aivaliotis et al., 2007; Kirkland et al., 2008b).
H.volcanii proteins are often cleaved by MAP and/or Na-acetylated.
Likewise, Na-acetylation appears to impact a large per-
PenultimateresiduesexposedbyMAPareoftensmallanduncharged
centage of the S. solfataricus proteome, based on the find- (Gly,Ala,Pro,Val,Ser,orThr).OftheN-terminithatareacetylated,a
ing of 17 Na-acetylated N-termini of 26 total detected by relatively equal divide exists between proteins Na-acetylated at their
MS (Mackay et al., 2007). Interestingly, to date, only a N-terminal Met vs. a residue exposed after MAP cleavage (with
single protein (the a subunit of the 20S proteasome) is Na-acetylation of exposed Ser and Ala residues common). Among
reported to be Na-acetylated among the methanogens, proteins with Na-acetylated N-terminal Met residues, most (over
80%) have the Met residue followed by a small, uncharged residue
suggesting that this form of modification may be rare in
(Gly,Ala,Pro,Val,Ser,orThr)typicallycleavedbyMAP.
this group of Archaea (Forbes et al., 2004; Zhu et al.,
2004; Enoki et al., 2011). cleaved form (103 : 1 ratio), in H. volcanii (Humbard
In H. volcanii, like other Archaea, many of the et al., 2009). Whether or not Na-acetylation efficiency is
Na-acetylated proteins appear to be generated by a NatA- also near 100% for other H. volcanii protein substrates
type activity [i.e. acetylation of penultimate Ser or Ala and/or can be altered by growth conditions remains to be
residues exposed after removal of N-terminal methionine determined.
residues by methionine aminopeptidase (MAP)] (Fig. 5). While proteins with Na-acetylated Met-Gln and Met-
Na-Acetylated N-terminal methionine residues with pen- Asn sequences are not prevalent in Archaea, 20S protea-
ultimate Asp and Asn residues are also detected, consis- some a-type subunits with these N-terminal sequences are
tent with the NatB-like activity of eukaryotes (Kirkland specifically Na-acetylated in the Euryarchaeota, H. salina-
et al., 2008b). Proteins with a Na-acetylated N-terminal rum, N. pharaonis, H. volcanii, and Methanothermobacter
methionine residue followed by a small penultimate resi- thermoautotrophicus (Falb et al., 2006; Humbard et al.,
due (Ser, Ala, Thr, Pro, and Gly) are also detected, sug- 2006; Aivaliotis et al., 2007; Enoki et al., 2011). Site-direc-
gesting that Na-acetylation can restrict their cleavage by ted mutagenesis of the N-terminal Met-Gln sequence of
MAP. Interestingly, many of the proteins of halophilic ar- the H. volcanii 20S proteasome a1 protein has been used
chaea (including those of H. volcanii) that are Na-acety- to further investigate this specific Na-acetylation (Hum-
lated are also readily identified by semi-quantitative MS bard et al., 2009). Variants of a1 were expressed in vivo
spectral counting in unmodified and/or MAP-cleaved and analyzed by MS to detect alterations in N-terminal
forms (Falb et al., 2006; Aivaliotis et al., 2007; Kirkland modification (i.e. Na-acetylation, MAP cleavage). A Q2A
et al., 2008b). Liquid chromatography-multiple reaction substitution rendered the a1 protein susceptible to cleav-
monitoring (LC-MRM) MS, a technique that provides a age by MAP followed by Na-acetylation of the penulti-
more accurate perspective on the abundance of the mate Ala by an apparent NatA-type activity similarly to
Na-acetylated state of a protein, reveals that the a1 most Na-acetylated proteins in haloarchaea. However,
proteins that form 20S proteasomes are primarily in an the N-termini of a1 proteins with the small penultimate
Na-acetylated Met form, as compared to the MAP amino acid residues Ser and Val were detected
ª2012FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolRev37(2013)583–606
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Proteinmodificationinthehaloarchaea 591
predominantly in the uncleaved forms, with example, proteins modified by Na-acetylation are often
Na-acetylated methionine residues intact. Alteration of over-represented in protein abundance profiles and
penultimate amino acid residues to Asp, Pro, and Thr comprise a high proportion of the proteome (Falb et al.,
resulted in a mixture of a1 protein in the Na-acetylated 2006; Aivaliotis et al., 2007; Kirkland et al., 2008b;
methionine,MAPcleavedand/orunmodifiedforms.Thus, Martinez et al., 2008). Furthermore, Na-acetylation
the enzyme(s) responsible for Na-acetylation of the a1 blocks the Na-amino group of a protein from further
N-terminal Met appears to have relaxed sequence specific- modification by destabilizing processes such as ‘linear’
itywithregard tothepenultimateresidueofthesubstrate. ubiquitylation (Meinnel et al., 2005). However, recent
Furthermore, only the Q2A substitution rendered the evidence suggests Na-acetylation can mark a protein for
N-terminus of a1 fully susceptible to MAP cleavage, sug- destruction by the ubiquitin-proteasome system using a
gesting that structural elements of a1 or interacting part- mechanism named the Ac/N-end rule (Hwang et al.,
ners/chaperones may mask primary N-terminal sequences 2010). The Ac/N-end rule is based on the finding that an
that are otherwise optimal for MAP cleavage (e.g. Met-Ser E3 ubiquitin ligase (named Doa10) can recognize proteins
of the a1 Q2S variant). Interestingly, Na-acetylation of a1 with acetylated N-termini and facilitate their ubiquityla-
Met appears important in gating the 20S proteasome, tion (at internal lysine residues) and degradation by pro-
based on the enhanced peptidase activity of 20S protea- teasomes in yeast (Hwang et al., 2010). To rationalize this
somes with the MAP-cleaved a1 Q2A variant and the discrepancy between Na-acetylation in protein stability
inabilityofthegeneencodingthea1Q2Avarianttocom- and degradation, Hwang et al. (2010) provide a model
plement the a1 mutation for growth at ‘low’ salt in which nascent proteins can ‘hide’ their acetylated
((cid:3) 1.3 M NaCl). Insight into archaeal enzyme(s) that N-termini by rapid folding, interaction with chaperones,
mediate Na-acetylation is provided through comparative and/or assembly into appropriate multi-subunit com-
genomics, in vitro reconstitution of Na-acetyltransferase plexes. Such sequestration of N-termini would render the
activityandcrystallography.MembersoftheGNATsuper- Na-acetylation-based degradation signals inaccessible for
family that use acetyl-CoAs to acylate their cognate recognition by the Doa10 E3 ubiquitin ligase, ultimately
substrates, including the Na-acetylation of proteins, are stabilizing the protein. In contrast, delayed or defective
widely distributed among Archaea (Vetting et al., 2005). protein folding would expose acetylated N-termini and
Sulfolobus solfataricus ssArd1 (SSO0209) is an archaeal allow for Doa10-dependent ubiquitylation and proteolysis
GNATrelatedtoNa-acetyltransferases thathas beendem- by proteasomes.
onstrated to acetylate the N-terminal residue (Ser) of the Insight into a potential archaeal Ac/N-end rule pathway
DNA-binding protein, Alba (Mackay et al., 2007). Much is provided by study of the a1 protein of 20S protea-
like the eukaryal Ard1 of NatA, SsArd1 preferentially somes in H. volcanii (Humbard et al., 2009; Varshavsky,
acetylates N-terminal Ser and Ala residues exposed after 2011). Here, the identity of the N-terminal penultimate
methionine removal (Mackay et al., 2007). SsArd1 also residue of a1 was found to dramatically alter the concen-
catalyzes appreciable Na-acetylation of N-terminal tration of a1 protein in the cell. In particular, the
Met-Glu and Met-Leu sequences, similar to Nat3 of NatB levels of N-terminal a1 variants that were partially non-
in eukaryotes (Mackay et al., 2007). While ssArd1 can Na-acetylated were remarkably higher than the levels of
Na-acetylate a variety of proteins in vitro, it shows prefer- Na-acetylated (wild type) a1 protein. Furthermore, most
ence for proteins with N-termini that are disordered in of the a1 proteins associated in 20S proteasomes had
crystal structures (Mackay et al., 2007). Thus, it is unclear acetylated N-termini. Thus, proteasomal partners are
whetherssArd1functionspost-orco-translationallyinthe predicted to obstruct recognition of the acetylated
cell. The archaeal ssArd1 is thought to represent an ances- N-terminal domain of a1 and prevent its proteolytic
tral form of some eukaryal Na-acetyltransferases based on destruction by an Ac/N-end rule pathway.
its relaxed sequence specificity. Recent crystallography of
SsArd1 now provides structural detail for analysis of this
Ne-acetylation
ancestralfunction(Okeet al.,2010).
The reversible and differential Ne-acetylation of proteins
can have a major impact on transcription, translation,
Na-acetylation and protein stability
stress response, detoxification, and carbohydrate and
Based on analogy to eukaryotes, Na-acetylation is energy metabolism (Hu et al., 2010; Jones & O’Connor,
predicted to regulate protein turnover in H. volcanii and 2011; Thao & Escalante-Semerena, 2011). In eukaryotes,
other haloarchaea. The long-held argument that histones are well known to be modified by Ne-acetyla-
Na-acetylation stabilizes a protein comes from several tion, in addition to Na-acetylation, methylation,
lines of indirect evidence (Meinnel et al., 2006). For phosphorylation, ubiquitylation, ADP ribosylation,
FEMSMicrobiolRev37(2013)583–606 ª2012FederationofEuropeanMicrobiologicalSocieties
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592 J.Eichler&J.Maupin-Furlow
glycosylation, and sumoylation (Shiio & Eisenman, 2003). unit of the elongator complex possessing acetyltransferase
Numerous bacterial proteins are also differentially Ne- activity, Sir2 is a class III HDAC homolog similar to
acetylated, with ‘K-acetylomes’ (subproteomes composed S. solfataricus Sir2, and HdaI is a class II HDAC homolog
of proteins with Ne-acetylated lysine residues) thought to related to yeast Hda1. Single deletion of sir2, pat1, pat2,
rival phosphoproteomes (Aka et al., 2011). In contrast, or elp3 genes or double deletion of pat1 with either pat2
Ne-acetylation of archaeal proteins is poorly understood. or elp3 was found to have no detectable impact on the
In Archaea, only a few proteins are known to be viability of H. volcanii cells. In contrast, hdaI appeared
Ne-acetylated. A couple of early studies, focused on deter- essential, based on the finding that the gene could only
mining the amino acid sequence of 2Fe–2S ferredoxins be deleted when a wild-type copy of hdaI was provided in
from the haloarchaea H. salinarum and H. marismortui, trans. Attempts to create an elp3 deletion in any of the
revealed that a lysine residue near the C-terminus of these pat2 null strains were unsuccessful, implying that these
proteins is conserved and Ne-acetylated (Hase et al., two mutations are synthetically lethal and affect a single
1978, 1980). Another archaeal protein that is Ne-acety- function or pathway (Altman-Price & Mevarech, 2009).
lated is Alba, a chromatin protein of S. solfataricus. Alba Thus, Elp3- and Pat2-mediated acetylation and HdaI-
is not only Na-acetylated on its N-terminal Ser (as dis- mediated deacetylation of lysine residues are predicted to
cussed above) but is also Ne-acetylated on lysine 16 (Bell be important in H. volcanii.
et al., 2002). Acetylation of Alba Lys16 is mediated by the
protein acetyltransferase, Pat (Marsh et al., 2005). Pat is a
Hypusine modification
homolog of Salomonella Pat (YfiQ) and a member of the
family of NDP-forming acetyl-CoA synthetase enzymes Hypusine [Ne-(4-amino-2-hydroxybutyl)-L-lysine] is
with GNAT domains (Starai & Escalante-Semerena, formed upon PTM of a conserved lysine residue and
2004). Alba Lys16 can be deacetylated by an NAD+- is found only in eukaryotic translation ‘initiation’ factor
dependent histone deacetylase (HDAC) class III homolog 5A (eIF5A) and the related archaeal aIF5A (Park et al.,
of S. solfataricus (Sir2) (Bell et al., 2002). Ne-Acetylation 1981, 2010; Schumann & Klink, 1989; Bartig et al., 1992).
of Alba reduces its binding affinity for DNA and RNA Hypusine is essential for the activity of eIF5A/aIF5A
and strongly prevents its ability to inhibit the DNA heli- (including that of H. salinarum), now considered impor-
case activity of the mini-chromosome maintenance pro- tant in translation elongation (Wagner & Klug, 2007;
tein (Bell et al., 2002; Jelinska et al., 2005; Marsh et al., Sainiet al.,2009;Parket al.,2010).Inhypusinemodifica-
2006). Thus, Ne-acetylation of Alba is thought to have a tion of eIF5A, deoxyhypusine synthase (DHS) transfers a
global impact on chromatin packaging and gene expres- 4-aminobutyl moiety from spermidine to the e-amino
sion in Crenarchaeota, such as S. solfataricus (Wardle- group of the conserved lysine residue to form a dexoxy-
worth et al., 2002; Zhao et al., 2003). hypusine intermediate, which is hydroxylated to a hypu-
Haloferax volcanii has served as a model for under- sine residue by deoxyhypusine hydroxylase (DOHH)
standing the importance of histone acetyltransferase (Fig. 6). In Archaea, DHS homologs are widespread,
(HAT) and HDAC gene homologs to archaeal cell func- suggesting the lysine residue of aIF5A is modified to a
tion. In particular, gene homologs for three HATs (Pat1, deoxyhypusine residue by an enzyme similar to eukaryotic
HVO_1756; Pat2, HVO_1821; Elp3, HVO_2888) and two DHS. In contrast, DOHH is an oxygen-dependent
HDACs (Sir2, HVO_2194; HdaI, HVO_0522) were tar- enzyme,anditshomologsarerarein(theoftenanaerobic)
geted for deletion from the H. volcanii genome (Altman- Archaea. Thus, generation of the hypusine-modified form
Price & Mevarech, 2009). Pat1 and Pat2 are related to the of aIF5A from its deoxyhypusine precursor likely involves
S. solfataricus Pat, Elp3 is related to the yeast Elp3 sub- asecondenzymedistinctfromDOHHinArchaea.
Deoxyhypusine Hypusine
H2N H2N
NH –OH Fig.6. HypusinemodificationofeIF5A.
2 NH NH
1,3-diaminopropane HypusineisauniversalmodificationinArchaea
andeukaryotesonasingletypeofprotein
Lys DHS Lys DOHH Lys (eIF5A)byasequentialseriesofenzyme
eIF5A eIF5A eIF5A reactions.DHStransfersa4-aminobutyl
O H O moietyfromspermidinetothee-aminogroup
2 2
ofaspecificlysineresidueoneIF5A.DOHH
donor-H acceptor
2 hydroxylatesthemodifiedlysinetoform
Spermidine
hypusine.
ª2012FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolRev37(2013)583–606
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