Posttranslational Protein Modification in Archaea

34 Pages·2005·0.34 MB·English

MICROBIOLOGYANDMOLECULARBIOLOGYREVIEWS,Sept.2005,p.393–425 Vol.69,No.3
1092-2172/05/$08.00(cid:1)0 doi:10.1128/MMBR.69.3.393–425.2005
Copyright©2005,AmericanSocietyforMicrobiology.AllRightsReserved.
Posttranslational Protein Modification in Archaea
Jerry Eichler1* and Michael W. W. Adams2
DepartmentofLifeSciences,BenGurionUniversity,Beersheva,84105Israel,1andDepartment
ofBiochemistryandMolecularBiology,UniversityofGeorgia,Athens,Georgia306022
INTRODUCTION.......................................................................................................................................................394
PROTEINGLYCOSYLATION.................................................................................................................................395
GlycosylatedArchaealProteins............................................................................................................................395
S-layerglycoproteins..........................................................................................................................................395
(i)S-layerglycoproteinsrevealuniqueaspectsofarchaealproteinglycosylation................................395
DDD
Flagellins..............................................................................................................................................................397
ooo
(i)Evidenceforflagellinglycosylation.........................................................................................................397 www
Otherproteins.....................................................................................................................................................397 nnn
ProcessofProteinN-GlycosylationinArchaea...................................................................................................398 lololo
aaa
Dolicholcarrier...................................................................................................................................................399 ddd
(i)Antibioticsthataffectdolicholprocessinginterferewitharchaealproteinglycosylation..............399 eee
ddd
(ii)Analysisofdolichol-boundglcyans........................................................................................................399
fff
EnzymesofN-glycosylation...............................................................................................................................399 rororo
(i)Genomicstudies........................................................................................................................................399 mmm
(ii)Biochemicalstudies.................................................................................................................................400 h h h
Subcellularlocalizationofglycosylation..........................................................................................................401 tttttt
ppp
RoleofProteinGlycosylationinArchaea............................................................................................................401 :::
///
Structuralroles...................................................................................................................................................401 /m/m/m
Functionalroles..................................................................................................................................................401 mmm
Glycosylationasanenvironmentaladaptation..............................................................................................401 bbb
LIPIDMODIFICATION............................................................................................................................................402 rrr
...
MembraneLipidsofArchaea................................................................................................................................402 aaa
sss
Lipid-ModifiedArchaealProteins........................................................................................................................402 mmm
Lipoproteins.........................................................................................................................................................402 .o.o.o
Isoprenylatedproteins........................................................................................................................................404 rrr
ggg
Acylatedproteins.................................................................................................................................................404 ///

ooo
GPI-anchoredproteins.......................................................................................................................................404
nnn
PROTEINPHOSPHORYLATION...........................................................................................................................404
JJJ
TargetsandFunctionsofProteinPhosphorylationinArchaea.......................................................................405 aaa
nnn
Phosphorylationofcomponentsinvolvedinsignaltransduction................................................................405
uuu
PhosphorylationofcomponentsinvolvedinDNAreplication,cellcycleregulation,andtranslation...405 aaa
rrr
Phosphorylationofotherproteins....................................................................................................................406 yyy

ArchaealProteinKinasesandPhosphatases.....................................................................................................406 444
,,,
Eucaryalproteinkinases...................................................................................................................................406
222
Histidinekinases.................................................................................................................................................406 000
111
Proteinserine/threoninephosphatases............................................................................................................407
999
Proteintyrosinephosphatases..........................................................................................................................407
bbb
ProteinkinasesandphosphatasesofThermoplasmaacidophilum................................................................407 yyy

PROTEINMETHYLATION......................................................................................................................................407 ggg
uuu
ProteinMethylationinResponsetoExternalStimuli......................................................................................407 eee
MethylationofMethyl-CoenzymeMReductase.................................................................................................408 sss
ttt
MethylatedProteinsinThermophilicArchaea...................................................................................................408
MethylationofArchaealDNA-BindingProteins................................................................................................409
MethylationofArchaealRibosomalProteins.....................................................................................................409
DISULFIDEBONDSINPROTEINS......................................................................................................................409
DisulfideBondsinCytoplasmicArchaealProteins...........................................................................................409
DisulfideBondsinExtracellularArchaealProteins..........................................................................................410
EnzymesInvolvedinDisulfideBondFormationinArchaea............................................................................410
PROTEOLYTICALLYPROCESSEDPROTEINS.................................................................................................411
ArchaealSignalSequences....................................................................................................................................411
ProteintranslocationinArchaea......................................................................................................................411
*Corresponding author. Mailing address: Dept. of Life Sciences,
BenGurionUniversity,P.O.Box653,Beersheva84105,Israel.Phone:
97286461343.Fax:[phone].E-mail:[email protected].
393
394 EICHLER AND ADAMS MICROBIOL.MOL.BIOL.REV.
Genomicsurveysofarchaealsignalsequences..............................................................................................411
Removalofarchaealsignalsequences.............................................................................................................412
Amino-TerminalMethionineRemoval.................................................................................................................413
InteinsinArchaealProteins.................................................................................................................................413
Carboxy-TerminalMaturationofArchaeal[NiFe]Hydrogenases..................................................................414
OTHERPOSTTRANSLATIONALMODIFICATIONSINARCHAEA...............................................................414
ProteinAcetylation.................................................................................................................................................414
ProteinUbiquitination...........................................................................................................................................414
Hypusine-ContainingArchaealProtein...............................................................................................................415
PROTEOME-WIDEANALYSISOFPOSTTRANSLATIONALMODIFICATIONSINARCHAEA...............415
CONCLUSIONS.........................................................................................................................................................415
ACKNOWLEDGMENTS...........................................................................................................................................416
REFERENCES............................................................................................................................................................416
D
INTRODUCTION ificationsandtheireffectsonproteinchemistryandcellbiology o
become even broader when one also considers the effects of w
With complete genome sequences appearing at an ever n
more rapid rate, attention is becoming increasingly directed additional,secondaryposttranslationalmodificationstepssuch lo
as the addition of organic (e.g., flavins) or inorganic (e.g., a
towards describing the protein complement of a given organ- d
metal groups) cofactors. Such modifications, however, lie be-
e
ism,i.e.,theproteome.Studiesofproteinsconductedbothat
yondthescopeofthisreview. d
the level of the individual polypeptide and cellwide have re- Long-knowntobewidespreadinEucaryaandBacteria,itis fr
vealed that the repertoire of expressed proteins can expand o
becomingclearthatposttranslationalmodificationofproteins m
beyond what is predicted by direct translation of the comple-
also takes place in Archaea. Best known in their capacities as
h
mentofopenreadingframescontainedwithinagenome.For
extremophiles,i.e.,microorganismsabletothriveintheharsh- tt
example, the proteome can assume additional levels of com- p
est environmental conditions on this planet, Archaea express :
plexity with differential expression of individual polypeptides //
proteinsthatenablethemtosucceedinsuchhabitats.Indeed, m
ormembersofproteinfamiliesasafunctionofdevelopmental
archaealproteinsareabletoremainproperlyfoldedandfunc- m
stage or in response to environmental cues. The various per- tional in the face of extremes of salinity, temperature, and b
mutations of protein-protein interactions possible further ex- otheradversephysicalconditionsthatwouldnormallyleadto r.a
pand the complexity of the proteome. However, one of the s
protein denaturation, loss of solubility, and aggregation. Al- m
most important and fundamental aspects of proteomic com-
thoughposttranslationalmodificationsmayhelparchaealpro- .
plexity comes from the various processing events that many o
teinsovercomethechallengespresentedbytheirsurroundings, r
proteins experience following their synthesis, i.e., posttransla- g
inmostcases,thereasonforposttranslationalmodificationof /
tionalmodification. o
aparticulararchaealproteinremainsunclear.Table1liststhe n
Proteinscanbemodifiedposttranslationallybycovalentat-
posttranslationalmodificationsthatarchaealproteinsmayex- J
tachmentofoneormoreofseveralclassesofmolecules,bythe a
perience.
formation of intra- or intermolecular linkages, by proteolytic n
Analysisofthevariousposttranslationalmodificationsexpe- u
processing of the newly synthesized polypeptide chain, or by a
riencedbyarchaealproteinshasservedtorevealnotonlynovel r
anycombinationoftheseevents.Bychemicallylinkingvarious y
protein modifications not previously observed in Eucarya or
modifying groups either permanently or temporarily and by 4
Bacteria but also variations of previously characterized post- ,
allowingforchangesinthemolecularcompositionofthemod- 2
translational modifications. By and large, however, archaeal 0
ifying moieties, covalent modifications can endow proteins 1
with properties that are very different from those that are 9

b
predicted by the encoding genes. Examples of such covalent
y
modifications include glycosylation, lipid attachment, phos- TABLE 1. Posttranslationalmodificationsofarchaealproteins g
phorylation,andmethylation. Posttranslational Comment ue
ThecovalentbondingofpairsofCysresiduestoformdisul- modification s
t
fidebridgesnotonlymodulatesthethree-dimensionalconfor-
Glycosylation N-glycosylation,O-glycosylation
mationofapolypeptidechainbutcanalsobeusedtomaintain Lipidmodification Lipoproteins,isoprenylation,acylation,
proteinsinmultisubunitcomplexes.Controlledreductionand GPIanchoring
reoxidationofproteindisulfidebondsisalsoemployedinelec- Phosphorylation Phosphoaspartate,phosphohistidine,
phosphoserine,phosphothreonine,
trontransferreactionsfundamentaltomanycellularprocesses.
phosphotyrosine
Proteolyticprocessingofnewlysynthesizedpolypeptidechains Disulfidebonds Cytosolicproteins
similarlyallowsthecelltocontrolthefoldingandfunctionof Proteolyticprocessing Signalsequencecleavage,intein
a protein. By removing specific targeting sequences or other excision,amino-terminalandcarboxy-
terminalmaturation
stretches of amino acid residues, the cell is able to control
Methylation Methylarginine,methylasparticacid,
where,when,andhowaproteinwillact.Assuch,posttransla-
methylcysteine,methylglutamicacid,
tional modifications can significantly modulate the physico- methylglutamine,methylhistidine,
chemicalandbiologicalpropertiesofaproteinthrougheffects methyllysine
on protein function, subcellular localization, oligomerization, Acetylation
Aminoacidmodification Hypusination,thiolation
folding,orturnover.Thedistributionofposttranslationalmod-
VOL.69,2005 ARCHAEAL POSTTRANSLATIONAL MODIFICATIONS 395
posttranslational modifications often resemble their eucaryal TABLE 2. Archaealspeciesreportedtocontainglycoproteins
or bacterial counterparts. Hence, elucidating such similarities
Evidencefor
provides insight into evolutionary relationships across the Species glycosylationa Reference(s)
threedomainsoflife.Moreover,themosaicprofileofeucaryal,
Haloarculajaponica G 299
bacterial, and archaeal traits that describes posttranslational
Haloarculamarismortui C 136
protein modification in Archaea also holds true when one ex- Halobacteriumsaccharovorum E 389
aminestheenzymesandmechanisticstepsinvolvedinarchaeal Halobacteriumsalinarum A,B 246,280
protein modification processes. Here too, examination of ar- Haloferaxmediterranei E 232
Haloferaxvolcanii A,B,D 98,421
chaeal systems has served to expand our understanding of
Methanobacteriumbryantii D,F 219
natural pathways or to underscore the similarities between Methanococcusdeltae F,H 27
archaeal, eucaryal, and/or bacterial biology. Nonetheless, nu- Methanococcusmazei D 481
merous aspects of archaeal posttranslational processing re- Methanococcusvoltae A,B 453
main poorly described. In the following review, what is cur- Methanosaetasoehngenii A 340
Methanospirillumhungatei E,F 406
rently known of posttranslational protein modification in D
Methanothermusfervidus A,B,E 196,204
o
Archaeaisconsidered. Methanothermussociablis A,B 41 w
Natrialbamagadii E 197 n
Pyrococcusfuriosus C,D,E 44,230,231, lo
PROTEINGLYCOSYLATION 455,464 a
d
Sulfolobusacidocaldarius A,B,E,G,H 146,147,161, e
Oneofthemoreprevalentposttranslationalmodifications 258,286 d
experienced by eucaryal proteins is glycosylation. Indeed, Sulfolobusshibatae E 112 f
r
protein glycosylation, which begins in the lumen of the en- Sulfolobussolfataricus D,E 106,146,262 o
Staphylothermusmarinus C,F 345 m
doplasmic reticulum and continues in the Golgi apparatus,
Thermococcuslitoralis E 44,145 h
is thought to be experienced by more than half of all euca-
Thermococcusstetteri E 184 tt
ryalproteins(12).Upontranslocationintotheendoplasmic Thermoplasmaacidophilum A,B 478 p
:
reticulum, proteins can be N-glycosylated, when branched Thermoplasmavolcanium E 112 //
m
oligosaccharide trees of 14 subunits are initially added to
aA, saccharide-amino acid linkage determined; B, glycan structure deter- m
selected Asn residues. O-glycosylation of Ser or Thr resi- mined;C,saccharidecontentdetermined;D,lectinbinding;E,glycanstaining;F, b
duesusuallytakesplaceintheGolgi.InEucarya,theglycan deglycosylation;G,inhibitionofglycosylation;H,aberrantSDS-PAGEmigra- r.
tion. Where A and/or B is true for a given glycoprotein, other evidence for a
moieties of glycosylated proteins fulfill a multitude of roles glycosylationisnotlistedforthatcase.Seetextfordetails. s
m
related to protein solubility, folding, stability and turnover,
.
and subcellular localization as well as participating in nu- o
r
merous recognition events (46, 157, 333, 409, 448). Long g
/
believed to be an exclusively eucaryal trait, it is now clear (345). Although the experimental evidence for glycosylation, o
n
thatbothBacteriaandArchaeaarealsocapableofattaching ranging from chemical characterization of the bound glcyan
J
glycanmoietiestoselectedproteins(285,292,381,425,445, moieties to glycol staining, is stronger in some cases than a
456). A list of those archaeal strains reported to contain others, it has been calculated that these S-layer glycoproteins n
u
glycosylated proteins is provided in Table 2. experience an overall degree of glycosylation of up to 15% a
r
(292,382). y

Like eucaryal glycoproteins, archaeal S-layer glycoproteins 4
GlycosylatedArchaealProteins ,
canundergobothN-andO-glycosylation.Incontrast,bacterial 2
0
S-layerglycoproteins.Thesurface(S)-layerglycoproteinof S-layerglycoproteinscontainonlyO-linkedglycans(285,445), 1
thehalophilicarchaeonHalobacteriumsalinarumwasthefirst although examples of N-glycosylation of other bacterial pro- 9

b
prokaryotic glycoprotein to be described in detail (246, 283). teinshavebeenshown(107,425,456).Analysisofthecompo-
y
Subsequently, S-layer glycoproteins have been studied in nu- sition of the N-linked glycan moieties of archaeal S-layer gly- g
merousprokaryotes(292,381–383).Servingasthemain,ifnot coproteins has revealed the wide variety of saccharides u
e
sole, component of the protein layer surrounding many ar- available for protein glycosylation in Archaea, including ga- s
t
chaeal cells (101, 382, 383) (Fig. 1), S-layer glycoproteins re- lactofuranose, galactouronic acid, glucose, glucuronic acid,
mainamongthebest-characterizedarchaealglycoproteins.In- iduronic acid, mannose, N-acetylgalactosamine, N-acetylglu-
deed, examination of the processes used for glycosylation of cosamine, and rhamnose (204, 280, 335, 421, 456). In many
archaealS-layerglycoproteinshasnotonlyservedtoenhance cases, these sugar subunits may themselves be modified by
our understanding of prokaryotic cell surface biogenesis but methylationorsulfation.Suchdiversityintherangeofsaccha-
has also provided insight into the general phenomenon of ridesusedinarchaealS-layerglycoproteinN-glycosylationex-
proteinglycosylationinArchaea. ceeds that seen in the bacterial and eucaryal N-glycosylation
While the glycosylated nature of S-layer proteins has been processes(425,456).
proposedinmanyarchaealspecies,experimentalproofforthis (i) S-layer glycoproteins reveal unique aspects of archaeal
posttranslational modification is limited to the S-layer glyco- protein glycosylation. Despite the proposed evolution of the
proteins of Halobacterium salinarum (246), Haloferax volcanii eucaryal N-glycosylation system from a precursor process in
(421), Haloarcula japonica (299), Methanothermus fervidus Archaea(46,157),studiesofarchaealS-layerglycoproteingly-
(204), Methanothermus sociablis (41), Sulfolobus spp. (146), cosylation,andinparticularglycosylationoftheHalobacterium
and components of the S-layer of Staphylothermus marinus salinarum S-layer glycoprotein, have revealed differences in
396 EICHLER AND ADAMS MICROBIOL.MOL.BIOL.REV.
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
m
m
b
r
.
FIG. 1. SchematicdepictionoftheglycosylationoftheHalobacteriumsalinarumS-layerglycoprotein.ThetopologyoftheS-layerglycoprotein, a
thepositionsofthe11AsnresiduesthatundergoN-glycosylation,andtheheavilyO-glycosylatedThr-richregionbetweenThr-755andThr-779 s
m
areindicated(246).Theinsetshowsthecompositionofthethreeoligosaccharidemoietiesboundtotheprotein(247).Abbreviationsused:G,
.
glucose; GA, glucaronic acid; Gal, galactose; GalA, galacturonic acid; Galf, galactofuranose; GalN, N-acetylgalactosamine; GN, N-acetylglu- o
r
cosamine;OMe,O-methyl;SO ,sulfate.Approximatelyathirdoftheglucaronicacidresiduesmaybereplacedbyiduronicacid. g
4
/

o
n

J
a
N-glycosylation in the two domains. Such differences are re- transferasesinthisspecies.Atpresent,itremainsunclearhow n
u
flected,forexample,inthefailurethusfartodetectantennary the cell would distinguish between the different N-glycosyla- a
r
structures in Archaea similar to those employed in eucaryal tionsites. y

proteinN-glycosylation(46,157,235,333,409,442),orinthe Finally,thelinkageofglycanmoietiestotheHalobacterium 4
,
identified amino acid sequence motifs recognized by the ar- salinarum S-layer glycoprotein at selected Asn residues 2
0
chaealN-glycosylationmachinery. through either N-acetylgalactosamine or glucose subunits 1
It was observed that replacement of the Ser residue of the (335)isincontrasttotheN-acetylglucosaminelinkagelargely 9
Asn-2-Ala-3-Ser-4 sequence of the Halobacterium salinarum b
employed in eucaryal N-glycosylation (46, 157, 235, 333, 409, y
S-layer glycoprotein with Val, Leu, or Asn did not prevent 442). In the case of the eucaryal protein laminin, however, g
N-glycosylation at the Asn-2 position (486). By contrast, the N-glycosylation involves a (cid:2)-glucosyl-Asn protein linkage ue
eucaryal system almost invariably recognizes the Asn-X-Ser/ s
(385). It is of note that laminin is a component of the extra- t
Thrsequencemotif,whereXisanyresidueapartfromPro(46,
cellular basement membrane, a structural layer surrounding
157,235,333,409,442),althougharareexceptionofN-glyco-
mammalian cells in a manner reminiscent of the archaeal
sylationatanAsn-Gly-Gly-Thrmotifhasbeenreported(211).
S-layer.
The ability of Archaea to glycosylate proteins at Asn residues
In addition to N-glycosylation, archaeal S-layer glycopro-
that are not part of the consensus Asn-X-Ser/Thr motif sug-
teinscanalsobemodifiedbyO-glycosylationofselectedSeror
gests that predictions of the glycosylation status of archaeal
Thr residues. In both Halobacterium salinarum and Haloferax
proteinsmayhaveoverlookedsimilarornovelN-glycosylation
volcanii,Thr-richregionsadjacenttothemembrane-spanning
sites. Moreover, the finding that the repeating sulfated pen-
tasaccharide moiety attached at the Asn-2 position of the domain of the protein are decorated at numerous positions
Halobacterium salinarum S-layer glycoprotein through an N- withgalactose-glucosedisaccharides(283,421).Interestingly,a
acetylgalactosamine link is chemically distinct from the sul- glycoprotein isolated from a eucaryal basement membrane
fated polysaccharide unit attached via glucose subunits found containsasimilardisaccharide(254).Presently,littleisknown
at the other ten N-glycosylation sites in the S-layer glycopro- ofthestepsinvolvedinarchaealO-glycosylationortherelation
tein (247) implies the existence of two different N-saccharyl- ofsuchstepstotheparalleleucaryalorbacterialprocesses.
VOL.69,2005 ARCHAEAL POSTTRANSLATIONAL MODIFICATIONS 397
Flagellins.InArchaea,cellmotilitymediatedbyflagellahas antibioticthatinterfereswithproteinglycosylation(seebelow)
been reported for representatives of the major phenotypic (247).Suchtreatmentresultedinmorerapidmigrationofthe
groups, i.e., the halophiles, the methanogens, the thermo- protein as reflected by SDS-PAGE analysis (27). Similar bac-
philes, and the hyperthermophiles, largely based on micro- itracin treatment, however, had no effect on the glycosylation
scopic investigation (20, 184, 436). Although fulfilling similar of Halobacterium salinarum flagellins, as gauged by migration
roles,archaealflagellabearlittleresemblancetotheirbetter- in SDS-PAGE, although incubation with EDTA, thought to
characterizedbacterialcounterparts(7,265)intermsofstruc- specificallyinhibitanexternallyorientedMg2(cid:1)-dependentoli-
ture or assembly. Such differences become evident when one gosaccharidetransferase (420), successfully modified flagellin
considerstheflagellarfilamentinthetwodomains.Ultrastruc- migration. By contrast, treating cells with EDTA did lead to
tural studies have shown that, unlike bacterial filaments, ar- the appearance of Methanococcus deltae flagellins of lower
chaeal flagellar filaments are not hollow structures (72) and apparentmolecularweight(27).Together,theseobservations
that the archaeal structures are generally thinner than their pointtodifferencesintheglycosylationmachineriesofthetwo
bacterialcounterparts(79,185,190,406). species.
Archaeal and bacterial flagella also differ at the level of Other proteins. While the bulk of attention on archaeal D
o
flagellin, the major structural component of the flagellar fila- proteinglycosylationhasfocusedonS-layerglycoproteinsand w
ment. Whereas bacterial flagella are, for the most part, com- flagellins, other archaeal glycoproteins have been identified. n
posed of a single type of flagellin, archaeal flagellar filaments Ofthoseadditionalglycoproteinswhoseidentitiesareknown, lo
a
are made up of several types of flagellins (with the possible the majority are membrane associated. In many instances, d
e
exceptionofSulfolobussolfataricus,wheregenomeannotation these are binding proteins involved in nutrient uptake (see
d
efforts have reported the existence of only a single flagellin- below),suchasthemaltose/trehalose-bindingproteinsofTher- f
r
encoding gene) (20, 184, 436). Indeed, archaeal and bacterial mococcuslitoralis,showntoreactwithglyco-stain(145)andof o
m
flagellins do not share sequence similarity (19). Moreover, Pyrococcus furiosus, shown to contain glucose-containing gly-

h
many archaeal flagellins are glycosylated (184), a posttransla- canmoietiesbylectinbindingandmolecularanalysis(231),or
t
t
tionalmodificationthatisconsideredrareforbacterialflagel- the Pyrococcus furiosus cellobiose-binding protein, which re- p
:
lins(95,139,291,384,435). acts with lectins and glyco-stain (230). Glyco-staining also in- //
m
(i) Evidence for flagellin glycosylation. Glycosylation has dicated the glycosylated nature of Pyrococcus furiosus CipA
m
beenreportedforflagellinsofnumerousarchaealstrains(112, and CipB, two ABC transporter binding proteins whose ex- b
184, 196, 197, 389, 436), including Halobacterium salinarum pression is up-regulated in response to cold shock in this hy- r.
a
(470),Methanococcusdeltae(27),Methanococcusvoltae(453), perthermophile (464). Glycosylation of pyrolysin, a thermo- s
m
and Methanospirillum hungatei (406). In most of these exam- stableserine-proteasealsoassociatedwithPyrococcusfuriosus
.
ples, the evidence for glycosylation comes from studies em- membranes, was proposed on the basis of sequence analysis o
r
ployingglycan-detectingstains,suchasthymol-sulfuricacidor that revealed the presence of numerous potential N-glycosyl- g
/
periodic acid-Schiff reagent. Such techniques, however, may ationsitesandsupportedbyglyco-stainingoftheprotein(455). o
n
notalwaysaccuratelyreflecttheglycosylatednatureofapro- Basedonlectinbinding,aseriesofglycosylatedsugar-bind-

J
tein (222). Hence, additional evidence for glycosylation is ing proteins, apparently containing mannose, glucose, galac- a
desirable. tose, and N-acetylglucosamine, was detected in Sulfolobus n
u
This has been achieved for the flagellins of Halobacterium solfataricus membranes (106). Sulfolobus acidocaldarius cyto- a
r
salinarum and Methanococcus voltae, for which the chemical chromeb wasshowntobeahighlyglycosylatedintegral y
558/566
compositions of the covalently linked glycan moieties have membrane protein, containing both O-linked mannose sub- 4
,

been elucidated. The Halobacterium salinarum flagellin con- unitsandN-linkedhexasaccharides(161).Analysisofthecom- 2
0
tains a sulfated glycoconjugate, N-linked through a glucose position of the latter glycan moiety revealed the presence of 1
bridgeandbasedonglucuronicoriduronicacid,similartothe glucose, mannose, and N-acetylglucosamine in addition to 9

b
glycan moiety found on the S-layer glycoprotein (420, 468). 6-sulfoquinovose (484). 6-Sulfoquinovose (or 6-deoxy-6-sulfo-
y
More recently, Methanococcus voltae flagellins have been glucose) is a rare acidic sugar, commonly found in the glyco- g
showntocontainanovelN-linkedtrisaccharide(453),despite lipids of chloroplasts and photosynthetic bacteria (177), but u
e
the fact that earlier glycoprotein staining-based studies had notpreviouslyfoundinaglycoprotein.Theglycosylatedchar- s
t
failed to detect flagellar glycosylation in this species (195). acterofamembrane-associatedSulfolobussolfataricusprotein
Analysisoftrypsin-generatedpeptidesderivedfromtheMeth- serine/threoninekinasewasconfirmedthroughprecipitationof
anococcus voltae S-layer glycoprotein also revealed modifica- a protein with kinase activity using lectin-conjugated agarose
tion by the same novel trisaccharide (453), suggesting a com- beads and by the decreased apparent molecular mass of the
monglycosylationprocessforthetwoproteins.Supportforthe protein and resistance to glyco-staining following treatment
glycosylationofMethanospirillumhungateiflagellabeyondgly- withchemicaldeglycosylationagents(262).
can staining was presented by chemical deglycosylation with Inadditiontomembraneproteins,secretedarchaealglyco-
trifluoromethansulfonic acid, a treatment that decreased mo- proteinshavealsobeendetected.Lectinbindingandchemical
lecular mass, as estimated by sodium dodecyl sulfate (SDS)- deglycosylation confirmed the glycosylated nature of the cop-
polyacrylamide gel electrophoresis (PAGE) (406). The same per response extracellular proteins secreted by the copper-
wasnotedforHalobacteriumsalinarumflagellinsuponsimilar resistantmethanogenMethanobacteriumbryantiiBKYH(219).
treatment(247). Indeed,differentialglycosylationisresponsiblefortheappear-
The glycosylated nature of Methanococcus deltae flagellins ance of multiple isoforms of the copper response protein. A
was indicated upon incubation of cultures with bacitracin, an secreted,induciblealkalinephosphatasepurifiedfromHaloar-
398 EICHLER AND ADAMS MICROBIOL.MOL.BIOL.REV.
TABLE 3. N-glycosylationofproteinsacrossthethreedomainsoflifea
Parameter Eukarya Archaea Bacteria(Campylorbacterjejuni)
Site ER(Golgi) Plasmamembrane Plasmamembrane
Saccharidedonors UDP-GlcNAc,GDP-Man, UDP-saccharide,GDP-Man?, UDP-saccharide
dolicholphosphate-Man/Glc dolicholphosphate-Man/Glc?
Lipidcarrier Dolicholpyrophosphate Dolicholphosphate, Undecaprenolpyrophosphate
dolicholpyrophosphate
Additionofsaccharides Yes No No
followinglipidflipping
Modificationoflipid-bound No Yes Yes
oligosaccharide
Finaloligosaccharide GlcNAc ManGlc Variable GalNAc(Glc)GalNAcBac
2 9 3 2 3
composition
Proteinglycosylationmotif Asp-X-Ser/Thr Asp-X-Ser/Thr/Val/Leu/Asp Asp-X-Ser/Thr
Linkingsugar GlcNAc Variable GalNAc D
Oligosaccharide-transferring Oligosaccharidetransferase STT3(isoforms?),additional Pg1B o
enzyme complex proteins? w
Oligosaccharide Yes ? No n
modificationfollowing lo
proteintransfer a
d
e
aAbbreviationsused:Bac,bacillosamine;Glc,glucose;GalNAc,N-acetylgalactosamine;GlcNAc,N-acetylglucosamine;Man,mannose;ER,endoplasmicreticulum. d

f
r
o
m
culamarismortuiwasshowntobeglycosylated,inpartthrough tinct from CipA and CipB and the expression of which is h
the use of radiolabeled glucosamine-containing growth me- relatedtogrowthtemperature(464). tt
p
dium (136). Quantitative analysis revealed that glycosylation :
/
/
accountedfor3%ofthemassoftheprotein.Basedonglyco- m
staining,asecretedenzymepossessingthermostableamylopul- ProcessofProteinN-GlycosylationinArchaea m
lulanase activity, i.e., capable of hydrolyzing both (cid:3)-1,6 link- b
r
ages in pullulan and (cid:3)-1,4 linkages in amylose and soluble In Eucarya, N-glycosylation begins on the cytoplasmic face .a
of the endoplasmic reticulum membrane, where nucleotide- s
starch, was detected in the growth media of both Pyrococcus m
furiosus and Thermococcus litoralis (44). Based on aberrant activated monosaccharides are sequentially added by mem- .
o
SDS-PAGE migration and sequencing data, it has been pro- brane-embedded monosaccharyltransferases to the saturated rg
posed that the partially secreted acid protease of Sulfolobus polyisoprenol-basedlipidcarrierdolicholpyrophosphate.This /
o
acidocaldarius,thermopsin,isalsoglycosylated(258). generates the heptasccharide core of the glycan structure ini- n
Inadditiontotheseidentifiedmembraneandsecretorygly- tially found on all eucaryal N-glycosylated proteins (46, 157, J
a
coproteins, numerous other glycoproteins, uncharacterized 235,333,409,442).Onceassembled,theglycan-chargedlipid n
apartfromtheirglycosylatednature,havebeenreported.Us- translocates (or “flips”) across the plane of the endoplasmic u
a
ing lectin-based purification techniques, a 152-kDa glycopro- reticulummembranebilayersothattheoligosaccharideisnow ry
teinwasisolatedfromThermoplasmaacidophilummembranes oriented within the endoplasmic reticulum lumen. The trans- 4
(478).Subsequentanalysisoftheglycanmoietyoftheprotein location of the glycan-charged dolichol pyrophosphate across , 2
revealedittobeahighlybranched,mannose-basedstructure, the membrane is catalyzed by an ATP-independent flippase 0
1
N-linked to the polypeptide chain through an N-acetylglu- (165),identifiedastheRTF1proteininSaccharomycescerevi- 9
cosamine subunit. Several lectin-binding proteins have been siae (159), with homologues reported in other Eucarya (158). b
y
observedinMethanococcusmazeiS-6,withthelevelsofthese Additional sugar subunits are then added to the lipid-bound
g
glycoproteins related to the adoption of morphologically dis- polysaccharide, transferred from flipped, lumen-facing doli- u
e
tinctformsbythecells(481).InHaloferaxvolcanii,membrane chol phosphate glucose or mannose carriers (158). The com- s
glycoproteins of 150, 98, 58, and 54 kDa, distinct from the pleted oligosaccharide is next transferred to appropriate Asn t
S-layer glycoprotein, were identified in lectin-based studies residuesofanascentpolypeptidechainenteringtheendoplas-
(98).Asecondstudyofthesamestrainnotedthepresenceof micreticulum(46,157,235,333,409,442).Thisismediatedby
glycoproteinsof105,56,and52kDainwhole-celllysates(489). oligosaccharidetransferase,amultisubunitcomplexassociated
Itremainstobeseenwhetheranyoftheproteinsidentifiedin withthetranslocon,themembraneproteincomplexresponsi-
the two studies are the same and whether the smaller glyco- bleforproteintranslocationacrosstheendoplasmicreticulum
proteinsarederivedfromtheheavierpolypeptides. membrane(392).
Relying on glyco-staining, lectin-binding techniques, and If,asproposed(46,157),theelaborateprocessresponsible
treatments with inhibitors of glycosylation or deglycosylating for protein N-glycosylation in Eucarya originated from a sim-
agents, the membranes of both Sulfolobus acidcaldarius and plerarchaealsystem,thenmanyofthefundamentalstepsand
Sulfolobussolfataricuswereshowntocontainunidentifiedgly- central components involved in eucaryal protein N-glycosyla-
coproteins distinct from the S-layer glycoprotein (147, 262). tion should also be present in Archaea. As summarized in
Glycoproteinstainingwasusedtoidentifyaseriesofglycosy- Table 3 and discussed in the following section, available evi-
lated proteins in Pyrococcus furiosus membranes that are dis- dencesuggeststhatthisisindeedthecase.
VOL.69,2005 ARCHAEAL POSTTRANSLATIONAL MODIFICATIONS 399
Dolichol carrier. Across evolution, isoprene-based lipids (ii) Analysis of dolichol-bound glcyans. Evidence for the
play essential roles in the glycosylation process by delivering involvement of dolichol phosphate-linked oligosaccharides in
theirboundglycancargotoselectedproteintargets(46,362). archaealproteinN-glycosylationalsocomesfromexamination
In Archaea, glucose-, mannose-, N-acetylglucosamine-, and of the carrier-bound glycan moieties. The transfer of radio-
sulfated tetrasaccharyl-containing phospho- and pyrophos- labeled glucose from UDP-[3H]glucose to Haloferax volcanii
phopolyisoprene(containing11to12isopreneunits)werefirst glycoproteins proceeds through a glucose-containing phos-
observedinHalobacteriumsalinarumbyionexchangeandthin- phopolyisoprenolintermediate(489).Thedolichol-linkedsul-
layer chromatography (281). Later studies (248) confirmed fatedpolysaccharidemoietyfoundinHalobacteriumsalinarum
thatthelipidmoieyofthesecompoundsisC dodecaprenol. is identical to glycan moieties found on the S-layer glycopro-
60
This is similar to the dolichol used in eucaryal protein N- teinandflagellininthisspecies(248,470).Ontheotherhand,
glycosylation (46) but distinct from undecaprenol, which is thesulfatedpolysaccharideismethylatedatthedolichol-linked
composed of 11 unsaturated isoprene units and used by Bac- stage, whereas no 3-O-methylglucose is detected in the pro-
teriaforproteinglycosylationandpeptidoglycansynthesis(362, tein-linkedpolysaccharide(249).
425). Mass spectrometry and nuclear magnetic resonance- Theimportanceofthistransientmethylationisillustratedby D
o
basedapproachesrevealedthepresenceofEucarya-likesugar the detrimental effect of inhibiting S-adenosylmethionine-de- w
carriers in Haloferax volcanii, including mannosyl-galactosyl- pendent methylation. Such treatment interfered with glyco- n
protein biosynthesis but did not affect either general protein lo
phosphodolichol, lesser quantities of a dihexosyl- a
phosphodolichol and a tetrasaccharyl-phosphodolichol con- biogenesis or the biosynthesis of sulfated phosphodolichol- d
e
boundoligosaccharides.Itthusappearsthatmethylationisan
taining mannose, galactose, and rhamnose, all linked to a d
dolicholcontaining11or12isopreneunits(242). essential step in the biosynthesis of the sulfated oligosaccha- fr
ride moiety prior to being transferred to its nascent polypep- o
(i)Antibioticsthataffectdolicholprocessinginterferewith m
tidetarget.Bycontrast,thehexasaccharidemoietyattachedto
archaeal protein glycosylation. The use of various antibiotics
h
the Methanothermus fervidus S-layer glycoprotein retains its
andothercompoundsknowntopreventproteinglycosylation tt
methylation (204). It is not clear whether such methylation is p
byinterferingwiththeprocessingofdolicholcarriershaspro- :
involved in the translocation of the sulfated oligosaccharide //
vided insight into the role of this lipid in archaeal protein m
phosphodolichol across the membrane or the subsequent
N-glycosylation. Tunicamycin hinders transfer of UDP-N- m
acetylglucosamine to polysaccharide-loaded dolichol carriers transferoftheglycanmoietytothenascentpolypeptidechain. b
(105).TreatmentwiththisantibioticinterfereswithSulfolobus InEucarya,chemicalmodificationofglycoproteinglycanmoi- r.a
etiesoccursonlyaftertheoligosaccharidehasbeentransferred s
acidocaldariusS-layerglycoproteinglycosylation(147).Incon- m
tothenascentpolypeptide(449).
trast, tunicamycin has no effect on the biosynthesis of the .
EnzymesofN-glycosylation.JustasarchaealN-glycosylation o
Haloferax volcanii S-layer glycoprotein (99) and accordingly, r
relies on the dolichol carriers implicated in eucaryal protein g
theglycanmoietyoftheHaloferaxvolcaniiS-layerglycoprotein glycosylation,Archaeaalsocontainhomologuesofmanyofthe o/
doesnotincludeN-acetylglucosamine(242,280).Bacitracinis n
enzymes involved in eucaryal N-glycosylation. These include

another drug that interferes with protein glycosylation via an J
thoseinvolvedinoligosaccharidechargingofthelipidcarrier, a
interruptionoftherecyclingofpyrophosphate-containingdoli- translocationofthedolicholcarrieracrossthemembrane,and n
chol species (420). Accordingly, in Halobacterium salinarum, u
transfer of the oligosaccharide entity to the nascent polypep- a
bacitracininterfereswiththeattachmentoftherepeatingsul- r
tidechain(Fig.2). y
fated pentasaccharide found at the Asn-2 position of the S- (i)Genomicstudies.AnalysisoftheNCBIproteindatabase 4
,
layerglycoprotein(284,469),althoughnotwiththeattachment
(www.ncbi.nlm.nih.gov) reveals the presence of genes encod- 2
ofthesulfatedpolysaccharidefoundattheotherN-glycosyla- 0
ing homologues of the staurosporine- and temperature-sensi- 1
tionsitesoftheprotein(469). tiveyeastprotein3(Stt3p)(425),anessentialproteinthought 9

Bacitracin also inhibits glycosylation of flagellins in Meth- b
tocontaintheactivesiteofthemultisubunityeastoligosaccha-
y
anococcus deltae (27) and slowed Sulfolobus acidocaldarius ride transferase complex (309, 493), in 18 archaeal strains. In g
growth, possibly through interference with the protein N-gly- Bacteria, such as Campylobacter jejuni, it is believed that the u
e
cosylationpathway(286).Incontrast,bacitracinhadnoeffect Stt3phomologuePglBistheonlycomponentneededfortrans- s
on the glycosylation of the S-layer glycoprotein or a second fer of glycans to Asn residues during protein N-glycosylation t
98-kDaglycoproteininHaloferaxvolcanii(99,232).Thefailure (425).
of the antibiotic to prevent Haloferax volcanii glycoprotein A close examination of the Archaeoglobus fulgidus genome
biogenesis is likely related to the fact that, unlike Halobacte- sequence revealed genes encoding STT3-like proteins within
riumsalinarum,inwhichbothmonophosphate-andpyrophos- two gene clusters encoding putative homologues of other en-
phate-containingdolichololigosaccharidecarriersarepresent zymesinvolvedinyeastproteinglycosylation(Fig.3)(46).One
(247), only bacitracin-insensitive monophosphate-containing oftheseclusterscontainsthreeadjacentopenreadingframes
oligosaccharide-dolichol intermediates are found in Haloferax (ORFs), one of which encodes a polypeptide that appears to
volcanii (242). Incorporation of glucose from UDP-glucose containamotifpresentintheyeastAlg1pandAlg2pglycosyl-
intoHaloferaxvolcaniiglycoproteinswas,however,inhibitedby transferase proteins. In the yeast proteins, this motif is in-
amphomycinandtwosugarnucleotideanalogs,PP36andPP55 volvedinthetransferofnucleotidesugarstothephosphodoli-
(489), compounds reported to block transfer of nucleotide- chol carrier (46). The other two ORFs putatively encode a
conjugated sugars to phosphopolyisoprenols in Eucarya (201, dolichyl-phosphoglucose synthase homologue and a homo-
202,336). logueofStt3p.OtherORFsinthisclustershowhighsequence
400 EICHLER AND ADAMS MICROBIOL.MOL.BIOL.REV.
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
FIG. 2. Schematic depiction of archaeal N-glycosylation. Step 1. A dolichol pyrophosphate (or monophosphate) species is glycosylated by //
m
transfer of saccharide subunits from nucleotide sugars (or possibly from lipid-bound sugar precursors). Step 2. Glycosylated phosphodolichol
m
“flips”acrosstheplasmamembrane,likelyinanenzyme-mediatedprocess.Step3.TheoligosaccharidestructureistransferredtoselectedAsn
b
residuesofanewlytranslocatedpolypeptide.Thefiguredoesnotconsidertherelationshipbetweenproteintranslationandproteintranslocation
r
ortherelationshipbetweenproteintranslocationandproteinglycosylation.Step4.Followingtransferoftheoligosaccharidemoietytoaprotein .a
target,thephosphorylateddolicholcarrierisrecycledtoitsoriginaltopology.Seereferences247,420,and468,thetext,andTable3foradditional s
m
information.
.
o
r
g
/
similarity to RfbA and RfbB, components of a transporter ORFs also encoding putative glycosyltransferase, dolichyl- o
familypresumablyinvolvedintheflippingofbacterialO-anti- phosphoglucosesynthase,andSTT3proteins,andliesnearsix n

J
gen (467) and lipopolysaccharides (364) across the plasma ORFsbearingsimilaritytogenesencodingproteinsinvolvedin a
membrane. While the functions of these putative gene prod- bacteriallipopolysaccharidebiosynthesis(46). n
u
ucts remain to be shown, it has been postulated that this Ar- (ii) Biochemical studies. In addition to such gene-based a
r
chaeoglobus fulgidus gene cluster encodes a functional unit predictions,enzymaticactivityhasalsobeendemonstratedfor y

involved in the assembly, translocation, and transfer of doli- some archaeal glycosylation-related proteins. Biochemical 4
,
cholphosphate-linkedoligosaccharidestoproteintargets(46). characterization of Pyrococcus furiosus UDP-(cid:3)-D-glucose py- 2
0
The second gene cluster in Archaeoglobus fulgidus includes rophosphorylase, responsible for UDP-glucose synthesis, rep-
1
9

b
y

g
u
e
s
t
FIG. 3. SchematicdepictionoftwoArchaeoglobusfulgidusgeneclustersputativelyinvolvedinproteinglycosylation.Putativegeneproductsare
givenaboveeachORF.Forfurtherdetails,seereference46.
VOL.69,2005 ARCHAEAL POSTTRANSLATIONAL MODIFICATIONS 401
resentsthefirstanalysisofanarchaealsugarnucleotidyltrans- riumsalinarumcellsintospheresledtotheproposedstructural
ferase (290). An N-acetylglucosamine transferase was also functionofarchaealproteinglycosylation(282).Infittingwith
partiallycharacterizedfrommembranesofHalobacteriumsali- a role for the sulfated S-layer glycoprotein oligosaccharide
narum (281). Dolichylphosphate mannose synthase, which is chains in maintaining the rod shape of Halobacterium salina-
abletotransferGDP-mannosetoadolicholphosphatecarrier, rum cells, it was noted that similarities exist in the overall
was purified from Thermoplasma acidophilum (490). Ampho- structuresoftheS-layerglycoproteinandproteoglycans,com-
mycin, an inhibitor of dolichylphosphate mannose synthases ponents of the extracellular matrix of animal cells (30, 468).
(202),blockedtheactivityoftheenzyme(490).Using5-azido- For example, iduronic acid, a major component of proteogly-
[32P]UDP-glucoseinaphotoaffinityapproach,asingle45-kDa cans (296), is found in the glycans decorating the Halobacte-
specieswasidentifiedinHaloferaxvolcaniihomogenatesthatis rium salinarum S-layer glycoprotein. Similarly, the O-glycosy-
thought to correspond to dolichylphosphate glucose synthase lationclustersituatednearthemembrane-spanningbaseofthe
(489). HaloferaxvolcaniiS-layerglycoproteinhasalsobeenassigned
Pyrophosphataseswiththeiractivesiteorientedtowardsthe astructuralsupportroleintheformationofaperiplasmic-like
cell exterior have been purified from the membranes of two space (217). In Thermoplasma acidophilum, an organism that D
o
different Sulfolobus acidocaldarius strains (8, 286). The pyro- lacksacellwall,ithasbeensuggestedthattheglycanmoieties w
phosphate-hydrolyzing activity of the enzymes, proposed to attached to the major glycosylated membrane-bound protein n
participate in the hydrolysis of dolicholpyrophosphate-linked species coating the cell surface act to either trap water mole- lo
a
oligosaccharides during protein glycosylation, was stimulated cules or allow the cell surface proteins to interact with each d
e
inthepresenceofSulfolobusmembranelipids.Sequenceanal- other.Ineitherscenario,glycosylationwouldcontributetothe
d
ysis of one of these pyrophosphatases has led to the identifi- rigidityofthecellsurface(478). f
r
cation of putative homologues in the genome sequences of Functionalroles.Theglycosylationofarchaealproteinshas o
m
Sulfolobus tokodaii and Solfolobus solfataricus as well as in also been implicated in protein assembly and function. In ar-

h
Methanobacteriumthermoautotrophicum(294).Thisstudyalso chaealflagellins,glycosylationisassociatedwithproperflagel-
t
t
revealed the presence of a strongly conserved phosphatase larassembly,sinceuponbacitracin-mediatedinterferencewith p
:
tripartite sequence motif, Lys–XXXXX-Arg-Pro-X -Pro- flagellinglycosylation,alossofMethanococcusdeltaeflagella- //
12-54 m
Ser-Gly-His-X -Ser-Arg-XXXXX-His-XXX-Asp, also de- tionwasobservedmicroscopically(196).InamutantHalobac-
31-54 m
tectedinLpp1pandDpp1p,Saccharomycescerevisiaeproteins terium salinarum strain in which underglycosylated flagellins b
showinghydrolyticactivitytowardsdolichylphosphate,dolich- areoverproduced,increasedlevelsofflagellaweredetectedin r.
a
ylpyrophosphate, and other isoprenoid phosphates/pyrophos- the growth medium, suggesting proper flagellin glycosylation s
m
phates(116). to be important for correct flagellar incorporation into the
.
Subcellular localization of glycosylation. Several lines of plasmamembrane(470).Thisexplanationis,however,incon- o
r
evidencesuggestthatarchaealglycosylationoccursattheouter sistent with the apparent nonglycosylated nature of other ar- g
/
cell surface, the topological equivalent of the luminal-facing chaealflagellins(184)ortheglycosylationofMethanospirillum o
n
leaflet of the endoplasmic reticulum membrane bilayer, the hungatei flagellins, which only occurs in low-phosphate media

J
siteofN-glycosylationinEucarya(46,157,235,333,409,442). (406).Similarly,evidenceagainstglycosylation’splayingarole a
Despite its inability to cross the plasma membrane of haloar- in protein function comes from bacterial expression of ar- n
u
chaea (284), bacitracin is nonetheless able to interfere with chaeal binding proteins. Normally glycosylated in their native a
r
Halobacterium salinarum protein glycosylation by preventing hosts, nonglycosylated heterologously expressed versions of y

transferofsulfatedoligosaccharidestotheS-layerglycoprotein theseproteinswerealsocapableofsubstratebinding(170,230, 4
,

(284,469).Theexternalorientationofthearchaealglycosyla- 231).Nevertheless,glycosylationcouldplayaroleinstabiliza- 2
0
tionapparatusisfurthersupportedbythedecorationofexog- tionagainstproteolysisorcouldaffecttheinteractionofbind- 1
enously added, soluble cell-impermeable hexapeptides con- ingproteinswiththecellmembraneorenvelope(4). 9

taining the Asn-based N-glycosylation motif with sulfated Glycosylationasanenvironmentaladaptation.Copingwith b
y
oligosaccharidesbylivingHalobacteriumsalinarumcells(248). the often harsh environmental conditions encountered by Ar- g
Otherobservationsalsofavortheassignmentofarchaealpro- chaeaservesasthebasisforyetanotherhypothesizedrolefor u
e
teinglycosylationtothecell’soutersurface.Theseincludethe archaeal protein glycosylation. In a comparison of the glyco- s
t
ecto-enzymaticnatureofaSulfolobusacidocaldariuspyrophos- sylation profiles of S-layer glycoproteins from the moderate
phatase (8, 286), the proposed specific inhibition of an exter- halophile Haloferax volcanii and the extreme halophile
nally oriented Mg2(cid:1)-dependent oligosaccharidetransferase by Halobacterium salinarum, it was noted that the latter experi-
EDTA,anon-cell-permeantreagent,andsubsequentinterfer- ences a higher degree of glycosylation than the former (280).
ence with Halobacterium salinarum flagellin glycosylation Moreover, the glycan moieties of the extreme halophile were
(420),aswellasstudiessupportingthecotranslationalmodeof enrichedinsulfatedglucuronicacidsubunitsasopposedtothe
membraneproteininsertioninArchaea(360). neutral sugars found in the moderate halophile. These prop-
ertiesendowtheHalobacteriumsalinarumS-layerglycoprotein
withadrasticallyincreasedsurfacechargedensityrelativetoits
RoleofProteinGlycosylationinArchaea
Haloferaxvolcaniicounterpart.
Structural roles. Given the seemingly routine glycosylation Theenhancednegativesurfacechargesarethoughttocon-
of archaeal proteins, one can ask what role is played by this tribute to the stability of haloarchaeal proteins in the face of
posttranslational modification in Archaea. The observation molar salt concentrations (266). Accordingly, the Halobacte-
that bacitracin treatment transformed rod-shaped Halobacte- rium salinarum S-layer glycoprotein also contains 20% more
402 EICHLER AND ADAMS MICROBIOL.MOL.BIOL.REV.
acidicaminoacidresiduesthandoesthecorrespondingprotein MembraneLipidsofArchaea
inHaloferaxvolcanii(246,421).Theenhancednegativesurface
One of the defining traits of Archaea that distinguish them
charge associated with protein glycosylation and the resulting
fromEucaryaandBacteriaisthechemicalcompositionoftheir
protection that this would afford in the face of acidic condi-
membranephospholipids(206,208).First,unlikeeucaryaland
tionshavebeenofferedastheroleofSulfolobusacidocaldarius
bacterial phospholipids which are built on a glycerol-3-phos-
cytochrome b glycosylation (161, 484). It has also been
558/566 phatebackbone,archaealphospholipidsarebasedontheop-
suggested that a significant amount of the protein surface is
posite stereoisomer, glycerol-1-phosphate. Second, archaeal
shielded from the (cid:4)pH 2 environment by the high degree of
phospholipidscontainpolyisoprenylsidechainsratherthanthe
glycosylation(484).Finally,glycosylationhasalsobeenimpli-
acylgroupsemployedbyeucaryalandbacterialphospholipids.
cated in the stabilization of thermophilic archaeal glycopro-
Third, archaeal phospholipids rely on ether bonds to link the
teins(4,258,455).
isoprenylsidechainstotheglycerol-1-phosphatebackbone.In
Eucarya and Bacteria, ester bonds link acyl side chains to the
glycerol-3-phosphate backbone. Of these three traits, the use D
LIPIDMODIFICATION
ofglycerol-1-phosphateisconsideredthemostdefining,since o
w
examplesofether-linkedlipidshavebeenobservedinEucarya
Lipidmodification,definedhereinasthepermanentortem- n
porary covalent attachment of lipid-based groups at various and Bacteria (172, 328) and non-ester-linked phospholipid lo
fatty acids and genes encoding components involved in the a
positionswithinapolypeptidechain,isacommonmodification d
metabolismoffattyacidshavebeenreportedinArchaea(127, e
experiencedbybotheucaryalandbacterialproteins.Anexam- d
inationofknownlipidmodificationsrevealsthatawidevariety 342). Indeed, free fatty acids have been observed in the lipid f
oflipidmoietiescanbedirectlyorindirectlylinkedtoaprotein phase of Methanosphaera stadtmanae and Pyrococcus furious ro
(51, 191). Finally, archaeal phospholipids are generally orga- m
atanyofnumerousattachmentsitesthroughtheuseofanyof
nizedintothebilayerstructurethatisalsopresentineucaryal h
several linkages (414). For instance, lipid modification can t
andbacterialcells,althoughtetraetherlipid-basedmonolayers tp
involve myristoyl or palmitoyl acyl groups (358), isoprenyl
can be found in thermophilic and hyperthermophilic Archaea :/
polymersofvariouslengths(393),oraminoglycan-linkedphos- (92,226). /m
pholipids(103).Thesecanbeaddedattheaminoterminus,the m
Whereasphospholipidsandotherpolarlipids(phosphogly-
carboxy terminus, or at internal residues via ester, thioester, b
colipids, glycolipids, and sulfolipids) account for the vast ma- r
thioether, or amide bonds, or through mediating elements, jority of archaeal membrane lipids, archaeal membranes also .a
suchasthephosphopantethenegroupoftheacylcarrierpro- s
containacetone-solublenonpolarlipidspecies,primarilyneu- m
tein(267). tralsqualenesandotherisoprenoid-basedpolymers(206,207, .o
Lipid modification of proteins is largely a posttranslational 334,439,440).InhalophilicArchaea,inwhichmembranelipid rg
event(115).Itservesavarietyofroles,themostobviousbeing compositionhasbeenmoststudied,pigmentedcarotenoids,in /
o
to enhance the membrane affinity of the modified protein. particularbacterioruberins,aremajorcomponentsofthenon- n
Accordingly,amino-terminalacylationleadstothelocalization polar lipid pool (243, 438). These have been implicated in J
a
ofnumerousproteinstotheoutermembraneofgram-negative affordingprotectionfromUV-induceddamage(390).Inaddi- n
Bacteria (156, 379), as exemplified by Braun’s lipoprotein in tion, many halophilic Archaea also contain retinal as part of u
a
Escherichiacoli(40).Similarly,otherwisesolubleeucaryalpro- bacteriorhodopsin,thepurpleretinal-containingproteincom- ry
teins also become membrane associated upon the covalent plexthatfunctionsasalight-drivenprotonpump(244). 4
attachmentofoneormorelipidmoieties(102,153,194,462). ,
2
Lipidmodificationcanalsomodulateprotein-proteininterac- 0
Lipid-ModifiedArchaealProteins 1
tions in Eucarya, as shown by the effects of myristylation or 9
prenylationupontrimericGproteinsubunitaffinity(124,178, b
InArchaea,lipid-modifiedproteinshavebeenreportedfrom
y
462), and in viruses, exemplified by the involvement of myri- a wide range of species. In many cases, modification involves g
stylation of the capsid proteins of human immunodeficiency uncharacterized lipid entities, whereas in others, direct proof u
e
virus type 1 and picornavirus in virion particle assembly and
for the presence of attached lipid groups remains lacking. s
secretion(65,142). t
Table 4 summarizes the various lipid-based modifications
Lipid modifications of eucaryal proteins has also been im- shown or presumed to exist in Archaea, while Fig. 4 offers a
plicated in a variety of other cellular events. These include schematic presentation of representative archaeal lipid-modi-
signal transduction (287), embryogenesis and pattern forma- fiedproteins.
tion (271), protein trafficking through the secretory pathway Lipoproteins. In the haloalkaliphile Natronobacterium
(297),andevasionoftheimmuneresponsebyinfectiouspar- pharaonis, halocyanin, a small blue copper protein, was pro-
asites (369, 461). Yet another role for lipid modification is posedtoundergoamino-terminallipidmodificationbasedon
exemplifiedbythebacterialtoxinhemolysinA,whichrequires thepresenceoftheso-calledlipoboxsequencemotifnearthe
fattyacidacylationonaninternalLysresidueforitsactivation startofpredictedaminoacidsequence(274).InBacteria,the
(414). Leu-Ala-Gly-Cyslipoboxsequencemotif(156)liesattheend
Giventheubiquitousdistributionandnumerousfunctionsof of the signal sequence, the short N-terminal extension not
lipidmodificationsineucaryalandbacterialproteins,itisnot foundinthemature,lipid-modifiedprotein(seebelow).Atthe
surprising that lipid-modified proteins have also been identi- membrane, the bacterial lipobox motif is sequentially recog-
fiedinArchaea. nizedandprocessedbythreeenzymes.Thesulfydrylgroupof