An pathway

TheEMBO Journal vol.13 no.10 pp.2289-2296, 1994

An alternative protein targeting pathway in Escherichia coli: studies on the role of FtsY

Joen Luirink1, Corinne M.ten Hagen-Jongman, Coen C.van der Weijden, Bauke Oudega, Stephen High2, Bernhard Dobberstein3 and Ron Kusters

IDepartment of Microbiology, Institute of Molecular Biological Sciences, Biocentrum Amsterdam, De Boelelaan 1087, 1081 HV Amsterdam, TheNetherlands, 2Departmentof Biochemistry, School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK and3Zentrumfir MolekulareBiologie Heidelberg, ImNeuenheimer Feld 282, D-6900 Heidelberg, Germany Communicated byB.Dobberstein

In Escherichia coli, a signal recognition particle (SRP) hasbeenidentifiedwhich bindsspecificallyto the signal sequenceofpresecretoryproteinsand which appears to be essential for efficient translocation of a subset of proteins.In this study we have investigated the function ofE.coliFtsY which shares sequence similarity with the a-subunit of the eukaryotic SRP receptor ('docking protein') inthe membraneoftheendoplasmic reticulum.

A strain was constructed which allows the conditional expression of FtsY. Depletion ofFtsYisshown to cause theaccumulation of theprecursorform of ,B-lactamase,

OmpF

andribose binding protein in vivo, whereas the processing of various other presecretory proteins is unaffected. Furthermore, FtsY-depleted inverted cytoplasmic membrane vesiclesareshown to bedefective inthetranslocation of

pre-,3-lactamase

using an in vitro import assay.

Subceliular

localization studies revealed that FtsYis located inpart atthecytoplasmicmembrane with which it seems peripherally associated. These observations suggest that FtsY is the functional E.coli homolog of the mammalian SRP receptor.

Key words: Escherichia coli/FtsY/protein targeting/signal recognition particle

Introduction

Thetargeting andtranslocation ofproteins into and across the membranes of the endoplasmic reticulum (ER) in eukaryotes and thecytoplasmic membrane ofprokaryotes occurby virtue ofahydrophobic N-terminal signalsequence.

The structural and functional conservation of the signal sequenceofeukaryoticand

prokaryotic

proteinssuggests that the basic mechanisms of membrane targeting and translocation may be similar in both cases (Hartl and Wiedmann, 1993; High and Stirling, 1993; Luirink and Dobberstein, 1994).

InEscherichia coli several factors have been

identified,

by both genetic andbiochemical means, which cooperate inthe so-called

general

secretory

pathway (for

areviewsee

Pugsley,

1993).

In this

pathway cytosolic

molecular chaperoneslike SecBfunctiontomaintain the translocation

competenceof preproteinsin thecytosoland to target them tothecytoplasmic membranewhere acomplexmachinery consisting of SecA, SecD-FandSecYassistsin membrane insertion and translocation.

Inmammalian cells,targetingof mostproteinstothe ER membrane is mediated by the signal recognition particle (SRP) whichconsists ofoneRNAmolecule(SRP7SRNA) andsixpolypeptides of 9, 14, 19, 54, 68 and72kDa

(for

a review, seeRapoport, 1992). The SRP binds via its 54 kDa subunit (SRP54) to the signal sequence of nascent presecretory proteins, thereby lowering their rate of translation. Thecomplex ofthe ribosome,nascentchain and SRPis then targetedtotheERmembraneby interaction with thedocking protein complex. TheSRP is released from the membrane-bound ribosome-nascent chain complex in a GTP-dependentmannerand the translationarrestisrelieved.

Theremaining ribosome-nascent chain complex associates withacomplexofmembraneproteins, thetranslocon,which catalyzesmembraneinsertion andtranslocation ofthe nascent chain.Thus, the SRP functions bothas acytosolic chaperone preventingprematurefoldingof thepreprotein bycoupling translation to translocation and as a

'pilot'

to guide the preproteintothe SRP receptor complexinthe membrane.

Genetic and biochemical evidence indicates that SRP- mediatedtargetingmayalso occurinE.coli (for reviewssee Hardand

Wiedmann,

1993;Luirink and Dobberstein, 1994), Bacillus subtilis (Honda etal., 1993) and Saccharomyces cerevisiae (Hann and Walter, 1991; Ogg etal., 1992). ^In E.coli, anSRP-likecomplex wasidentified whichconsists ofoneprotein (P48or Ffh) andoneRNAmolecule (4.5S RNA) thatarehomologoustothe SRP54 andSRP7SRNA constituents of the eukaryotic SRP, respectively (Poritz etal., 1990; Ribes etal., 1990). Depletion of either the RNA or the protein component ofthe E.coli SRP affects theexportofseveralsecretoryproteins (Ribeset

al.,

1990;

Phillips and Silhavy, 1992). Moreover, P48 interacts specifically with the signalsequenceofnascentpresecretory proteinsas wasshownbyphotocross-linkinginacrude E.coli cell lysate (Luirink etal., 1992) and with the use of a

reconstituted chimeric SRP

(Bernstein

etal.,

1993).

The role of the E.coli SRP in protein secretion is not known. Thus, the SRP may support co-translational translocation ina separate secretory

pathway

ormayform part of the

general

secretory

pathway.

If theSRPfunctions in a separate targeting pathway, one would expect a membrane receptor for the SRPtoexist. In thisrespectit isof interest that theC-terminal

region

of the E.coli

protein

FtsYdisplaysstrikingsequence

similarity

with the

cx-subunit

ofthecanine

docking protein, leading

tothe

hypothesis

that FtsY may function as amembrane-bound receptor forthe E.coli SRP

(Bernstein

et

al., 1989;

Romischet

al., 1989).

Originally,

FtsY has been

implicated

in celldivision because itsgeneis located inanoperon

together withftsE andftsX,

inwhich temperaturesensitive mutationshavebeenidentified

J.Luirink et a!.

pi; r9 pEkT9-FLsY pET9-FtsY

+ + + pET9-FtsY

pLysE plvysE pLysS

A ^{_ + _ + _ + + 1PTG}

|t

_t}^r'maW__

~~~~~~~~.

^.-

-g ~

1 2 3 4 5 6 7 8

B

40- FLYs'i

1 2 3 4 5 6 7 8

C

94 _

*

W ^" FtsY

-

^FtsY

3-i.%

43 .-.::. -

29-

1 2 3 4 5

Flg. 1.Overexpressionandpurificationof FtsY. BL21(DE3)cells carryingthe indicatedplasmidsweregrownfor 1 h inYTwith 0.4 mMIPTGorwithout inductionasindicated. Proteinswereidentified by SDS-PAGEfollowed by CoomassieR-250staining (A)and immunoblotanalysisusing antiserumagainst FtsY(B). (C)Purification of FtsY.Samplestaken^atdifferent stages of thepurificationprocess wereanalyzed bySDS-PAGEandCoomassieR-250staining. Lane 1, molecularweightmarkers;lane2, totalcell lysateafterinduction of FtsY; lane3, peakfractionafter thefirst runoverMonoQ Sepharose;

lane4, peakfraction after the second runoverMonoQSepharose;

lane5, finalproduct, peakfractionaftergelfiltration using Superose-12.

that cause cell

filamentation

at the non-permissive temperature (Gill and Salmond, 1986). However, nofts mutations have been mapped in ftsY despite localized mutagenesis oftheftsYEXgenecluster(Gibbset

al.,

1992).

Inthis study, wedemonstrate that FtsY isin part located at the cytoplasmic membrane and that depletion and overexpression ofFtsY affects both

cell

morphology and protein export.

Results

Overexpression

and

purification

ofFtsY

To examine the effects ofoverexpression of FtsY and to facilitate its

purification,

the gene

encoding

FtsY was

subcloned into the

expression

^vector

pET9

under control of the T7 promoter. For

expression,

the

resulting

construct

(pET9-FtsY)

was transferredto E.coli

BL21(DE3)

which contains ^a chromosomal copyof the T7

polymerase

gene under control of the lac

promoter/operator.

As shown in

Figure

IA

(lanes

7 and

8),

cells

harboring pET9-FtsY expressed

^a

polypeptide

which

migrates during

SDS-PAGE

as a

characteristically 'bulged'

band at 92 kDa. Gill and Salmond have

previously

shown that FtsY

migrates

asa92 kDa

polypeptide although

themolecular

weight

ofFtsYas

deduced from the DNA sequence is 54 kDa (Gill and

Salmond, 1990).

Thebandwas

positively

identifiedasFtsY

by

means of

immunoblotting using

an antiserum raised

against

^a

synthetic

C-terminal

peptide

ofFtsY

(Figure 1B,

lanes 7 and

8).

pET9-FtsY

^wasdifficulttomaintain

stably

in

BL21(DE3), probably

dueto the detrimental effect ofuninduced FtsY

expression (Figure

IAand

B,

lane

7).

Toreduce the basal

expression

of

FtsY,

the

compatible plasmids pLysE

and

pLysS

were introduced which encode T7

lysozyme,

an

inhibitor of T7 RNA polymerase activity (Studier et

al., 1990).

Both

pLysE

and

pLysS,

which differ in the

degree

ofT7

lysozyme expression,

wereabletostabilize

pET9-FtsY

in

BL21(DE3)

andreduce both induced and non-induced

expression

ofFtsY

(Figure

IA and

B,

lanes

3-6).

The FtsY

protein

was purified

by

anion

exchange chromatography

and

gel

filtration. At different stages in the

purification procedure, samples

weretaken andanalyzed

by

SDS-PAGE

(Figure IC).

As shown in lane

5,

a second

protein

co-purified with theFtsY protein. To identifythis proteinandtoconfirm theidentityofFtsY,theN-terminal amino acidsequenceof bothproteinswasdetermined

(data

not

shown).

ThefiveN-terminalamino acidresidues of the upperbandwereidenticaltothepredictedsequenceofFtsY.

The lower bandappearedtorepresentFtsYmissing 14 N- terminal amino acid

residues,

probably as a result of proteolyticcleavage occurring duringthepurification. The overall

purity

ofboth bands together ^was estimated to be

>95%.

Remarkably,

FtsY elutes from the gel filtration columnasa

single peak

withanapparentmolecular

weight

of >200 kDa. Gelfiltration inthe presence of8 M urea

did not change the elution profile, suggesting that the unexpected apparent molecular weight is not due to

oligomerization. Thereasonfor theaberrantmobilityofFtsY in both

gel

filtration and SDS-PAGE is unknown.

Effects of FtsYoverexpression

Induction of FtsY expression by growth of

BL21(DE3)

harboring pET9-FtsYandpLysE inthepresenceofIPTG had a negative effect on cell growth and caused cell filamentation onlyatlatetimepoints when cellgrowthwas

alreadyaffected. StrongoverexpressionofFtsYbygrowth ofBL21(DE3) harboring pET9-FtsY inthe presenceofIPTG led to inclusionbody formation (data not shown).

Toexamine theeffectsof FtsYoverexpressiononprotein export, theaccumulation ofprecursorformsofOmpAand ,B-lactamasewasmonitoredinvivobyimmunoblotting. IPTG

was added to BL21(DE3) harboring pET9-FtsY and

Roleof FtsY in protein targeting in E.coIi

3 4 5- 6

ittsY -

7 8 9 I1 ¹²

i.- lL .&l

-p Bla -.m

OmpA *,, _ Ono _m_ _i _ __ - m

Fig. 2. Processing of presecretory proteins in cells overexpressing FtsY. Strain BL21(DE3) carrying either pACYC177AHaeII (lanes 1-4)or acombination ofpACYCl77AHaeII andpET9-FtsY (lanes 5-12)wasgrown inYTwith 0.4 mM IPTG (lanes 1-4 and9-12) orwithout induction (lanes5-8). Samples were taken 1 h (lanes 1, 5 and9), 2 h (lanes 2, 6 and 10), 3h(lanes3, 7and 11) and4h (lanes 4, 8 and 12) after induction and analyzed by immunoblotting using antisera against FtsY, (3-lactamase(Bla) andOmpA as indicated attheleft side of the blot panels. The positions of the precursor and mature formsof the secretory proteins are marked with 'P' and 'M' respectively, at the right side of the blot panels. The position of the precursorform of OmpA was identified inaSecA(Ts) strain grown at thenon-permissivetemperature (not shown).

A

II FtsY I| tsE

p_~

ArB

IFtsY'

AraC Dim pAral4-FtsY'

oil

p_ ~~~~~orl_

py

BpB_DR322

PAraL

B

1:c

s

Z

r.

c

I.- 1.0

pACYC177AHaeII (encoding 3-lactamase,seeMaterials and methods) at the early log phase ofgrowth. Samples were

takenatvarioustimepointsafterinduction andanalyzed by immunoblottingtodetermine theextentofFtsY induction.

The level of FtsY was drastically increased ¹ h after induction andremainedveryhigh throughout the induction period (Figure 2, upperpanel, lanes 9-12). Intermediate andlow(wild-type) expressionwasobserved in the absence of inducer and in the absence ofpET9-FtsY respectively (Figure 2,upperpanel, lanes 1-8).Inaddition, OmpAand 3-lactamase were identified in the samples by immuno- blotting. A strong accumulation of pre-3-lactamase was

observedfrom 1hafter inductionofFtsY(Figure 2,middle panel, lanes 9-12). Even the intermediate uninduced FtsY expression resulted in the appearance of trace pre-3-

lactamase(Figure 2, middle panel,lanes5-8).Incontrast, no pre-OmpA could be identified even after prolonged overexpression of FtsY (Figure 2, lower panel). These observations areindicative ofaratherspecificeffect of FtsY overexpression onproteinexport resembling the effects of overexpression of P48 (Ribesetal., 1990)and depletion of 4.5S RNA (Poritz etal., 1990; Ribes etal., 1990), the constituents of the E.coli SRP.

O 2 4 6

Timefhⁱ

C

_ _ _

o} 2 4

arabinose +

time(h) after shift Construction ofa mutant with conditional FtsY

expression

To study therole ofFtsY in moredetail, a mutantE.coli strain was constructed in which the expression of FtsY is undertheregulationofthearaBpromoterandoperator. The construction is shown schematically in Figure 3A and described in detail in Materials and methods. E.coliN4156, deficient in DNA polymerase I, was transformed to

ampicillinresistance withpAral4-FtsY'whichcontained the 5' end of FtsY downstream from the araBpromoter/operator region. SincepAral4-FtsY'containsaColE 1 typereplicon which is unableto replicateinpolA strains, transformants

canonlybeobtained whenpAral4-FtsY' integratesinto the chromosome. Integrationwill takeplaceinto thehomologous chromosomalftsYgenethereby disruptingtheftsYEXoperon

andplacingacompleteftsYcopyunder control of the araB

Fig.3. Conditional expressionof FtsY. (A) Construction ofstrain N4156::pAral4-FtsY' by integrationofpAral4-FtsY' into theftsYEX

geneclusterof theE.coli N4156 chromosome resultinginPAraB

controlledexpressionoftheftsYgene. P, promoter; Bla, gene encodingj3-lactamase. (B) GrowthcurvesofN4156::pAral4-FtsY'.

Cellsweregrownovernightin YTsupplemented with0.4% fructose and0.2% L-arabinose, collected bycentrifugation, washedonce inYT and usedtoinoculate YTcontaining 0.4% fructoseonly (L) or a

combination of 0.4% fructoseand0.2% L-arabinose(O) at0 h. (C) Extent of FtsYdepletion.At theindicatedtimepoints after the shiftto mediumsupplementedwithorwithout L-arabinose(see underB), samples weretaken andanalyzed by immunoblotting usingantiserum againstFtsY.

promoter/operator. Correct integration was confirmed by Southernblottingof HindII-orXmnl-digestedchromosomal DNA extractedfromintegrantsusing thecloned5' endof

ftsYas a hybridization probe (data not shown).

FtY I1

*m&.wo A-

- A

J.Luirinketal.

A

B

"IW

Fig.4. Fluorescencemicrographs(1600x) ofFtsY-depleted(A) or

'wild-type' (B)cells. StrainN4156::pAral4-FtsY' wasdepletedof FtsYasdescribed in thelegendtoFigure3B. Micrographsweretaken 5 hafter the shifttomedium supplementedwith L-arabinose(B) or not supplemented (A).

Effects of FtsYdepletion

N4156::pAral4-FtsY' is unable to form colonies in the absence of the inducer arabinose(not

shown) indicating

that FtsY is essential for cell viability.

The effects of FtsY depletion on cell growth in

liquid

medium are shown in

Figure

3B. When strain N4156::

pAral4-FtsY',

pregrown inYTinthepresenceofarabinose, wasshiftedto YTlackingarabinose, the optical density of the culturelagged thatof

arabinose-supplemented

cultures indicating impaired cell growth. In order to evaluate the degree of FtsY expression upon removal of the inducer arabinose, sampleswere taken atvarious timesafter the shift and analyzed by

immunoblotting

(Figure 3C). A strong reduction inthe amount ofFtsY was detected as soon as 2 hafter theshift, indicating efficient depletionoftheculture for FtsY.

ThemorphologyofFtsY-depleted cells was examined by phasecontrastandfluorescence microscopy after nucleoid staining of cells fixed with OSO4. As shown in Figure 4, cells

depleted

forFtsYform short

filaments

of swollen

cells

with large spherical bulges unevenly distributed along the filaments. This characteristic phenotype is visible from 3 h after the shifttoarabinose-free medium. In the bulging cells the nucleoids appear to spread out. Concomitant with the change in phenotype, a mild induction of the heat shock

proteins DnaK and GroELwasobservedby

immunoblotting (data

not

shown).

If FtsYfunctions like theyeastandmammalian

docking

protein in targeting of presecretory proteins to the cytoplasmic membrane, one would expect this process to bedisruptedupondepletion of FtsY (Oggetal., 1992). To test this notion, the accumulation of various presecretory proteinswasmonitoredby immunoblottingupondepletion of FtsY in N4156::pAral4-FtsY' (Figure 5A). A strong accumulation of pre-3-lactamase is observed which is apparent as soon as 2 h after the shift to arabinose-free medium.Inaddition,precursorforms of OmpF and ribose binding protein (RBP)canbedetected from 2 and 3 h after thestartofdepletion respectively. The weakaccumulation ofpre-OmpF(and pre-,-lactamase in longerexposures, not shown), observed in the arabinose-supplemented cells,might be due to aslightoverproductionof FtsY(seealsoFigure 2).

The processing of pre-OmpA, pre-OmpC and pre-MBP (maltose binding protein) seemed unaffected by depletion of FtsY.

If the accumulation ofpreproteinsinFtsY-depleted cells is causedby atargeting defect onewould expectthem to remain untranslocated. To test this hypothesis, the accessibilityofpre-3-lactamaseforproteinaseK was tested inN4156::pAral4-FtsY' cells whichhad beendepletedfor FtsY. The cells were treated with Mg2+, EDTA or EDTA+Triton X-100, incubated in the presenceorabsence of proteinase K and subjected to SDS -PAGE and immunoblotanalysis (FigureSB). EDTApermeabilizes the outer membrane which allows access of theproteasetothe

periplasm.

Pre-3-lactamasewas not

degraded by

proteinase K incells treated with EDTA(Figure5B,lane4)consistent with theexpectation that thisformis inside thespheroplast.

Under these conditions, OmpA (which has a protease- sensitive periplasmic domain) was degraded (Figure 5B, lane 8) leaving a protected outer membrane embedded domainof -20 kDa asexpected (Schweizeret

al.,

1978),

indicating

that

proteinase

Khadaccess tothe

periplasm

of the spheroplasted cells. In the lysed (Triton-treated) spheroplasts, pre-(3-lactamase was

degraded (Figure

5B, lane6)confirming that this form isnotintrinsicallyresistant toproteinase K

digestion

incontrastto mature

,3-lactamase

(Minskyetal., 1986).Intheabsence ofproteinaseK some pre-,B-lactamase was degraded in the lysed spheroplasts (Figure 5B, lane5),

possibly

by endogenous E.coli proteases.

Inorder toobtain direct insight into the involvement of FtsY inproteintargeting,an in vitrotranslocation assaywas applied. Translation of

pre-,3-lactamase

wascarriedout in anFtsY-depleted cell-freeextract.Five minutesafter thestart of translation, inverted cytoplasmic membrane vesicles (IMVs) with either a wild-type level of FtsY (Figure SC, lane 1)or with anundetectable level of FtsY (Figure5C, lane2-7)wereadded.Simultaneously, the reaction mixture wassupplemented with purified FtsY to a final concentration ranging from0 to0.8 ,iM (Figure SC, lanes 1-7). After proteinaseKtreatment, the amount of protected protein was determined. As can be seen in FigureSC (lanes 2-7), translocation almost doubled upon addition of FtsY. Optimal translocationefficiency was reached at 0.2

tiM

FtsY. This concentration is within the range in which SecA is effective instimulating in vitro translocation (Kusters et al., 1989).

These results indicate that FtsY directly stimulates

Role of FtsY in protein targeting inE.coli B

2 3 4 5 6 8 9

Prot.K

EDTA

Triton Bla

- _m - -P

Rla _Vom m an, - as -N1

(OnpF _4 &bd m_ __.o-

MD embeome MN

C

OmpA

fe

"-NJ

OmpA -P

C ..M___0~ _

OmpC m_'Wim_ domm4m_ M sso--PNJ

INMV \VT 0-Ftsl depleted e

-p

_____p -M

addedFtsl'pN4 ^{- - 0.05 0.10} 0.20 0.40 0.80 rel.transloc. 100 35.9 38.4 47.7 66.5 44.8 40.41

Fig 5. (A) Invivoprocessing ofpresecretoryproteins upondepletionof FtsY. CellsofN4156::pAral4-FtsY' weredepleted forFtsYasdescribed in thelegend of Figure 3B. Samplesweretakenat0h (lanes 1), 2h(lanes2and 6), 3 h (lanes 3and7), 4 h (lanes4 and 8) and 5 h(lanes5 and 9) afterthe shifttomediumsupplemented with L-arabinose (lanes2-5)ornotsupplemented(lanes6-9) and analyzedby SDS-PAGE and

immunoblotting using the indicated antisera. The positions of theprecursorandmature formsof thesecreted proteins aremarkedwith 'P' and 'M', respectively, attheright side of the blotpanels. The positions of theprecursorformswereidentifiedinasecA(Ts) straingrownatthenon-

permissivetemperature(not shown). Thebandsmigratingbelow themature form of Bla andabovetheprecursorformofOmpArepresent crossreactingprotein speciesofunknownorigin (not shown). (B) Protease accessibilityofpre-,B-lactamasein FtsY-depleted spheroplasts. Cellsof N4156::pAral4-FtsY' weredepleted of FtsYfor 4 hasdescribed inthelegendofFigure 3B.Thecells wereincubatedintheabsence(lanes 1, 3, 5 and7)orpresence(lanes2, 4, 6 and8)ofproteinaseK in 100 mMTris-HCI, 250 mM sucrose(pH 8.0)with either 10 mM MgCl2(lanes1 and 2), 5 mMEDTA(lanes3, 4, 7and 8)or5 mM EDTA + 1%TritonX-l00 (lanes5and 6). Subsequently,thesampleswereTCAprecipitatedand analyzedasdescribed in(A). TheproteinaseK-resistantfragmentofOmpA isindicatedattherightsideofthe blotpanel (*). (C)Invitro

translocation ofpre-,3-lactamase. 35S-labeledpre-3-lactamasewassynthesized byin vitrotranscriptionand translation in thepresenceofanFtsY- depletedS-135 cell-freeextract. Translocation acrossinvertedcytoplasmic membrane vesicles (IMVs) derivedfromawild-type (lane1)orFtsY- depletedstrain (lanes2-7) wasdetermined byproteolytic degradationofallnon-translocatedproteins. Purified FtsYwasaddedtothereaction mixtureattheindicated concentration. The translocation efficiencywasexpressedas apercentageof thetranslocationacrosswild-type(WT) IMVs (takenas 100%). Thispercentage iscorrectedforvariation intheamountofproteinsynthesized. The positions of the precursorandmatureforms of ,B-lactamasearemarked with 'P' and'M', respectively, attherightsideofthegel panels.

translocation. Thedecrease intranslocation observed above theoptimalFtsYconcentrationmaybeduetonon-productive interactions of FtsY withthepreproteinorcomponents of the translocation apparatus, like the SRP. In vivo, overexpressionof FtsY alsoprovokesthespecificaccumul- ation ofpre-3-lactamase (see above).

Subcellular localization of FtsY

Strain BL21(DE3) expressingFstY atwild-typeorelevated levels was subjected to subcellular fractionation using immunoblottingtoidentifyFtsYinthe fractions. As shown inFigure6A,FtsYexpressedinwild-typeamountsis found inboth the soluble andcytoplasmicmembrane fraction. In cellsoverexpressingFtsY(Figure 6B) relativelymoreFtsY is found in the soluble fractionwhichmight suggestthatthe

number of membranebinding sites for FtsY is limited.

Thecellular distribution of FtsY was alsoexamined by immunoelectron microscopyusingaffinitypurifiedanti-FtsY antiserum and colloidal gold-labeled second antibody on

ultrathin cryosections. Unfortunately, we were unable to

detect FtsY expressed at wild-type levels. In cells

overexpressing FtsY, the gold particles were primarily locatedinthe innerpartofthe cellenvelopecorresponding

to thelocation ofthecytoplasmic membrane (Figure 6C).

Thegoldparticleswere more orless randomlydistributed bothalong thelengthand thepolesof the cells withoutany

visible concentration at constriction sites (not shown).

The apparent discrepancy between the localization of overproducedFtsYbyfractionationversusimmunoelectron microscopy might be caused by a release of membrane- associatedFtsY into the soluble fractionduring disruption of the cells prior to the fractionation procedure.

Alternatively, cytoplasmicFtsYmighthaveaconformation inultrathincryosectionswhich ispoorly recognized bythe anti-FtsY antiserum.

Toexamine thenatureofthe associationof FtsYwith the

cytoplasmic membrane, inverted cytoplasmic membrane

vesiclesderived fromBL21(DE3) expressingFtsYat wild- typelevelswereextractedwith1 MNaCl,4Murea or0.2 M Na2CO3 to remove peripherally associated proteins.

Theseextractionprocedures solubilizedmostbut notall of

the membrane-associated FtsY (Figure 6D). Under these

conditions all SecY which is an integral inner membrane

A

+ + +

+ + + + + +

RBI' -

+ +

1 2 3 4 5 6

7 8

1 2 3 4 5

NIBP

6 7

- p

-M

J.Luirinketat.

.k.A-ild-t.-P

'I 1) S As1 M INI I | I

I 9'.7 _-' _

B. FtsY-overproducer

T 1) S M OM IM

..u..

-h

proteinwasrecovered in the insoluble

(membrane)

fractions (not shown).

Takentogetherthese studiessuggestthat FtsY is located inpart at the cytoplasmic membrane to whichmost FtsY seems peripherally bound.

Discussion

67

4NMW

.*3,..

2( -w- si.4

..

.w^,..

*

2 3 4 5 6

Fig. 6.

Sucellular

localization of FtsY. (A)Fractioi

BL21(DE3) carrying pET9andpLysE(wild-typeF

(B) Fractionationofstrain

BL21(DE3)

carrying pE' (FtsYoverexpression). Cellsweregrownin YTto 660 nmof0.3, induced with 0.4 mM IPTG and^c(

induction. Fractions derived from 0.25

OD6W

units

immunoblotting

using ^anantiserumagainstFtsY.T

D,cellular debris(pelletof low speedcentrifugatio

S,solublefraction; M, totalmembrane fraction; O0 fraction;

IM,

cytoplasmic membrane fraction. (C) I

microscopy ofcellsoverexpressing ^FtsY. Strain BL

pET9-FtsYandpLysSwasgrowninYTtoanabs*

of0.3, inducedwith 0.4 mMIPTGandcollected3 induction. Sectionsofthese cellswereimmunolabel

purifiedanti-FtsY. (D) Solubilizationofperipheral i

proteins. IMVsof strain N4156

(wild-type

level of

wereextracted with 1M NaCl (lanes 1 and2), 4 h

4) and0.2M

Na2CO3

^{(lanes5 and}6). Insoluble

(l4

andsoluble (lanes2,4and6)proteinfractionswer

immunoblotting

usinganantiserum againstFtsY.

E. coli FtsY has beenimplicated both in cell division and in

protein

export.A role in cell division was proposed based onthe location

offtsYin

alocus which affects cell division (Gill and Salmond, 1986; see below). On the other

hand,

the

striking similarity

between the C-terminalregionsof FtsY and the ca-subunit of the mammalianSRPreceptorargues in favor ofa role for FtsY in thedocking and

subsequent

* >: releaseof the

recently

identified E.coli SRP

(Bernstein

et

al., 1989; Romisch

et

al.,

1989).

To examine the function ofFtsY,strains were constructed in which the intracellular levelof FtsY can be varied. Both

2 3 4 5 6

depletion

and

overexpression

of FtsY induced the accumulation of precursor forms of several secreted

proteins

invivo, indicative of

impaired protein

translocationacross the

cytoplasmic

membrane. Precursor accumulation upon FtsY

depletion, appeared

to be specific for

,B-lactamase, OmpF

andRBP and wasalready apparent after 2-3 h of

depletion, suggesting

that it is not an indirect effect of

impaired

cell

growth

orheat shock induction which are

only apparent

at later time

points.

Pre-3-lactamase also accumulated upon

overproduction

ofFtsYwhich could be due to

non-productive

interactions between FtsY and the E. coli SRP.

Furthermore,

f3-lactamase was translocated with reduced

efficiency

in anin vitro translocation assay in the absence of FtsY.

Replenishment

of

cytoplasmic

membrane vesicles

depleted

of FtsY with

purified

FtsYpartiallyrestored the translocation defect inaconcentrationdependent

fashion, giving strong support

tothe notion that FtsY has a function in

protein

translocation.

Depletion

of FtsY also affected cell

morphology.

The

'bulging'

cell

shape

observed is similartothatreportedfor double mutants with defects in both cell division

IftsA(Ts), ftsQ(Ts)

or

ftsI(Ts)]

and cellelongationsystems

[rod4(Ts)

or

pbpA(Ts)] (Begg

and

Donachie, 1985).

It is conceivable that

depletion

of FtsY

indirectly

affects cellshape by

having

aneffectonthe insertionof

proteins

involved in cell division and

elongation

into the

cytoplasmic

membrane.

In

bothftsE andftsX,

whicharelocated in

theftsYEX

gene nationof stran cluster, temperature sensitive mutations have been mapped 'tsYexpression). which cause filamentation at the

non-permissive

temperature T9-FtsYandpLysE

(Gill

andSalmond, 1986; Gibbset

al., 1992). However,

the

anabsorbanceat

classification offtsE

asacelldivision gene is debatable since

ollected

2 hafter the ftsE(Ts)

mutant

forms

filaments

at the non-permissive

wereanalyzed by

'total ell lysate-

temperature only in rich medium (Taschner etal., 1988).

Inafter

cell

lysis).

Furthermore,

filamentation is not

exclusively

correlated with M,outermembrane

cell

division defects and is also observed upon aberrant

[mmunoelectron

expression of factors involved in protein export likeSecA

r21(b

)caty mg (Oliver and Beckwith, 1981) and the constituents ofthe

3o ^min after

E.coli SRP, P48

(Phillips

andSilhavy, 1992) and4.5SRNA led using

affinity

(Poritz etal., 1990;Ribeset

al., 1990). Interestingly,

FtsE inner

membrane

wasshowntobehomologouswith the ATP bindingcassette

FtsY expression) family,

agroup ofprokaryotic and eukaryotic nucleotide

anes1,

^{3 and5)}

binding

proteins that are involved ina

variety

oftransport

eanalyzed by processes(Higginset al., 1990).It is

tempting

tospeculate that FtsY and FtsE

(and perhaps

FtsX), which are all

I

l-

Role of FtsY in proteintargetingin

E.coIi

cytoplasmic membraneproteins, cooperate in the reception and insertion of a subset of proteins at the cytoplasmic membrane.However, we did not observe any accumulation ofpre-3-lactamaseinanftsE(Ts) mutant grown at the non- permissive temperature(data not shown). Thus, elucidation of the functionofFtsE awaits further analysis.

Anothercandidate forperforming a role in theinsertion ofproteins into the cytoplasmic membrane is FtsH which is also amembrane-associatedputative ATPase (Tomoyasu etal., 1993). Thermosensitivefilamentation of aftsH mutant wasshown to becorrelatedwith adecrease in theinsertion of PBP3 into the cytoplasmic membrane (Ferreira etal., 1987). It would be interesting to gain knowledge about the effectsof this mutation on theinsertionand secretion of other proteins.

FtsY appeared to be located in part in the cytoplasmic membranewhich is in agreementwith localization studies by Gill and Salmond (1987) using a maxicell expression system.InspectionoftheFtsYsequence reveals noobvious membrane spanningsegments (Gill andSalmond, 1990). It is conceivable that FtsY interacts with other membrane componentslike FtsE, FtsX or perhaps an as yetunidentified E.coli homolog of the (3-subunit of the mammalian SRP receptor. This might explain the limited number of associationsites in the membrane to which most FtsY seems looselybound. We cannot,however,exclude thepossibility ofa direct interaction of FtsY with phospholipids as was alsoobservedfor theoverallnegatively charged SecA protein (Breukink etal., 1992).

Inconclusion,ourobservationssupport the hypothesis that FtsY is involved in an alternative pathway of protein targeting inE.coli,presumablyas acognate receptor for the SRP at the cytoplasmic membrane. The fact that the constituents ofthe SRP, P48 and 4.5S RNA, and FtsY are all essential in E.coli suggests that this pathway may be crucialforcorrecttargeting of asubset of proteins. In this respect it isinteresting thatproteinsthat do not depend on SecBfor efficient export, like ,B-lactamase and RBP, were most strongly affected by depletion of P48 (Phillips and Silhavy, 1992), suggesting different ways ofchaperoning presecretory proteins in the cytoplasm. Depletion of FtsY seemstobring aboutasecretiondefect of similarspecificity.

A notable exception is pre-OmpF which on the one hand bindsSecB(KumamotoandFrancetic, 1993) but on the other handaccumulatesinFtsY-depletedcells. However, the effect ofdepletion of P48 ontheprocessingofpre-OmpF has not yet been reported (Phillips and Silhavy, 1992).

Inanalogy with themammaliandocking protein, FtsY may also play a role in the co-translational insertion of inner membrane proteins which would prevent the cytoplasmic exposure ofhydrophobic regions in nascent polypeptides.

Weintendtoinvestigate thispossibilitywithspecialemphasis on inner membrane proteins involved in cell division. In addition, futurestudies will concentrate on the interaction ofthe SRP and FtsY and on thepossibleinterplaybetween componentsoftargeting pathways in E.coli.

Materials and methods

Strains,plasmidsandmedia

E.coli HMS174(F-hsdR recARifr)and BL21 (F- hsdSgal) (DE3)were usedforinitialsubcloningand forexpressionofftsY respectively(Studier

etal, 1990). StrainN4156(poUendthy gyrA)wasused fortheconstruction ofastrain withconditionalexpression offtsY (Gellertetal.,1977). Strain

LMC515 [ftsE118(Ts)zhg-1::TnJO] was used to study the effects of inactivating FtsE(Taschner et al., 1988). Strain MM52, aderivativeof MC4100 (F- AlacUl69 araD 136 rpsL thi relA) carrying a secAts5l mutation (Oliver and Beckwith, 1981), was used for theidentificationof precursorformsofseveralpresecretory proteins. StrainJM109was used in routine cloningprocedures (Sambrooket al., 1989).

Plasmids pET9a, pLysE, pLysS (Studier et al., 1990),pAral4(Cagnon etal., 1991) andpDB1(Gill andSalmond, 1990) were used forsubcloning andcontrolledexpressionofftsY.Tostudy the effect of FtsYoverexpression onthetranslocationof,3-lactamase, pACYC177AHaeII wasconstructed bydeletion of the 1.4 kb Haellfragment frompACYC177 (Chang and Cohen, 1978) which iscompatible with pET9a derivatives.

To facilitate in vitro expression, the geneencoding (3-lactamase was subcloned inPET9a-adt,aderivative of pET9a which contains an extended multiple cloning site (MCS) downstream from the T7 promoter. Plasmid pAL2carrying the,B-lactamasegene (LamninetandPluckthun, 1989) was cut withAlwNI and treated with DNApolymerase^{I to}create blunt ends.

Subsequently, the plasmid was cut with NdeI and the resulting small NdeI-AlwNIfragmentencompassingthecomplete(3-lactamasegene was ligated intopET9a-adttreatedwithNdeIandStuIwhich both cut in the MCS.

Cellswereroutinely grown in M9 or in YT mediumsupplementedwith 0.4% glucose. N4156 derivatives were grown in M9 or YT medium supplementedwith0.4% fructose and 0.2% L-arabinosewhen indicated.

Ifrequired,antibioticswereadded to the culture medium(Sambrooket al., 1989).

Generalmethods

RecombinantDNAtechniques were carried out as described by Sambrook etal. (1989). A digoxygeninlabeling and detection kit(Boehringer)was used to probeSouthernblots. DNAsequencingwasperformedusing the Taq Dye Primer Cycle Sequencing Kit and the 373AAutomated DNA SequencerofAppliedBiosystems.

Protein wasdeterminedaccording to Bradford (1976) with bovine serum albumin as standard. SDS-PAGEandimmunoblotting were carried out as described by Bollag and Edelstein (1991). Bound antibodies were visualizedonimmunoblotsbyenhancedchemiluminescence (Amersham).

Overexpressionandconditionalexpressionof FtsY

The T7expression system was used for high level expression of FtsY(Studier etal.,1990).TbeftsYgenewassubclonedfrom pDBI,apBR322derivative whichcontains the complete ftsYEX operon. An NdeIrestrictionsitewas created at thestartingATGofftsYby sitedirectedmutagenesisinM13mpl9 and theresultingNdeIfragmentencompassingthecompleteftsYwascloned into theexpressionvectorpET9a. The resulting plasmid was designated pET9-FtsY.

Astrain which allows theconditional expressionofftsY wascreated by

transformingN4156 toampicillinresistancewithpAral4-FtsY'whichcarries the 5' endofftsY under control of the araBpromoter-operatorcomplex.

Inorder toconstructpAral4-FtsY',thefirst 495 bp of the FtsY coding sequence were amplified by PCR using pET9-FtsY as a template. Primers weredesigned to introduceNcoIandHindmrestrictionsites at the 5' and 3'endsrespectively.Thefragmentwassequenced and cloned between the NcoIandHindmIsites ofpAral4resulting inpAral4-FtsY'.

Purification ofFtsY

FtsYwaspurified fromoverproducing cells. StrainBL21(DE3)carrying pET9-FtsY and pLysE was grown in 11 of YT medium to an optical density at 660nmof0.4andinduced for FtsYexpressionbytheaddition of 0.4 mMIPTG. After 2 h ofinduction, the cells wereharvested,resuspended in 10 ml of 50 mMTris-HCI (pH 7.5) and 10% glycerol (buffer A), frozen inliquidnitrogen and stored at-80'C.Thecellsuspension was thawed andpassedtwicethrough a French pressurecellat8000p.s.i. Celldebris andmembranes were removed in twocentrifugationsteps (5 min at 15 000 gfollowedby 30 min at 165 000 g). Thesupematantwasapplied twice to anFPLC MonoQ anionexchange column(Pharmacia)andeluted with anon-linear gradient of NaCl in buffer A. FtsY eluted at 390 mM NaCl and was furtherpurified by gel filtration using a Superose-12 column (Pharmacia). Themain peak was recovered and analyzed.

In vitro translocation

The invitro transcription,translationandtranslocationreactionswerecarried out basically as described (De Vrije et al., 1987). T7 polymerase (Boehringer)was used fortranscriptionofplasmidpET9-PAL2.TheS-135

extractused fortranslation waspreparedfromN4156::pAral4-FtsY'grown in the absenceofarabinose for 3hwhichreducedtheamountof FtsYto

undetectablelevels. Thesamestrainandconditionswereused for theisolation ofinvertedcytoplasmicmembrane vesicles(IMVs)depletedof FtsY. IMVs