The methionine-rich domain of the 54 kDa subunit of signal recognition particle is sufficient for the interaction with signal sequences

The methionine-rich domain of the 54 kDa subunit of signal recognition particle is sufficient for the interaction with signal ^sequences

Henrich Lutcke, Stephen High,

Karin Romisch', Anthony J.Ashford and Bernhard Dobberstein

Cell BiologyProgramme, European Molecular BioloVy Laboratory, Meyerhofstrasse 1, 6900Heidelberg, Gernany, and Department of Molecular and Cell Biology, 401 Barker Hall, U.C. Berkeley, Berkeley, CA 94720,USA

Communicated by B.Dobberstein

The signal recognition particle (SRP) binds to signal sequences whenthey emerge from a translating ribosome andtargetsthe complex ofribosome, nascent chain and SRP to the membrane of the rough endoplasmic reticulum (rER) allowing the co-translational translocation of thenascent chain. Byphoto-crosslinking ithasbeenshown that the signalsequence ofpreprolactin (PPL) only interactswith themethionine-rich(NI) domain of the 54 kDa protein subunit (SRP54)of SRP. Here we show that (i) a signal-anchor sequence is likewise crosslinked onlytothemethionine-richdomain of SRP54, (ii) free SRP54 can interact with signal sequences independently oftheothercomponents ofSRP, (iii) its Mdomainsufficestoperformthisfunction, and (iv) an essentially intact M domain is required for signal sequencerecognition.Alkylationof the N + G domain in intact SRP54 with N-ethyl maleimide

(NE),

but not after cleavage with V8protease, prevents the binding of a signal sequence to the M domain. This suggests a proximitybetween the N+ G and M domains of SRP54 andraises the possibilitythat the roleof the N+ G domain maybetoregulatethebindingand/or the release of signal sequences.

Key words: GTPbinding protein/methionine-rich domain of

SRP54/photo-crosslinking/signal

sequencebinding/signal recognition particle

Introduction

Threesequentially performed functions have been attributed

tothe signal recognition particle (SRP): (i) Recognition of andbindingtosignalsequencesofproteins destinedtoenter the 'secretory pathway' upon their emergence from a

translatingribosome(WalterandLingappa, 1986),(ii)arrest or retardationof the elongationofthe nascentpolypeptide (Siegel and Walter, 1985; Wolin and Walter, 1989) until (iii)theinteractionof thecomplexofribosome,nascentchain andSRPwiththedockingprotein(DPca) (orSRPreceptor)

onthemembrane of theroughendoplasmicreticulum(rER) (Gilmoreetal., 1982; Meyer etal., 1982). Upon binding

toDPo! thesignalsequenceisdisplacedfromSRP inaGTP- dependent process (Connolly and Gilmore, 1989; High

etal., 1991a), the elongation arrest is released and translocation of thenascentpolypeptide is initiated(Walter

(C) Oxford University Press

and Lingappa, 1986). SRPis

subsequently

releasedfrom the membrane of the rER in a steprequiring GTP hydrolysis (Connolly etal., 1991).

SRP consists ofa 7S RNA and six different proteins: a 19 kDa(SRP19)and a54 kDa(SRP54)protein both of which are RNA bindingproteins (Lingelbachetal., 1988; Ribes etal., 1990;

Romisch

etal., 1990; Zopfetal., 1990)and twoheterodimers of 9/14kDaand68/72kDarespectively, which each bythemselvesalso bind to 7S RNA(Walter and Blobel, 1983). The 9/14 kDaheterodimeris necessary for the elongation arrest function ofSRP (Siegel and Walter, 1985) while the68/72kDaheterodimerhas beenimplicated in thedockingprocess(Siegel and Walter, 1988). By photo- crosslinking (Kurzchalia etal., 1986; Krieg etal., 1986;

High et al.,

1991b)

and by chemical modification (Siegel andWalter, 1988) SRP54has beenshowntobe theprotein responsible for the interaction of SRP with the signal sequences of nascent secretory and membrane proteins.

Signal sequences for rER targeting and insertion differ widely in primary structure and have as the only common feature anoverall hydrophobic nature (vonHeijne, 1988).

HowSRP54recognizesand binds tosuch signalsequences is poorly understood. The investigation ofthis interaction may serve as aparadigmforothersignal-mediatedprocesses.

Based on

amino

acid sequencehomologies SRP54has been proposedtocontainatleast twodomains, an N+Gdomain (SRP54N +G)whichcontainsconsensus motifs forbinding GTP and a methionine-rich or M domain (SRP54M) (Bernstein etal., 1989;

Romisch

etal., 1989). The postulated domain structure is supported by the results of analyses using limitedproteolysisof SRP andidentification of the resulting fragments with

peptide-specific

antisera against SRP54

(Romisch

etal., 1990; Zopfetal., 1990).

Theseexperimentslocated the RNAbindingsiteofSRP54 tothe Mdomain. Photo-crosslinkingof thesignal sequence of PPL to SRP, combined with limited proteolysis, fractionation and antibody mapping of the crosslinked products showed that SRP54M is also the domain which interactswith thissignal sequence(Zopfetal., 1990; High and Dobberstein, 1991). SRP54M contained in proteolytically cleaved SRP was found still to be capable of recognizing the signal sequence of PPL (High and Dobberstein, 1991). However, the experiment does not exclude thatSRP54N+Gwas

non-covalently

associated with SRP after proteolysis and therefore

required

for

signal

sequence recognition.

Usingasimilarcrosslinking

approach

tothat used before (HighandDobberstein, 1991),weherereport the

mapping

of the binding site on SRP54 for three

signal

sequences.

Lysines modified with a

photo-activatable crosslinking

reagentwere incorporatedat different

positions

relativeto the

hydrophobic

coreof these

signal

sequences.

Thus,

the results were

independent

of the

position

of the

photo-

activatable crosslinker within the nascent chain. In vitro

synthesized

SRP54 was found to interact with

signal

1543

sequences with anefficiency similartothatofSRP54present aspart of thepurifiedSRPcomplex. Theeffect ofN-ethyl maleimide(NEM)modificationandlimitedproteolysisupon the interaction between SRP54 and signal sequences was tested. Using mutant SRP54proteins, deleted atthe NH2- or

COOH-terminus,

it was possible todefine the minimal regionoftheproteinnecessaryforsignalsequencebinding.

Results

The Mdomain ofSRP54 interacts with different types ofsignal sequences

By a

photo-crosslinking

approach (Krieg ^etal., 1986;

Kurzchalia etal., 1986) it has been shown

previously

(Zopf etal., 1990; High and

Dobberstein, 1991)

that thecleavable signal sequence of nascent PPLinteracts with theMdomain ofSRP54. A nascentchain-ribosome

complex

wasformed by translation ofa truncated PPL mRNA ina wheatgerm cell-free translation system.

[35S]methionine

was included as alabel for thenascentchain. Lysyl-tRNA modified with theUV-activatable

crosslinking

reagent

4-(3-trifluoromethyl- diazarino)benzoic

acid (TDBA) was

incorporated

into the nascentchainas ameansto

identify interacting

components by

photo-crosslinking.

The site of

crosslinking

was then mappedbylimited proteolysisand

immunoprecipitation

with peptide-specific antibodies.

As the arrested peptideof PPL contains lysine residues onlyatpositionspreceding thehydrophobiccoreofthePPL signal sequence (Figure 1) it remained unclear whether lysines in other positions in this orother

signal

sequences would interactwith, andthereforebecrosslinked to, other parts of SRP54(High and

Dobberstein, 1991).

Totestthis we usedthe SRP-arrested translation product ofthetype I membrane protein

IMC-CATA27

which contains an uncleaved signal-anchor (SA) sequence close to its NH2-terminus

(Figure

1,

High

et

al.,

199

lb). Crosslinking

reagent is incorporated in this protein

exclusively

COOH- terminal of the hydrophobic core of the SA sequence (Figure 1). Truncated

IMC-CATA27

mRNA codes for the NH2-terminal 103 amino acids of

IMC-CATA27

(IMC-

CAT103A27).

When the mRNA was translated in the presence of SRP one major polypeptide of the expected molecular mass of11 kDa was synthesized,andupon UV irradiation amajor specific crosslinkedproduct of 65 kDa was seen(Figure 2,lane 1) which wasabsentwhensamples were notirradiated (notshown). Asthe 65 kDacrosslinked productcould be

immunoprecipitated

with antisera specific forpeptidesderivedfrom SRP54(Figure 2, lanes3-7)but notwith a control serum(lane 2)itrepresents acrosslinked product between IMC-CAT

03A27

and SRP54 (see Figure lb for the locations of peptides used to raise the antibodiesandRomischetal., 1990).Toidentify theportion of SRP54 interacting with the SA sequence of IMC-

CAT,O3A2,

'we

digested

thepurified crosslinked complexes ofnascentchain, ribosomeand SRP(Figure 2,lane 1) with V8 protease after dissociation of ribosomes with EDTA (lanes 8-14). These conditions had resulted in complete cleavage of SRP54 into its M and N +G domains when PPL86

(NH2-terminal

86 amino acids ofproprolactin) was the binding partner (High and Dobberstein, 1991). After digestionwith V8proteasethemajorcrosslinkedportion of SRP54 had a relative molecular mass

(Mr)

of -21 kDa.

Thiswasdeducedbysubtractionofthe 8 kDacontribution ofthecleaved

IMC-CAT,O3A27

^fromthecrosslinked product 1544

of 29 kDa. This

corresponded

to an

essentially

intact M domain as

judged by

its

immunoprecipitation

with thetwo

anti-SRP54M

(lanes

13 and

14)

and thelack of

precipitation

withthe threeanti-SRP54N+G

(lanes

10-

12)

antisera

(cf.

Figure Ib).

A minor crosslinked

product migrating

^at -43 kDa contained the entire Mdomain

(precipitation by

antisera 982 and

831)

andcontainedaCOOH-terminal

portion

of the G domain as it was also

precipitated by

antiserum 981

(lane 12).

Note that upon

cleavage by

V8 protease IMC- CAT

103A27

lost -3kDa of its

NH2-terminus (lane

8). This

wasdeducedfrom the fact that the

proteolysed

nascentchain

migrated

^{at -}8 kDa andwas no

longer precipitated by

an

antibody

raised

against

its

NH2-terminal

30 amino acid residues

(not shown).

Consistentwith this

cleavage

isalso the

slightly

increased

mobility

ofthe crosslinked

product containing

residual

undigested

SRP54

(Figure

2, lane

8).

Free SRP54 interacts with

signal

sequences

Since

only

SRP54 could be crosslinked to

signal

sequences it seemed

possible

that itwassufficient for

binding

of

signal

sequences. We therefore tested ifSRP54

present

^as free

Fig. 1.(a) Outlineofthe nascentchains used in thisstudy: the synthesis ofthe nascentchains ofpreprolactin (PPL)andIMC-CAT

wasarrestedataminoacids 86(PPL86)and 103 (IMC-CAT103), respectively. Blackboxes indicate the hydrophobiccoresofcleavable

signal ^orSA sequences. Thearrow onthe PPL sequencepointstothe site ofcleavage by signal peptidase. Lysineresidues ( * ) presentinthe

nascentchains ofCOOH-terminallytruncated PPL

(PPLI8),

IMC-CAT (IMC-CATt ) andvariantsofIMC-CAT (IMC-CATIO3-7 and IMC-

CAT103A27 )are foundatpositions 4, 9, 72 and 78ofthePPL86and atpositions27,69, 72, 73, 75,77 and 101 ofIMC-CAT,03. Inthe mutantIMC-CAT-27 thelysineresidueatposition 27is replaced by a

glutamicacidresidue. InIMC-CATP27.46 the lysine residue atposition 27 is replaced byaglutamicacid residueand residue 46 isreplaced byalysine. (b)Antisera raisedagainstpeptides ofSRP54 andused for allimmunoprecipitations in thisstudy: SRP54and its N, G (SRP54N+G)and M (SRP54M) domainsaredelineated bythe borderingaminoacidpositions(numbersin italics). The positions of peptidesusedto raiseanti-peptide antisera 730, 832, 981,982 and831 (Romischetal., 1990)areindicated byarrowheads. Theapproximate positionofcleavagebyV8 protease isindicatedby thearrow

(Romischetal., 1990).

protein, rather than as acomponent of a ribonucleoprotein (RNP) particle, could be photo-crosslinked to PPL86 (Figure 3). SRP54 and, as a control, SRP19 were synthe- sized in the wheat germ system in the absence of [35S]

methionine. In a separate reaction PPL86 was synthesized in the presence of [35S]methionine and NE-4-(3-trifluoro- methyldiazarino)benzoyl-lysyl (e-TDBA-lysyl) tRNA. The two translation mixtures were combined and incubated, whereuponcrosslinking was induced by UV irradiation. In acontrol experiment PPL86(Mr = 9kDa) wascrosslinked tothe SRP54 subunit of added intact SRP (0.5 pmol) to yield the expected 63 kDa product (Figure 3, lane 1; High and Dobberstein, 1991). In thepresence of in vitro synthesized SRP54(Figure 3, lane 5) the majorcrosslinked product co- migratedwiththe 63 kDa of the control and was of similar intensity (Figure 3, compare lanes 1 and 5). However, when the translation mixture ofSRPl9wasincubatedwithPPL86 only a less intense 61 kDa crosslinked product was seen (Figure 3, lane 3,arrowhead). This was also observed when wheat germ lysate which had not been programmed with any mRNA was added (not shown). It corresponds to crosslinked product containing the SRP54 homologue

(SRP54WG)

of the endogenous wheat germ SRP (Prehn etal., 1987). The result suggested that free SRP54 could interact with a signal sequence.

Toexcludethat the in vitrosynthesizedSRP54 hadbound toapartially assembled wheat germ SRP, wereleased the crosslinked complexfrom the ribosome by treatment with puromycinandseparated free SRP54 from SRP by sucrose gradient centrifugation. We chose the centrifugation

conditions such that SRP54 would remain on top of the gradient while SRP would move to the middle of the gradient. When PPL86 crosslinked to SRP was thus analysed the 63kDacrosslinkedcomplexwas,asexpected, found in an llS particle in the middle of the gradient (Figure 4a). In contrast, when in vitro synthesized SRP54 was usedin thecrosslinkingassaythe63 kDacomplexwas found on top ofthe gradient(Figure 4b). Asmall amount of the 61 kDa crosslinked complex containing the endogenous

SRP54WG co-migrated

with the 1iS SRP (Figure 4b, arrow). From thisexperimentweconclude that in vitrosynthesized SRP54doesnotassemble intoanRNP particle and is capable ofrecognizing the signal sequence of PPL86 as a free

protein.

Free SRP54, like its SRP-bound form, interacts with signal sequences via its M domain

Inorder to test whether the interaction between the signal sequences of PPL or IMC-CAT and free SRP54 was the same asthatwithSRP-bound SRP54,wemapped the signal sequence binding domain of free SRP54 by photo- crosslinking and subsequentdigestionwithV8protease. The crosslinked product of PPL86and free SRP54 (Figure 5, lanes 1-8) was digested with V8 protease (lanes9-16), and the signal sequence binding domain was identified by

-IP.

&

I .:,.A ri1A

..:

l

Fig.2. The SA sequenceofIMC-CAT103isexclusivelycrosslinked to

theM domain of SRP54. The SRP- and truncation-arrested nascent chain ofIMC-CATA27 (IMC-CAT1O3A7, 11 kDa band in lane 1) was

photo-crosslinked toSRP54. The total crosslinked productsare shown after purificationbycentrifugation throughasucrosecushion (lane 1).

The purified material afterproteolysiswith 10Ag/ml V8protease in the presence of25 mM EDTA is shownin lane8. Crosslinked productswereimmunoprecipitated before (lanes 2-7)orafter proteolysis (lanes 9-14) with thefollowingantiseraasindicated: non-

related serum(NRS), lanes 2 and9; anti-SRP54peptide antisera730, lanes 3and 10;832, lanes4and 11;981, lanes5and 12; 982,lanes 6 and 13; 831, lanes7and 14.

Fig. 3. Cell-free synthesized SRP54 interacts with the nascent chain of PPL8S. PPL86was synthesized in the presence ofE-TDBA-lysyl tRNA and[

5S]methionine.

SRP(lane 1)orunlabelled cell-free translation mixturesof mammalian SRP19 (lanes2 and3)orSRP54(lanes4and 5)wereadded, and photo-crosslinking was induced byUV irradiation (lanes 1, 3 and5)ornot(lanes 2 and 4). TheUV-dependent crosslinkedproductsofPPL86(9 kDa)toSRP54 contained in SRP (lane 1)oraftertranslationinvitro (lane 5)areindicated (63kDa).

The 61 kDacrosslinked product ofPPL86tothe endogenouswheat germhomologue of SRP54 is indicatedin lane 3 (arrowhead).

1545

Fig. 4. Cell-free synthesized SRP54 isnotassociatedwithanRNP particle. Crosslinked complexes between PPL86and SRP(a) orPPL86 and cell-free synthesizedSRP54(b) werereleased fromtheribosome by puromycin/high salt andcentrifugedthrougha5-20%sucrose

gradient. Equal fractionswerecollected from thetop(fraction 1)to thebottom(fraction 12) ofthegradientand analysedby SDS-PAGE and fluorography. Only theportionsof thefluorograph containing the crosslinked complexes of PPL6with freeorSRP-contained SRP54 (63 kDa)orendogenous SRP54WG(arrow)areshown. Numbers belowthe lanes indicate fraction numbers.

immunoprecipitation using five SRP54anti-peptideantisera (cf. Figure lb).

As expectedimmunoprecipitationwithallfive antisera of theundigested crosslinkedmaterialyieldedaproductof63 kDa (Figure 5, lanes4-8)whichwasalsoprecipitatedby

a prolactin-specific antiserum (lane 2). Digestion with V8 proteasedid notalterthe apparentMrofPPL86 (Figure 5,

compare9kDa band in lane 10 with that in lane2)although

most ofthe 63 kDa crosslinked product was proteolysed (lane 10). As had been observed with SRP (High and Dobberstein, 1991;andFigure 2)the63 kDacomplexwas

digested to a major product of 30 kDa. This must be composed ofthe9 kDa PPL86 peptide and the 21 kDa M domain ofSRP54as itwas recognized only by antibodies specific for SRP54M (Figure 5, lanes 15 and 16) but not by those specific forSRP54N+G (Figure 5,lanes 11-14, cf. Figure lb). A weaker crosslinked complexof41 kDa comprises theentire MdomainandtheCOOH-terminalpart ofthe N+Gdomain asit wasalso immunoprecipitatedby both Mdomain-specificantisera andbyantiserum 981 which recognizes theCOOH-terminal portionofthe N +G domain (Figure 5, lanes 14-16; cf. Figure lb). An experiment using IMC-CAT103A27 as the crosslinking partner, instead ofPPL86, yielded similarcrosslinking products containing SRP54M(notshown).Thus the site ofinteractionofasignal

sequencewithfreeSRP54orwithSRP-boundSRP54 is the

same.

The Mdomain of SRP54 alone is sufficient for interaction with signal sequences

So far allassaysthat tested thebindingofSRP54tosignal

sequencescontainedthe N+G domain eitheraspartofintact SRP54^orpresent in the reactionmixture afterproteolytic cleavage (High and Dobberstein, 1991). Thus, a non-

covalentrequirementforSRP54N+Gduringtherecognition ofsignal sequences couldnotbeexcluded. In ordertotest ifsignal sequencescouldbe recognized bySRP54M alone

we removed or inactivated SRP54N+G.

Proteolysis of SRP using V8 protease liberates SRP54N+G from SRP54M which remains bound to the RNP particle and can be quantitatively separated from 1546

SRP54N+G

by absorption

toDEAE-

Sepharose (Romisch

et

al., 1990; Zopf

et

al., 1990).

Whenwetested

proteolysed

SRP

lacking

SRP54N +G the M domain was

efficiently

crosslinkedto

PPL86 (not shown)

as

previously

found for theunfractionated material

(High

and

Dobberstein, 1991).

This

suggested

that the N +G domain of SRP54 was

dispensable

for the interaction of SRP54M with

signal

sequences.

Inordertodetermine if SRP54M

alone,

i.e. in theabsence of 7SRNA and the other SRP

proteins,

was

capable

of

signal recognition

we

synthesized

unlabelled SRP54 in vitro as

above

(Figures

3 and

4)

and

digested

itwith V8protease.

After

crosslinking

to

PPL86,

the

interacting

componentwas

identified

by immunoprecipitation using

antibodies

recognizing

both mammalian and wheat germSRP54N +G

(antiserum 730)

or

specific

for the COOH-terminus of mammalianSRP54M

(antiserum 831). Figure 6,

lanes 5 and 6 show that the 30 kDa

proteolytic fragment

of the crosslinked

complex

between SRP54 and

PPL86

was

recognized by

antiserum 831

(Figure 6,

lane

6),

butnot

by

antiserum 730 which

only immunoprecipitated

the intact

endogenous SRP54WG (lane 5, arrow).

Thus we conclude thatSRP54M of V8 protease

digested

SRP54caninteract with the

signal

sequence of

PPL86

while SRP54N +G cannot.

The N+G domain was stillpresent in themixture after

proteolysis

and couldthushave assisted in

signal

sequence

binding.

In ordertoinactivatethisdomainwemadeuseof theobservationthat intactSRP54isinactivated

by alkylation

of

cysteine

residues with NEM

(Siegel

andWalter,

1988),

and that the five

cysteine

residues present in mammalian SRP54areexclusivelylocated in the N+G domain

(Romisch

et

al., 1989;

Bernsteinet

al., 1989).

ReconstitutionofSRP with NEM treated SRP54

(SRP54NEM)

had rendered it inactive in assays for elongation arrest and

protein

translocation and prevented its binding to

polysomes containing

PPL nascentchains (Siegel and Walter,

1988).

Consistent with these

observations,

we found that

PPL86

could notbe

photo-crosslinked

to NEM treated SRP

(not shown).

As the M domain does not contain any

cysteine

residues the NEM modified N+G domain was likely to

impair

the

binding

of

PPL86

to the M domain.

To address this possibility, cell-free synthesized SRP54 was mock treated (Figure 6, lanes 1 and2), treated with NEM

(lanes

3 and

4),

withV8protease (lanes5 and 6)or with both

(lanes

7 and 8) and assayed for the capacity to bind the

signal

sequence of

PPL86

by crosslinking. The

resulting

complexeswereimmunoprecipitatedwithantisera 730

(Figure 6,

lanes

1,

3, 5 and7) and 831 (lanes 2, 4, 6 and

8).

Both antisera immunoprecipitated the 63 kDa crosslinked product of PPL86 and in vitro

synthesized

mammalian SRP54(Figure 6, lanes 1 and2). Thisproduct

was not observed when the translation mixture ofSRP54 wastreatedwith NEM(Figure 6, lanes3 and 4). The wheat germ

homologue

of SRP54 was present in all of the crosslinkingassays inanunmodified form due to the addition of the

PPL86

translation mixture. This gave rise to the 61 kDa crosslinked complex which was

immunoprecipitated

under allconditions

by

antiserum 730 (Figure 6, lanes 1,

3,

5 and 7, arrow). As already mentioned, digestion of SRP54with V8 proteaseresulted in a30 kDa crosslinked product of PPL86 and SRP54M which was immuno- precipitatedbyantiserum 831 (Figure 6, lane 6) but not by antiserum730 (lane 5). NEM treatment of the V8digested

e

^- .

Fig. 5. The Mdomain of cell-free synthesized SRP54interacts with thesignal sequence of PPL86. Crosslinkedcomplexesbetween nascent PPL86 and cell-free synthesized SRP54were madeasdescribed inthe legendtoFigure 3and purifiedbycentrifugation through asucrose

cushion. Thetotal purified material (lane1)wasmock-digested (lanes 1-8) ordigestedwith 10 tg/ml V8protease inthepresenceof 25 mMEDTA (lanes 9-16) andimmunoprecipitated withthefollowing antisera:anti-prolactin (lanes 2 and 10); pre-immune serum831 (lanes 3 and 11); the anti-SRP54 peptide antisera 730(lanes4and 12); 832 (lanes5and 13); 981 (lanes 6 and 14); 982(lanes7 and 15);and 831 (lanes8and 16); noimmunoprecipitation (lanes 1 and9). See Figure lbforthelocations of the epitopes recognized bythe antibodies. Thetotalandimmunoprecipitatedmaterial analysedby SDS-PAGE andfluorography is shown. Thecrosslinked products between PPL86 and intact SRP54(63 kDa) orproteolytic fragments thereof(41 and 30kDa)areindicated. Lanes3-8 and 11-16were

fluorographedfive timeslonger than lanes 1, 2, 9 and 10.

mixturedidnotimpaircrosslinkingofSRP54M (Figure 6, lane 8),whereas itcompletely inhibitedanycrosslinkingto either intactSRP54 (lanes3and 4)orresidual intact SRP54 presentafter V8digestion (lanes5 and 6) andvisible in the non-alkylated samples after long ^exposure (not shown).

Thus, the NEMmodified N+Gdomain canonly interfere with thebinding ofasignalsequence if it is^partofSRP54, consistent with the N+Gdomain playing nodirectrole in signal ^sequence binding.

The regions of SRP54Mrequired forbinding to 7S RNA andsignalsequences largely overlap

Besides being sufficientforinteractingwithsignalsequences,

SRP54Malone is alsocapableofbindingto7SRNA inthe presence of SRP19 (Romisch etal., 1990; Zopf etal., 1990). Thebindingsites have beenproposedtobe formed byfouramphipathichelicesinteractingviatheirhydrophobic faces with the highly variable signal sequences (Bernstein

etal., 1989) and viatheirhydrophilic ones with 7S RNA

(HighandDobberstein, 1991). Inorderto mapthebinding

Fig. 6. NEM alkylationofSRP54butnotof V8cleaved SRP54 preventsthebindingofPPL86toSRP54M. SRP54synthesized inthe wheatgermcell-free systemwas mock-digestedorcleaved intoits N+G and Mdomains by incubationwith V8 proteaseafter(lanes

1-4) orbefore(lanes5-8) inactivationby diisopropyl

fluorophosphate, respectively. Both, SRP54and proteolysedSRP54

were treated with NEMafter (lanes 1, 2, 5 and6) orbefore(lanes3, 4, 7 and 8) the addition ofdithiothreitol. Afterphoto-crosslinking to nascentPPL86 crosslinkedproductswereanalysed by

immunoprecipitation withantiseraspecificforSRP54N+G (antiserum 730, lanes 1, 3, 5 and 7)orSRP54M (antiserum 831, lanes 2, 4, 6 and 8), SDS-PAGEand fluorography. Crosslinked productsofPPL86 withmammalian SRP54 (63 kDa) orSRP54M(30kDa) and

endogenousSRP54WG (arrows)are indicated. Note thatthe wheat germextract in whichPPL86had been synthesized was nottreated with NEM.

sites for 7S RNA and signal sequences more closely we

deleted various portions of the M domain of in vitro synthesizedSRP54and thentestedthedeletedproteinsfor thesetwobindingactivities. Figure7adepictsthedeletions ofSRP54and thepositionsofthe fourpredicted amphipathic helices (Bernstein etal., 1989). SRP54 truncated from its COOH-terminus by 19, 42, 65, 90 and 140 amino acids ('54-2' to '54-6') or a segment ('54-7') comprising the

COOH-terminal 154 amino acidsofSRP54M(aminoacids 351-504) were used in both tests. Inaddition, we tested the in vitro synthesized M domain ('54-M', amino acids 297-504) which had been sufficient for binding to 7S

RNA-SRP19 (Romisch etal., 1990) for its capacity to interact with signal sequences.

Binding ofSRP54, and truncations thereof, to 7S RNA

was monitored by batch absorption ofassembled RNPto

DEAE^-Sepharose as described previously (Lingelbach 1547

O

S

Fig. 7. DeletionmappingofSRP54M. (a) SRP54 andits domiiains areschematically representedasin Figurelb. Theapproximate position of cleavage byV8 protease is indicated byan arrow (cf'. R6nmischet fl., 1990). The positions ot' peptides used toraiseanti-peptide antisera730, 832.

981, 982 and831 areindicated byarrowheads. COOH-terminal (54-2 to54-6) and NH,-terminal (54-7and 54-M)truncationsofSRP54 (54-1)are represented by lineswith numbers in italicsindicating the positions of the lastor first and lastamino acids. Proposed amphipathic helices(Bernstein etal., 1989) aredepicted by short lines labelled h to h3b. Thebindingactivities to 7SRNA and signal sequences (SS) ofthetruncated SRP54 proteinsareindicated by + and -. The + indicates inefficient binding. * BindineL to7S RNA ofin vitrosynthesizedSRP54M('54-M') had been demonstrated previously (R6rmischet of., 1990). (b) Binding ofSRP54 ('54-1')and truncated SRP54 proteins ('54-2' to '54-5')inthe presenceof SRPl9 (arrow)to 7S RNA orbovine liver tRNAwas tested by batch absorption ofRNP particlesto DEAE-Sepharose. 20% of the total input ('T'), all of the DEAE-unbound material ('U')and all ofthe bound andeluted material ('B') were TCA precipitatedandanalysed by SDS-PAGE and fluorography. Numbersonthe right indicate the relative molecular imlassesofmarkerproteins. Incomplete cleavage resulted inthe residual full- length SRP54 observed with deletion 544 which howecer, did notcompetewith binding of54-4. (c) Quantificationofthe relative amountsof DEAE-unbound (U) and -bound (B)material: the original fluorograph wasscanned with anLKB Ultroscan XL laserdensitometer, andbindingto tRNA-SRPl9 (blackbars)or 7SRNA-SRPl9 (hatched bars) wasexpressedasthe percent ofthecombined bound and unbound fractions for each SRP54deletion (% bound ⁼ 100 B[U+B]-1, ordinate). Deletion mutants (54-2 ^to 54-7)^ofSRP54 (54-1) ^are indicated^ontheabscissa. The more

efficient bindingto 7S RNA ofSRP19 (mean + SD: 65.0% + 2.5%)than ofSRP54 (mean SD of 54-1 to 54-4: 40.5% + 4.4%) has been consistently observed in repeated experimentsindependently oftheamount ofproteinsynthesized and has beendiscussed previously (R6mischetal.,

1990).

etal., 1988; Romisch etal., 1990). Briefly, 7S RNA or tRNA (as anegative control) andSRPl9 were mixed in an estimated 16-fold molarexcesswith SRP54 oritsdeletions and allowed to form RNPs. An aliquot of the 'total' (Figure 7b: T) andthe DEAE-'bound' (B) and -'unbound' (U) material were analysed by SDS-PAGE and

fluorography.

The results of the RNA binding assay are 1548

displayed in Figure 7b, quantified by scanning of the fluorograph inFigure 7c andsummarizedinFigure 7a('7S RNA'). BindingofSRP54to7SRNA-SRP19 still occurred when the COOH-terminal 65 amino acids were removed fromitsMdomain(Figure 7a-c, '54-2' to '54-4')but was no longer possible when 90 or more amino acids were deleted(Figure 7a-c, '54-5' and '54-6'). While the entire

Fig. 8. Photo-crosslinking ofsignal sequencesto SRP54 and its truncations: PPL.6^wasphoto-crosslinked to unlabelled cell-free

synthesized SRP54 (54-1) anditstruncations (54-2 to54-7. outlined in

Figure7a) (lanes3-8. 11 and 12). toendogenous components of the wheatgerm lysate (lanes I and9)and toSRP (lanes 2 and 10).

Crosslinked complexes ^were immunoprecipitated withanti-peptide antiserarecognizinganepitope inthe NH,-terminal (982. lanes 1 -81

orthe COOH-terminal region (lanes 9- 12) ot SRP54M andspecific forthe mammalian SRP54. Theproducts wxereanalysed hb

SDS-PAGE and fluorography. Positions of molecular maiL.ss imiark-ers are indicatedonthe right.

M domain ('54-M') had been found to bind to 7S RNA-SRPl9(R6mischetal.. 1990). bindingnolongerwas

possible when the 53 NH,-terminal amino acids of SRP54M were removed (Figure 7a and c. '54-7').

The same truncated SRP54 proteins were also tested in

crosslinkingassays (inthe absence of 7S RNAand SRP19) for their abilities to interact with the signal sequences of PPL86(Figure 8), IMC-CAT103 and

IMC-CAT103<A27,46

(not shown). Crosslinkedproductswereimmunoprecipitatedwith theappropriateantisera, i.e. antiserum982 for SRP54-1 to

54-6 (lanes 3-8) and antiserum 831 for SRP54- 1 and SRP54-7 (lanes 11 and 12). Since neither antiserum

precipitatestheendogenouswheatgerm SRP54homologue

nocrosslinked complexes wereobserved when onlywheat germlysate(WG)wasadded(Figure 8, lanes 1 and9). Both antisera precipitated SRP54 which had been added to the

crosslinking reactions as a positive control and was either

part ofpurified SRP ('SRP', Figure 8. lanes 2 and 10) or

derivedfromtranslation in vitro('54-1', Figure 8. lanes 3

and ¹1). Of theCOOH-terminally truncatedproteins. only SRP54-2 interacted with the signal sequences of PPL86 (Figure 8, lane4), IMC-CATt) orIMC-CAT0 ;'2746(not shown). Since in SRP54-2 only the COOH-terminal 19

amino acids had been removed from SRP54 only a small shift in Mr of -2 kDacan be seen (Figure 8, cf. lanes 3

and 4; see also Figure 7b). All further COOH-terminal

truncations (54-3to54-6)abolished thecapabilitytointeract witheither of the three

signal

sequences tested. The minor crosslinked product seen inlane 6of Figure 8 (63 kDa) is that of incompletely truncated full-length SRP54 (cf.

Figure

7b

'54-4').

The signal sequence binding capacity of in vitro synthesized SRP54M

('54-M',

Romisch et al., 1990) was foundto be low (notshown) whiletheCOOH-terminal 154

amino acids

of SRP54

('54-7T)

were

incapable

of binding any of the signal sequences (not shown and Figure 8, lane 12). Since the M domain derived from in vitro synthesized SRP54by V8proteolysis showed efficient signal sequence binding (Figure 6) this suggests that in vitro synthesized M

domains

may not be able to fold into the correct

conformation for

optimal signal sequence binding.

Discussion

Photo-crosslinking was

combined

with limited proteolysis and mapping with site-specific antibodies or deletion

mutagenesis

to characterize the interaction between

signal

sequences and SRP54. Previously, thisinteractionhadonly been investigated

for

the cleavable signal sequence of

preprolactin.

and the M domain of SRP54 was the only detectable site ofinteraction (Zopfetal., 1990; High and Dobberstein, 1991). SinceuncleavedSA sequences, which can mediate the

membrane

insertion oftype I and type II

membrane

proteins, also interactwithSRP54 (High etal.,

1991

b) it wasof interestto know ifthey alsocontactedthe same

domain

ofSRP54. Forthe type I SAsequenceofIMC- CAT

we

showherethat it also interactswith the Mdomain ofSRP54. Thus, both acleavable signal sequence and an

uncleaved

SA sequence are only in contact with SRP54M and not

with

SRP54N+G.

In PPL the lysine residues are found only NH,-terminal of thehydrophobiccore of thesignalsequence. Bycontrast, in IMC-CAT 27the lysine residues from whichcrosslinking canoccurareonly foundCOOH-terminalofthehydrophobic core of the SA sequence. Thus, crosslinking from these

molecules

canoccurfromeitherside of thehydrophobiccore

of

the signal sequence. As both proteins were crosslinked to the M domain but not to the N+G domain, it is likely that over their

entire

length signal sequences are only in contact with the M domain of SRP54.

Sincetheonly component of SRPdetectedtointeractwith signal sequences was

SRP54.

it was conceivable that free SRP54 alone was sufficient to perform this function. This was indeed found to be the case since proteins bearing a cleavablesignal sequence oraSA sequencewereefficiently crosslinked to

ini vitro

synthesized free SRP54. SRP54 synthesized in this cell-freesystem was not part ofaRNP particle. This wasclearlydemonstrated by sucrosegradient analysis

which

showed that in vitrosynthesized SRP54did not

migrate

as an 1 IS RNP particle but as a free protein.

The site of interaction ofsignal sequences with free SRP54

was found

to be the

same

as that with SRP-bound SRP54 since again only SRP54M was found in contact with the signal sequencesof PPLorIMC-CAT. Thecrosslinkingof

signal sequences

tofreeSRP54 andSRP-bound SRP54was of roughly similar efficiency, since comparable molar quantities ofin

*vitro synthesized

and

particle-bound

SRP54 (as tested by

immunoblotting;

^not shown) resulted in crosslinked products of similar intensities.

SRP54M retained intheRNP

particle

andevenin its free 1549

form was found to interact with signal sequences. The presence of SRP54N+G was not required for this interaction, although alkylation of theN+Gdomainof intact

SRP54

inhibited signal sequence binding. In a

previous

study, alkylation of SRP54 with NEM

(SRP54NEM) followed

by itsreconstitution intoSRPrendered this

particle

unable to promote elongation arrest of PPL or to bind to acomplexofribosomeandnascent chain (Siegel and

Walter,

1988). This was interpreted as

SRP54NEM

being unable to recognize signal sequences. Byphoto-crosslinking we show that this is indeedthe case, and that free SRP54NEM unlike its unmodified form is unable to interact with

signal

sequences. The physical separation of SRP54N+G from SRP54M bycleavage with V8 protease priortoalkylation allowedthe subsequent cross-linking ofSRP54M to

PPL86.

In canine SRP54 only the N+G domain can be

alkylated

by NEM since all cysteine residues, the targets for modification by NEM, arefound in this domain and none are present in the M domain.

What could be the mechanism by which the NEM modified N+G domain inhibits the binding of a

signal

sequence to SRP54M? At leasttwo scenarios can

explain

this finding. (i) TheN+Gdomaincouldbeclosetothe

signal

sequence binding siteofthe Mdomain such that alkylation could then block the entry of a signal sequence into its binding site on SRP54M by steric hindrance.

(ii) Alternatively,

alkylation ofany one cysteine residue could interfere with a GTP-related function oftheN+G domain which contains consensus motifs typical for GTPases (Bernstein et al., 1989;Romisch et al., 1989). In vivo this domain may function to regulate the recognition and/or binding of signal sequences to the M domain

by

conformational changes induced by its GTP/GDP

binding

state. In either case, the removal of SRP54N+G

by

proteolysis would allow SRP54M to resume its function.

Fine mapping of the signal binding site on SRP54 using COOH-terminally deleted SRP54 proteins revealed that nearly the entireM domainwasrequired forbinding tothree different signal sequences. Since the binding of a

signal

sequence wasdeduced from its ability to becrosslinked we used several nascent chains with lysine residues at various positions. This madeit unlikely thata truncated M domain capable of signal sequence binding could notbecrosslinked to the nascent chain and could therefore escape detection.

For all signal sequences tested only the 19 most COOH- terminal amino acid residues ofSRP54 could be deleted without destroying its signalsequencebindingfunction. For binding to 7S RNA-SRPl9 a slightly larger fragment comprising the 65 most

COOH-terminal

amino acids of SRP54 wasfounddispensable. SRP54M createdby cleavage ofSRP54withV8proteaseefficiently boundtoboth

7S

RNA (R6misch et al., 1990; Zopf et al., 1990) and to signal sequences. Based on secondary structure predictions the primary amino acid sequence ofSRP54Mhasbeenproposed to form four amphipathic helices the

hydrophobic

faces of which might constitutea binding pocketforsignal sequences (Bernstein et al., 1989). It was furthermore proposed that the hydrophilic faces of the helices could interact with 7S RNA (High and

Dobberstein,

1991). This implies that the RNA andsignalsequencebinding functionsofSRP54would overlap on SRP54M.

Alternatively,

the RNA and signal sequencebinding functionscouldbeperformedbyseparate

sub-domains.

Ourresultsareconsistentwith theformerview

namely

that

largely overlapping regions

of SRP54M are

essential for both RNA and

signal

sequence

binding.

Our

finding

that

free,

i.e.

RNP-unbound SRP54

can

interactwith

signal

sequencesraises the

possibility

that the function ofSRP may

require

the

sequential assembly

and

disassembly

ofSRP54 with therestof the RNP

complex.

The

possibility

thatafreeformofSRP54hasarole inSRP- mediated

targeting

^as

proposed by Rapoport (1990)

should

now be considered.

Materials and methods

Materials

T7 RNA polymerase, RNase H, V8 protease, Klenow and restriction enzymeswerefromBoehringerMannheimGmbH(Mannheim,Germany).

Taq polymerase was from Perkin Elmer Cetus Co. (Emeryville, CA).

[35S]Methionine was from Amersham Buchler GmbH (Braunschweig, Germany). Cycloheximide,emetine,7-methyl-guanosine 5'-monophosphate

and puromycinweresupplied by SigmaChemical Co. (StLouis, MO).

DEAE-Sepharose CL 6B was from Pharmacia. 4-(3-trifluoromethyl- diazarino)benzoic acid(TDBA)was agiftfrom DrJosefBrunner,Swiss Federal Institute of

Technology,

Zurich, Switzerland.

SRP54 deletions

Deletion mutants 54-2 to54-4^were made by annealingcomplementary oligonucleotidesinlargemolarexcesstothe invitrotranscriptofSRP54 atthepositiontobecleaved and

incubating

it intranslationbuffer with RNase H (0.5 U/25 itl) for 15 minat37°C. Asdeduced from thepositions of the

complementary

oligonucleotides the SRP54proteins, 54-2, 54-3 and 54-4,werearrestedattheCOOH-terminiofaminoacids484,462 and442,

respectively.Deletions54-5 and 54-6wereconstructedonthecDNA level

bysubstitutingafragment^3'oftheuniqueBglIIsite inpSRP54-1 (Romisch

etal., 1989) withcorrespondingPCRfragmentscoding forstopcodons

atamino acidpositions415 and363, respectively. Deletionmutant54-7

wasconstructedbyremovingthe cDNAsequencecodingforSRP54N+G uptotheBglIIsite inpSRP54-1 (Romischeral., 1989). The first codon formethionine

following

theBglIIsite(aminoacidposition351)wasvery

efficientlyused astheinitiationsite in the wheat germtranslation system

yielding

a 17 kDaprotein (not shown).

Transcriptionandtranslation

Transcription of pSRP54-1 and deletion constructs thereof with T7

polymerasewasperformedasrecommendedbythemanufacturer. For the

photo-crosslinking assaystruncated mRNAcodingforthe NH,-terminal

regionsofpreprolactin (PPL86)and theartificialtypeImembranesignal

anchor protein IMC-CAT and its variants (Figure 1) was prepared as

previouslydescribed(Highetal., 1991b).Translationsin the wheatgerm cell-free translationsystemwerecarriedoutaspreviouslydescribed(Stueber

etal., 1984).

Photo-crosslinking

analysis

For

photo-crosslinking

translationwasperformed^asdescribedbyStueber etal. (1984)inthe presenceof3.75pmol E-TDBA-Lys-tRNAper25 izl oftranslationmixture.TheE-TDBA-Lys-tRNAwaspreparedasdescribed

by Wiedmannetal. (1987)except that the modified Lys-tRNA wasnot submitted ^to benzoyl DEAE-cellulose chromatography. Cell-free translations of

PPL86

and

IMC-CAT,03

werepulsed (Kriegetal., 1986) byadditionof7-methylguanosine5'-monophosphatetoafinal concentration of2mMafter 10 min ofincubation.Translationswerecontinued forafurther 5 min and thencycloheximide added to 2mM to prevent further chain

elongation. Translation oftruncated

nmiRNA

yieldsnascent chains which due to the lack ofastopcodon largely remainboundto theribosomes.

Whenpurified SRPwas used it was present duringthetranslation at,

oradded after thecompletionoftranslationto,afinal concentrationof25 nM. When unlabelled SRPproteins made by cell-free translation were

includedtheywereaddedatanequalvolume of translationmixturetothat ofthecompleted synthesis ofPPL86^or

IMC-CAT103.

After the addition of SRPorcell-freetranslationproductstothenascent

chain^-ribosome complexthe mixturewas incubated at 25°C for5 min and then chilledonice. Subsequentirradiationofthesamplesonicewas

aspreviouslydescribed(Highetal.,199lb).After irradiationthesamples

were TCAprecipitated fordirect analysisor immunoprecipitatedunder denaturingconditionsusingpeptide-specific antisera (Romischetal., 1990).

When crosslinked productsorribosome-nascentchaincomplexeswere

1550