An ATP-binding membrane protein is required forprotein translocation across the endoplasmicreticulum membrane
Deborah L. Zimmerman and Peter WalterDepartment of Biochemistry and BiophysicsUniversity of California, Medical SchoolSan Francisco, California 94143-0448
The role of nucleotides in providing energy forpolypeptide transfer across the endoplasmic retic-ulum (ER) membrane is still unknown. To addressthis question, we treated ER-derived mammalianmicrosomal vesicles with a photoactivatable ana-logue of ATP, 8-N2ATP. This treatment resufted ina progressive inhibition of translocation activity.Approximately 20 microsomal membrane proteinswere labeled by [a32PJ8-N3ATP. Two of these wereidentified as proteins with putative roles in trans-location, a signal sequence receptor (SSR), the 35-kDa subunit of the signal sequence receptor com-plex, and ER-pi80, a putative ribosome receptor.We found that there was a positive correlation be-tween inactivation of translocation activity andphotolabeling of aSSR. In contrast, our data dem-onstrate that the ATP-binding domain of ER-pI80is dispensable for translocation activity and doesnot contribute to the observed 8-N3ATP sensitivityof the microsomal vesicles.
Introduction
Protein transport across cellular membranes isfundamental for organelle biogenesis and cellgrowth. The transfer of large hydrophilic pro-teins across the lipid bilayer is thermodynami-cally unfavorable, and therefore energy must beexpended in the process. In many cases, partof the energy appears to be provided by thehydrolysis of ATP. For example, ATP is requiredfor protein import into chloroplasts (Grossmanet aL, 1980; Flugge and Hinz, 1986) and mito-chondria (Pfanner and Neupert, 1986; Eilers etaL, 1987) and for translocation across bacterialmembranes and the endoplasmic reticulum(ER)' membrane (Hansen et al., 1986; Rothblatt
and Meyer, 1986; Waters and Blobel, 1986;Chen and Tai, 1987; Lill et al., 1989).
Part of the requirement for ATP can be at-tributed to the need to keep substrate proteinsin a "translocation competent" or "unfolded"state, as has been demonstrated for mitochon-drial import (Pfanner et al., 1987) and post-translational translocation across the ER mem-brane in the yeast S. cerevisiae (Chirico et al.,1988; Deshaies et al., 1988). It is likely that thereis a further requirement for nucleotide hydro-lysis to provide the energy for polypeptide chaintransfer across the membrane. However, it hasbeen difficult to address this problem experi-mentally.Translocation across mammalian ER occurs
in at least four discrete steps: signal sequencerecognition by signal recognition particle (SRP),targeting to the ER via the SRP receptor, na-scent chain insertion into the membrane, andsubsequent translocation of the polypeptidechain (Rapoport, 1990). Both SRP and SRP re-ceptor bind GTP (Connolly and Gilmore, 1989;Miller and Walter, unpublished data), and GTPbinding is required to complete the first threesteps of translocation (Connolly and Gilmore,1986; Connolly et al., 1991). Thus, multiplerounds of GTP binding and hydrolysis may en-sure the proper vectorial delivery of the nascentchain to the site of translocation. However, GTPhydrolysis by SRP and SRP receptor probablydoes not contribute to the vectorial movementof the remainder of the nascent chain acrossthe membrane (Connolly et al., 1991).
In studies that further elucidate the nucleotiderequirements for protein translocation, Garciaand Walter (1988) found that there is a require-ment for ATP to translocate pre-elongated na-scent chains across the ER membrane. Simi-larly, Mueckler and Lodish (1986) found that
romycin-treated, salt-washed microsomal membranes;PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodiumdodecyl sulfate-polyacrylamide gel electrophoresis; SRa, thea subunit of the SRP receptor; SRP, signal recognition par-ticle; SSR, signal sequence receptor; TEA, triethanolamine;TpKRMs, trypsinized pKRMs; UV, ultraviolet.
ATP hydrolysis is required to translocate andinsert an integral membrane protein. Thesestudies do not distinguish whether ATP is re-quired by a cytosolic protein that unfolds thepre-elongated nascent chains or whether ATPis used by an ER membrane protein that actsduring translocation. If the second case is true,then there should be at least one ATP-bindingprotein in the ER membrane that is required fortranslocation. We have tested this directly byusing a photoactivatable analogue of ATP, 8-N3ATP, to cross-link the ATP binding proteinsin the membrane and to assess their role intranslocation.
- + + +A- - lx 2x 3x 3x *V- NI IN,* \ !
PIL_ -_w T3I
1 2 3 4 5 t
Bw _gII0-4_
w SSRsS iz
Results
Microsomes photolabeled with 8-N3ATP areinhibited for translocation activity8-N3ATP is an ATP analogue that can be usedto photocross-link ATP-binding proteins. Onexposure to ultraviolet (UV) light, the azidegroup on the probe becomes activated to a ni-trene, and the nucleotide analogue becomescovalently attached to the protein to which it isbound (Potter and Haley, 1983). Thus, ATP-binding proteins that require nucleotide hydro-lysis for activity might be inactivated by thisprocedure. To determine if an ATP-binding pro-tein in the ER membrane is required for proteintranslocation, we asked whether microsomesphotocross-linked with 8-N3ATP are impairedfor translocation activity (Figure 1 A).As shown in Figure 1 A, full length preprolactin
synthesized in a reticulocyte lysate translationextract was efficiently processed to prolactinwhen untreated microsomes were added to theextract (Figure 1A, compare lanes 1 and 2).However, after photocross-linking with 8-N3ATP, microsomes had a reduced capacity fortranslocation; thus, they were 68% active com-pared with untreated membranes (Figure 1 A,compare lanes 2 and 3). After continued pho-tocross-linking, their activity compared withuntreated membranes was reduced to -250/oand finally 3%, as assessed by a decrease inprocessed prolactin and an increase in full-length preprolactin (Figure 1A, lanes 4 and 5).These results suggest that there are micro-somal components involved in translocationthat are sensitive to photocross-linking with 8-N3ATP. Mock-treated microsomes exposed toUV in the absence of 8-N3ATP were almost fullyactive for translocation compared with un-treated membranes (Figure 1A, lane 6), indi-cating that neither UV irradiation alone nor sub-sequent handling of the microsomes resulted
2 3 4 5 6Figure 1. Inhibition of protein translocation activity by 8-NATP correlates with photolabeling of aSSR. (A) Trans-lation/translocation reactions were carried out in the ab-sence of EKRMs (lane 1) or presence of EKRMs that wereeither untreated (lane 2), treated with 5 mM 8-N3ATP (lanes3-5), or mock treated by UV irradiation (lane 6). EKRMswere UV irradiated for 1 x, 2x, and 3x 5 min as indicated.Samples were analyzed by SDS-PAGE. The precursor pPLand processed form of preprolactin (PL) are indicated. (B)The microsomal membranes used in (A) were analyzed byWestern blotting with antiserum against aSSR. aSSR andphotolabeled aSSR (aSSR*) are indicated. When activatedand quenched 8-N3ATP was added separately to a trans-location assay, no effect on translation or translocation wasobserved (data not shown). Thus, the effect observed is nota primary effect on translation or a nonspecific inhibitiondue to the presence of the activated 8-N3ATP.
in a significant reduction in translocation ac-tivity.
If 8-N3ATP is binding to bonafide ATP-bindingsites, then the binding should be competed forby ATP or other ATP analogues. Using a three-fold excess of ATPyS, we found that micro-somes photolabeled with 8-N3ATP were >50%protected from inhibition compared with micro-somes treated in the absence of ATPyS (Figure2A, compare lanes 3 and 4). In this experimentsome degree of inhibition of translocation ac-tivity is expected because binding of 8-N3ATPis irreversible during the time of irradition,whereas binding of ATPyS is reversible. Whenmicrosomes are irradiated with UV in the pres-ence of 5 mM ATP alone, no affect on translo-cation activity was observed (data not shown),thus the inactivation caused by treatment with8-N3ATP requires the presence of the photoac-tivatable azido group.
a signal sequence receptor (SSR) andERp-180 cross-link to 8-N3ATPOur results indicate that at least one ATP-bind-ing protein in the membrane causes an inhibition
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- - - + ATPyS
_ + + 8-N3ATP
pPL= PL
1 2 3 4
BaSSR*aSSR
2 3 4
Figure 2. ATP'yS inhibits 8-N3ATP cross-linking to mem-brane proteins. (A) Translation/translocation assays werecarried out in the absence (lane 1) or presence of EKRMs.EKRMs were mock treated by UV irradiation for 15 min (lane2) or photolabeled with 5 mM 8-N3ATP in the absence (lane3) or presence of 10 mM ATPyS (lane 4). The precursor pPLand processed PL are indicated. (B) The microsomal mem-branes indicated in (A) were analyzed by Western blottingwith antiserum against aSSR (lanes 2-4). aSSR and pho-tolabeled aSSR (aSSR*) are indicated.
of translocation activity when it is cross-linkedby 8-N3ATP. To identify the 8-N3ATP-bindingproteins in the membrane that are the potentialtargets for the inhibition, microsomal mem-branes were photolabeled with [a32P]8-N3ATP,and the profile of labeled proteins was examinedby sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). Approximately 20membrane proteins were cross-linked with theATP analogue (Figure 3, lane 1). All the photo-labeling observed can be competed for by ex-cess unlabeled 8-N3ATP (Figure 3, lane 2), in-dicating that the binding sites for [a32PJ8-N3ATPare saturable. Moreover, ATPyS competed outnearly all photolabeling by [a32P]8-N3ATP (Fig-ure 3, lane 3), indicating that the binding of 8-N3ATP to these proteins was specific.Two of the major [a32PJ8-N3ATP-labeled pro-
teins approximately comigrate with proteinsthat are thought to be involved in protein trans-location: aSSR, a 35-kDa subunit of the SSRcomplex (Wiedmann et al., 1987) and the 180-kDa protein, which we term ER-pl180, identified
as a putative ribosome receptor by Savitz andMeyer (1990). We tested the identity of the[a32P]8-N3ATP-labeled products by immuno-precipitation with antibodies raised againstthese proteins.SSR is an integral membrane glycoprotein
comprised of a 35-kDa a subunit (aSSR) and a22-kDa ,B subunit (I3SSR) (Wiedmann et al., 1989;Gorlich et al., 1990). aSSR was identified byphotoaffinity labeling to be in close proximity to
Mr 1 2 3 4 5 6 7
205O- -ER-p180
116 -
97
66 -
45 - W
aSSR
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I
Figure 3. Analysis of [aIPJ8-N3ATP-labeled microsomalmembrane proteins. EKRMs were photolabeled for 5 minwith 25 zM [a32P]8-N3ATP in the absence (lane 1) or pres-ence of either 15 mM unlabeled 8-N3ATP (lane 2) or 1 mMATPyS (lane 3). EKRMs photolabeled with 25 MM [a32P]8-N3ATP were treated with proteinase K and were analyzedby SDS-PAGE either directly (lane 4) or after immunopre-cipitation with antiserum raised against aSSR (lane 5).EKRMs were photolabeled with 25 MM [a'P]8-N3ATP andprepared for immunoprecipitation with antibodies againstaSSR (lane 6) or ER-pl 80 (lane 7). Note that the degree oflabeling was equivalent for all lanes and the exposure timesare comparable between lanes 1-4 and lanes 5-7. aSSR,ER-pI80, and protein standards (Mr) x 10-3 are indicated.No proteins were labeled when the samples were incubatedwith [a32P]8-N3ATP but not exposed to UV light or when the[a32PJ8-N3ATP was activated and quenched before beingincubated with the membranes (data not shown). Most pro-teins became cross-linked when the exposure time to ac-tivating light was between 1 and 5 min (data not shown),the time scale that is indicative of specific binding (Potterand Haley, 1983). No additional proteins were labeled whenthe time of UV exposure was increased to 15 min (data notshown). Thus, none of the labeling seen is due to the pres-ence of a long-lived reactive group or a secondary reactivegroup created by extended exposure to UV light.
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the nascent chain as it is being translocatedacross the membrane (Krieg et al., 1989; Wied-mann et al., 1987). Although its function is stillunknown, it is thought that SSR is actively in-volved in translocation and may comprise partof a protein translocation channel (Simon andBlobel, 1991). Antibodies raised against aSSR(Gorlich et al., 1990) immunoprecipitate the 35-kDa [a32P]8-N3ATP-labeled product, suggestingthat aSSR itself is an ATP-binding protein (Fig-ure 3, lane 6). aSSR is predicted to have a singletransmembrane spanning domain and a car-boxy-terminal cytoplasmic tail of -5 kDa thatis sensitive to degradation by proteolysis (Prehnet aL, 1990). When [a32P]8-N3ATP-labeled mi-crosomes are treated with proteinase K beforeimmunoprecipitation, a photolabeled product ofaSSR is no longer detected (Figure 3, lane 5),indicating that 8-N3ATP cross-links to aSSR inthe cytoplasmic domain.
In a similar manner we confirmed the identityof the 1 80-kDa cross-linked product as ER-pl 80(Figure 3, lane 7). ER-pl180 was originally iden-tified as a ribosome receptor because a solubleproteolytic fragment derived from this proteininhibits ribosome binding to microsomal mem-branes (Savitz and Meyer, 1990). However, ex-periments done in our lab show that this proteindoes not fractionate with the majority of ribo-some binding sites that can be assayed for inmicrosomal membranes (Nunnari et al., 1991).Thus, the role for ER-pl180 in translocation, ifany, remains to be determined. However, it isintriguing that two of the major [a32P]8-N3ATP-labeled proteins in the ER membrane are impliedto function during translocation and thus arepotential targets for the inhibition of translo-cation activity observed.No photolabeled products were immunopre-
cipitated by antibodies that recognize fSSR, thea subunit of SRP receptor (SRa), signal pepti-dase, or Ig heavy-chain binding protein (BIP)(data not shown). BIP is a soluble protein resid-ing in the ER lumen that is known to bind to ATP(Kassenbrock and Kelly, 1989) and thus mightbe expected to cross-link 8-N3ATP. However,all the cross-linked sites are sensitive to deg-radation by exogenously added protease (Figure2, lane 4), indicating that they are all cytoplas-mically exposed. Thus, under the conditionsused, [a32P]8-N3ATP labels only ATP-bindingsites exposed to the cytoplasm.
Inactivation of translocation activity by 8-N3ATP correlates with photolabeling of aSSRWe observed that when cross-linked to 8-N3ATP, aSSR undergoes a mobility shift when
analyzed by SDS-PAGE (Figure 1 B). We tookadvantage of this mobility shift to assess theextent to which aSSR is modified in membranescross-linked by 8-N3ATP and to compare thiswith the amount of inhibition of translocationactivity observed. Thus, when microsomesphotolabeled with 8-N3ATP were analyzed, wefound that the extent of aSSR cross-linkedqualitatively correlates with the amount of in-hibition of translocation activity observed (Fig-ure 1 B). Thus, after one round of 8-N3ATP la-beling, -30-400/o of aSSR was cross-linked(Figure 1 B, compare lanes 2 and 3). Moreover,the percentage of aSSR cross-linked increasesto >90% after three rounds of 8-N3ATP labeling(Figure 1 B, lane 5). As expected, when micro-somes were mock treated, no mobility shift wasdetected, indicating that the altered migrationis indeed due to cross-linking by 8-N3ATP (Fig-ure 1 B, lane 6). With respect to the role of aSSRin translocation, these findings are only a qual-itative correlation and do not demonstrate thataSSR is the 8-N3ATP-sensitive target requiredfor translocation.We have already demonstrated that the pres-
ence of ATPyS during photocross-linking pro-tects the membranes from the inhibition oftranslocation activity caused by 8-N3ATP cross-linking. Thus, we compared the extent of aSSRcross-linked in membranes photolabeled in thepresence and absence of 8-N3ATP and ATPTS(Figure 2B). Accordingly, when microsomeswere photolyzed in the presence of 8-N3ATPalone, all aSSR was shifted compared withmock-treated membranes (Figure 2B, comparelanes 2 and 3). Moreover, when microsomeswere photolabeled in the presence of both 8-N3ATP and 10 mM ATPyS, the amount of aSSRcross-linked was greatly reduced (Figure 2B,compare lanes 3 and 4).
ER-p180 is proteolyzed from trypsinizedmicrosomesER-p180 has a large cytoplasmic domain thatis extremely sensitive to proteolysis (Savitz andMeyer, 1990; Nunnari et al, 1991). To furthercharacterize this protein with respect to8-N3ATP labeling, we used mild proteolysisconditions to cleave this domain from the mem-brane. Puromycin-treated, salt-washed micro-somal membranes (pKRMs) were treated witha low concentration of trypsin, and the mem-branes were fractionated away from solubleproteolytic fragments by salt extraction andcentrifugation. The protein composition of bothfractions was analyzed with respect to ER-pl180
CELL REGULATION854
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by Western blotting with antibodies raisedagainst this protein (Nunnari et al., 1991; Zim-merman and Walter, unpublished data) (Figure4). All of ER-p180 was recovered in the mem-brane pellet after mock treatment of the micro-somes (Figure 4, compare lanes 2 and 3),whereas three proteolytic fragments were re-covered in the supernatant fraction after trypsintreatment (Figure 4, lane 4). Moreover, neitherintact ER-pl180 nor any detectable degradationproducts pelleted with the microsomes aftertrypsinization (Figure 4, lane 5).To map the site of [a32P]8-N3ATP cross-link-
ing to ER-pl 80, microsomes were photolabeledwith [a32P]8-N3ATP and then treated with tryp-sin as described. The [a32P]8-N3ATP label wasfound to be cross-linked to the trypsin-derivedfragments (Figure 4, lane 8). Thus, the site of[a32P]8-N3ATP binding to ER-pl180 is in the pro-tease-sensitive cytoplasmic domain. In contrastto ER-p180, many other sites cross-linked by 8-N3ATP are unaffected by mild trypsinization(Figure 4, compare lanes 6 and 7). For example,aSSR, which is less sensitive to proteolysis thanER-pl 80, is photolabeled in trypsinized pKRMs(TpKRMs) (Figure 4, compare lanes 9 and 10).
Trypsinized microsomes are sensitiveto 8-N3ATPWe showed above that TpKRMs no longer havethe 8-N3ATP-binding domain of ER-p180, and
thus, this protein should no longer be a targetfor 8-N3ATP in TpKRMs. Therefore, if photola-beling of ER-p180 leads to the inhibition oftranslocation activity that we observe, thenTpKRMs should not be inhibited for transloca-tion activity by 8-N3ATP. To test this, theTpKRMs and mock-treated pKRMs that weredepleted of proteolytic fragments of ER-p180as described above (Figure 4, lanes 1-5) werephotolabeled as described and were assayedfor translocation activity (Figure 5). Because thea subunit of the SRP receptor is required fortranslocation (Walter et al., 1979), but is itselfvery protease sensitive, we used an assay thatallows activity to be restored to membranes de-pleted of SRa by mild trypsinization (Walter eta!., 1979; Andrews et al., 1989). Thus, TpKRMswere inactive for translocation compared withpKRMs, as assessed by protection of prolactinby exogenously added protease (Figure 5, com-pare lanes 2 and 7), but when TpKRMs weresupplemented with SRa translated from syn-thetic RNA, translocation activity was restored(Figure 5, lane 9). A similar result was observedwhen UV-irradiated TpKRMs were supple-mented with SRa RNA (data not shown).As expected, unproteolyzed membranes were
inhibited for translocation by 8-N3ATP treat-ment (Figure 5, lanes 3-5), whereas TpKRMswere inactive for translocation activity both be-fore and after treatment with 8-N3ATP (Figure
rT+ - + + _
Mr T S P s P
+ Trypsi n
205
Figure 4. ER-pl80 is sensitive tomild proteolysis. Material derivedfrom 10 eq of pKRMs (lane 1) or ofsupernatant (S) and pellet (P) frac-tions of mock-treated (lanes 2 and 3)or trypsin-treated pKRMs (lanes 4and 5) was analyzed by Western blotwith antibodies against ER-pl 80.pKRMs were photolabeled with[a32P]8-N3ATP and were preparedfor SDS-PAGE (lane 6) or immuno-precipitation with antibodies againstaSSR (lane 9). [a'P]8-N3ATP-labeledpKRMs were treated with trypsin andwere prepared for SDS-PAGE (lane7) or immunoprecipitation with an-tibodies raised against ER-pl 80 (lane8) or aSSR (lane 10). aSSR and ER-p180 are indicated. Trypsin-derivedfragments of ER-pl 80 are indicatedby asterisks. Protein standards areindicated (Mr) x 10-3.
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D.L. Zimmerman and P. Walter
pKRM1s
----- - + + + + TpKRMs
- Ox Ix 2x 3x 3x Ox 3x Ox 3x 8-N3ATPxr in'
+ + SRa.
Ip
I 2 7, 4 5 6 7 8 9 10
Figure 5. Trypsinized microsomal membranes are inhibited for protein translocation activity by 8-N2ATP. Translocationreactions were carried out in the absence (lane 1) or in the presence of pKRMs (lane 2); pKRMs treated with 5 mM 8-N3ATPfor 5 min one (lane 3), two (lane 4), and three times (lanes 5 and 6); trypsinized pKRMs (lanes 7 and 9); or trypsinized pKRMstreated for a total of 15 min with 8-N3ATP (lanes 8 and 10). Translation reactions were supplemented with SRa whereindicated. All reactions were treated with proteinase K before being prepared for SDS-PAGE. The processed form of prolactin(PL) is indicated.
5, lanes 7 and 8). In contrast to uncross-linkedTpKRMs, translocation competence was notrestored to 8-N3ATP-treated TpKRMs whenSRa was added back to them (Figure 5, comparelanes 8 and 10). Thus, TpKRMs that no longerhave the 8-N3ATP binding site of ER-p180 arestill sensitive to 8-N3ATP treatment, and it isunlikely that photolabeling of this protein is re-sponsible for the inhibition of translocation ac-tivity that we observe.We showed above that TpKRMs are depen-
dent on newly added SRa for translocation ac-tivity (Figure 5, compare lanes 7 and 9). Sup-plying new SRa does not restore translocationcompetence to either TpKRMs (Figure 5, lane10) or pKRMs (Figure 5, lane 6) after treatmentwith 8-N3ATP. Because SRa binds GTP in itstrypsin-sensitive cytoplasmic domain and thisdomain is restored to TpKRMs after 8-N3ATPtreatment, then these data further demonstratethat photolabeling of SRa does not cause theinhibition of translocation activity that we ob-serve.
Discussion
We have shown that microsomes photolabeledwith 8-N3ATP are inactive for translocation. Therequirements for photolabeling are those ex-pected if inhibition is due to 8-N3ATP cross-linking to one or more ATP-binding proteins thatfunction during translocation. Our results fur-ther demonstrate that the target protein(s) is aresident membrane protein of the ER, becausemicrosomes stripped of all ribosomes and
loosely bound cytosolic factors are sensitive to8-N3ATP treatment. We find that there are up-ward of 20 substrates for 8-N3ATP, any of whichcould be responsible for the inactivation ob-served. However, it is intriguing that two of themajor targets for 8-N3ATP are proteins previ-ously proposed to have roles in translocation:aSSR and ER-p180.aSSR is an integral membrane glycoprotein
that forms a complex with another 25-kDa gly-coprotein (Gorlich et aL, 1990). Two types ofexperiments have implicated this protein com-plex in translocation. Photocross-linking studieshave demonstrated that aSSR is in close prox-imity to the nascent chain during translocation(Wiedmann etal., 1987, 1989; Krieg etal., 1989)and monovalent F.b-fragments produced fromantisera raised against aSSR block proteintranslocation in vitro (Hartmann et al., 1989).We have found that aSSR is quantitatively cross-linked in membranes inactivated for transloca-tion. Moreover, when microsomes are partiallyinactivated for translocation activity, the amountof aSSR cross-linked correlates with the inhi-bition of translocation activity observed. Al-though this result is intriguing, more experi-ments will need to be done to determinewhether photolabeling of aSSR causes the in-hibition of translocation activity that we observe.Moreover, it will be important to determinewhether the purified SSR complex has an in-trinsic ATPase activity or whether anotherclosely associated protein actually binds to 8-N3ATP in the membrane, putting the photoac-tivatable azido group in close proximity to aSSR.
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The second 8-N3ATP-binding protein that wehave identified, ER-pl180, has a putative role asa ribosome receptor (Savitz and Meyer, 1990).However, we have demonstrated that micro-somes that have been mildly trypsinized nolonger have the ATP-binding domain of ER-pl 80yet they remain sensitive to 8-N3ATP treatment.Thus, it is unlikely that photolabeling of ER-pl 80causes the inhibition that we observe after pho-tolabeling. Moreover, these results raise doubtsabout whether ER-pl180 plays an essential rolein protein translocation in general. As shownabove, translocation competence is restored toTpKRMs when they are supplemented with SRa.Thus, under the conditions used, the only tryp-sin-sensitive protein required for translocationis SRa. Because the proteolytic products de-rived from ER-pl180 that we can detect range insize from 70-100 kDa (Figure 4, lane 4), it canbe concluded that proteolysis of at least half ofthis protein does not impair microsomes fortranslocation activity.
Previous studies have shown that ATP is re-quired for protein translocation across themembrane of mammalian ER when pre-elon-gated nascent chains are used as a substrate(Mueckler and Lodish, 1986; Perara et al., 1986;Garcia and Walter, 1988). However, these stud-ies could not distinguish whether the ATP re-quirement involved a cytosolic component or amembrane protein. Thus, our results are the firstdemonstration that a putative ATP-binding pro-tein in the ER membrane is required for trans-location.Connolly and Gilmore (1986) found that, in
contrast to the longer chains used in the otherstudies, an 86 amino acid truncated form ofpreprolactin requires GTP, but not GTP hydro-lysis, for translocation. Because GTP is neededfor nascent chain targeting and signal sequenceinsertion (Connolly et al., 1991), then proper de-livery of these chains to the membrane may besufficient to ensure their subsequent translo-cation into the lumen. Thus, it might be ex-pected that 8-N3ATP treatment of membraneswould not affect translocation of short nascentchains. In contrast, we found that 8-N3ATP-treated membranes are blocked for transloca-tion at the level of signal sequence insertion(data not shown). Thus, it is possible that theATP-binding protein(s) that is cross-linked is re-quired for translocation of both long and shortnascent chains. However, more steps might berequired at the level of the membrane to trans-locate the longer chains, and ATP binding andhydrolysis may not be required until a later step.
Our results demonstrate that cross-linking ofan ER protein by 8-N3ATP renders microsomalmembranes inactive for translocation activity.Thus, it might be possible to restore translo-cation competence to 8-N3ATP-treated mem-branes by adding back uncross-linked protein,thereby providing an assay to purify the proteininvolved. We are currently using affinity chro-matography to purify the ATP-binding proteinsfrom the ER membrane and will use the recon-stitution assays currently available (Yu et aL,1989; Nicchitta and Blobel, 1990; Zimmermanand Walter, 1990) to try to complement 8-N3ATP-inactivated microsomes with the purifiedproteins and identify the required component.
Materials and methods
Reagents8-N3ATP, [a!32P, was purchased from ICN Biomedicals (Ir-vine, CA); 8-N3ATP and puromycin were from Sigma (St.Louis, MO); the ECL Western blotting detection system wasfrom Amersham (Arlington Heights, IL).
Preparation of microsomal membranesSalt-washed and EDTA-stripped microsomes were preparedas previously described (Walter and Blobel, 1983) exceptthat stocks of microsomes were stored in a buffer containing10 mM triethanolamine (TEA)-HOAc, pH 7.5, 250 mM su-crose, 100 MM Mg(OAc)2 (buffer A) at a concentration of 3eq/al. One equivalent is defined as the material derived from1 gl of rough microsomal membranes that are at a concen-tration of 50 A 280 units/ml (Walter and Blobel, 1983).
Preparation of pKRMs was adapted from a procedure byAdelman et al. (1973). Rough microsomes were brought toa final volume of 0.5 eq/,l in a buffer containing 50 mM TEA,250 mM sucrose, 10 mM Mg(OAc)2, 500 mM KOAc, pH 7.5,1 mM dithiothreitol (DTT), 1 mM puromycin and incubatedon ice for 1 h, followed by successive incubation for 10 minat 370C and room temperature. The membranes wereloaded on top of a 2-ml cushion [1.8 M sucrose, 50 mMTEA, 1 mM DTT, 100 mM KOAc, pH 7.5, 5 mM Mg(OAc)2]and centrifuged at 40C for 20 h at 40 000 rpm in a SW40rotor (Beckman, Palo Alto, CA). The membranes sedimentingat the interface were collected and resuspended in twicetheir original volume in a buffer containing 50 mM TEA, 250mM sucrose, 1 mM DTT (buffer B). The membranes werepelleted to remove excess sucrose and were resuspendedto their original volume in buffer B. Rough microsomes wereextracted twice with this procedure.
Photolabeling with 8-N3ATPReaction volumes ranged from 50-200 ,l in a buffer con-taining 10 mM TEA, 250 mM sucrose. Mg(OAc)2 was equi-molar with the final nucleotide concentration, and 0.5 mMGTP was included in all reactions. Microsomes were in-cluded in the reaction at a final concentration of 1.5 eq/,l.For each reaction all components except nucleotides and/or 8-N3ATP were mixed together and kept on ice. GTP and/orATPyS were added to the reaction mix just before additionof 8-N3ATP. The samples were transferred to siliconizedwells of a 1/1 6" S/P serological ring slide placed on ice andirradiated with UV light of 366 nm by a hand-held lamp (Min-
Vol. 2, October 1991 857
D.L. Zimmerman and P. Walter
eralight model UVGL-25 from UVP, San Gabriel, CA) at adistance of 3 cm for 5 min. After UV irradiation the reactionswere quenched by addition of an equal volume of buffercontaining 10 mM TEA, 250 mM sucrose, 60 mM DTT andwere transferred to centrifuge tubes fitting a TLA 100.2 rotor(Beckman). The ring slide plate was rinsed with an equalvolume of buffer, and this was added to the correspondingsample. The membranes were pelleted by centrifugation at70 000 rpm for 10 min (trypsinized/mock-trypsinized micro-somes were centrifuged for 15 min). Pelleted microsomeswere resuspended in 3 times their original volume in bufferA and pelleted again under the same conditions. The mi-crosomes were resuspended to 3 eq/til in buffer A and sub-jected to two more rounds of 8-N3ATP treatment as de-scribed. Aliquots of microsomes were saved at each stepfor analysis.
Photolabeling with [a32P]8-N3ATPPhotocross-linking with [a332P]8-N3ATP was carried out asdescribed above with the following differences. The finalreaction volumes ranged from 5-20 ul, and microsomeswere included in the reactions at a final concentration of1.5-2 eq/,Al. Before addition, an aliquot of anhydrous [a'P]8-N3ATP was dried under a gentle stream of nitrogen, resus-pended at 40C in buffer A to a final concentration of 100-200 MM, and immediately diluted into the reaction mixtureto the appropriate final concentration. Where included, nu-cleotides were added to the reaction mix just before additionof [a32PJ8-N3ATP. Samples were UV irradiated, and the re-actions were quenched as described above. Samples wereprepared for SDS-PAGE (Garcia and Walter, 1988) or im-munoprecipitation as described (Krieg et at., 1986).
Translation/translocation assaysRabbit reticulocyte translation extracts were prepared aspreviously described (Jackson and Hunt, 1983). Translationswere programmed with synthetic preprolactin RNA or SRaRNA as described (Andrews et at., 1989). Translocation as-says were as described (Andrews et at., 1989). Reconsti-tution of trypsin-treated microsomes with SRa was as pre-viously described (Andrews et at., 1989).
Protease treatment of microsomespKRMs at a concentration of 2 eq/Al in buffer B were ad-justed to 2 gg/ml of trypsin or 100 yg/ml proteinase K andincubated on ice for 1 h. The protease was inactivated byaddition of phenylmethylsulfonyl fluoride (PMSF) to a finalconcentration of 2 mM, and incubation was continued foran additional 15 min. Trypsinized membranes were pelletedby centrifugation at 40C in a TLA 100.2 rotor at 75 000 rpmfor 10 min and resuspended to 1 eq/,l in a buffer containing50 mM TEA, pH 7.5, 100 mM sucrose, 1 mM PMSF. Themembrane suspension was diluted with an equal volume ofbuffer containing 1 M KOAc, pH 7.5, 50 mM TEA, pH 7.5,1 mM PMSF, underlayered with a cushion of 50 mM TEA,pH 7.5, 500 mM sucrose, and centrifuged for 1 h at 70 000rpm. Membrane pellets were resuspended in buffer B to 0.5eq/al and pelleted again for 60 000 rpm for 1 h. The TpKRMswere finally resuspended in buffer B at a concentration of3 eq/tl.
Immunoprecipitations and Western blottingImmunoprecipitations (Krieg et at., 1986) and Western blot-ting were performed as described (Fisher et at., 1982) withthe following exceptions. The primary antibodies were de-tected using the enhanced chemiluminescent Western
blotting detection system (Amersham). Blots were incubatedwith horseradish peroxidase-labeled secondary antibodiesat a dilution of 1 :10 000 and were detected as described inthe Amersham manual.
AcknowledgmentsWe thank Tom Rapoport for anti-SSRa serum and PabloGarcia and Dieter Zopf for critical reading of the manuscript.D.L.Z. was supported by a grant from the Lucille P. MarkeyCharitable Trust. The work was supported by grants fromthe NIH and the Alfred P. Sloan Foundation.
Received: June 18, 1991.Revised and accepted: July 29, 1991.
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