Stop-and-Move of a Marginally Hydrophobic SegmentTranslocating across the EndoplasmicReticulum Membrane

Yukiko Onishi†, Marifu Yamagishi†, Kenta Imai, Hidenobu Fujita, Yuichiro Kida and Masao Sakaguchi

Graduate School of Life Science, University of Hyogo, Kouto Ako-gun, Hyogo 678–1297 Japan

Correspondence to Masao Sakaguchi: sakag@sci.u-hyogo.ac.jphttp://dx.doi.org/10.1016/j.jmb.2013.05.023Edited by J. Bowie

Abstract

Many membrane proteins are cotranslationally integrated into the endoplasmic reticulum membrane via theprotein-conducting channel, the so-called translocon. The hydrophobic transmembrane segment of thetranslocating nascent polypeptide chain stops at the translocon and then moves laterally into the membrane.Partitioning of the hydrophobic segment into the membrane is the primary determinant for membraneinsertion. Here, we examined the behavior of a marginally hydrophobic segment at the translocon and foundthat its stop-translocation was greatly affected by the C-terminally attached ribosomes. The marginallyhydrophobic segment first stops at the membrane and then moves into the lumen as long as the nascent chainis attached to translating ribosomes. When it is released from the ribosome by the termination codon, themarginally hydrophobic segment does not move. Puromycin or RNase treatment also suppressed movement.The movement was reversibly inhibited by high-salt conditions and irreversibly inhibited by ethylenediami-netetraacetic acid. There is an unstable state prior to the stable membrane insertion of the transmembranesegment. This characteristic state is maintained by the synthesizing ribosome.

© 2013 Elsevier Ltd. All rights reserved.

Introduction

Polypeptide chains synthesized by membrane-bound ribosomes are cotranslationally translocatedacross and integrated into the endoplasmic reticu-lum (ER) membrane via a protein-conducting chan-nel, the so-called translocon.1 Its functional core isthe Sec61 complex, comprising Sec61α, Sec61β,and Sec61γ subunits.2,3 Based on studies of thecrystal structures of archaeal4 and bacterial5 SecYcomplexes, which are homologs of the eukaryoticSec61 complex, the 10 transmembrane (TM) helicesof a single SecY molecule form an aqueous porethrough which a wide variety of hydrophilic polypep-tide chains can move. The pore can also openlaterally to allow the polypeptide chain to contact themembrane lipid. 6 Oligosaccharyl transferase(OSTase) and signal peptidase flank the translocon.ERmembrane proteins, such as translocating-chain-associated membrane protein and translocon-associated protein complex, are also suggested to

0022-2836/$ - see front matter © 2013 Elsevier Ltd. All rights reserve

flank the Sec61 complex7 and to promote thematuration of multi-spanning membrane proteins.8

Cotranslational ER targeting and membrane in-sertion are initiated by a signal sequence on thenascent chain emerging from the ribosome. Thesefunctions of the signal sequence are primarilydetermined by its hydrophobic segment, which isrecognized by a signal recognition particle andsubsequently by the translocon.9 The gating motifof the Sec61 molecule is critical for discrimination ofthe signal sequence.10 The membrane orientation ofthe signal sequences is mainly determined by theflanking charges11,12; if the N-terminus is rich inpositive charges, it is retained on the cytoplasmicside and the C-terminal side is translocated into thelumen to generate a type II signal-anchor sequencewith the Ncytoplasm and Clumen orientation. If theC-terminal side is rich in positive charges, theN-terminal side is translocated to form the type Isignal-anchor sequence with an Nlumen and Ccytoplasmorientation. In this case, a domain of more than 200

d. J. Mol. Biol. (2013) 425, 3205–3216

3206 Movement of Hydrophobic Segment

residues at the N-terminus can be translocated by thetype I signal-anchor sequence.13 During membraneinsertion, the signal sequence itself serves to gener-ate the motive force for movement of the polypeptidechain.14,15 After the translocation initiation by thesignal peptide and the type II signal-anchor sequence,the ongoing movement of polypeptide chains throughthe translocon is interrupted by a hydrophobicsegment on the chain, which is functionally termedthe “stop-transfer sequence”.16 In this process, thehydrophobicity of the segment is the primary determi-nant for membrane insertion and the downstreamflanking positive charges affect the stop-translocationefficiency.16–18 Simple partitioning of hydrophobicsegments into the lipid phase through the transloconis a major motivation for the stop-translocation andmembrane insertion.19,20 Even a marginally hydro-phobic segment (mH-segment) that is insufficientfor membrane insertion by itself can span themembrane when accompanied by its downstreampositively charged residues.18,21 In addition, werecently demonstrated that the positive chargesalone can arrest translocation at the entrance of thetranslocon, even in the absence of a hydrophobicsegment.22

The TM segment of a simple type I membraneprotein stops the ongoing translocation initiated bythe N-terminal signal peptide and moves laterallyinto the membrane lipid. A type I signal-anchorsequence is released into the membrane environ-ment generating the translocation motive force.15 Atype II signal-anchor sequence is also released intothe membrane after it reorients in the translocon.23

In the case of multi-spanning membrane proteins,various TM segments are sequentially inserted intothe membrane via the translocon, but some aretentatively retained near the translocon.24 In variouscases, membrane insertion of the TM segmentinvolves multiple stages. After the stop-translocationof a simple type I membrane protein, the TMsegment remains flanking Sec61α and TRAM untilthe nascent chain is released from the ribosome.25

Multiple TM segments are accommodated in thetranslocon as long as the polypeptide chain isretained in the ribosome.26 These findings suggestthat the ribosome controls the release of the TMsegment from the translocon.We examined the stop-translocation and move-

ment of 20-residue-long mH-segments and foundthat mH-segment of the translocating nascent chainfirst stops at the membrane but then moves into theER lumen as long as the nascent chain is retained inthe ribosome. When the nascent chain is releasedfrom ribosome, the mH-segment is statically retainedat the membrane. Movement into the lumen isreversibly inhibited by high-salt conditions andirreversibly suppressed by ethylenediaminetetraa-cetic acid (EDTA). The movement of the hydropho-bic segment can be regulated by the ribosome sit on

the translocon. Based on these findings, we proposethe existence of an unstable state of stop-transloca-tion in which the mH-segment can move dependingon the synthesizing ribosome.

Results

Ribosomes affect the stop-translocation of themH-segment

To analyze the extent of the stop-translocation ofthe mH-segment, we used a constructed proteinincluding the backbone of rat serum albumin (RSA),a model sequence consisting of 20 hydrophobicamino acid residues flanked by the insulating motifs(GGPG and GPGG), and two potential glycosylationsites, as indicated (Fig. 1a; full amino acid andnucleotide sequence of the 3L16AC construct isshown in Fig. S1). The model sequences are termedaccording to the constituents. We used the 3L16ACand 4L15AC segments as the mH-segment and the10L9AC segment as a sufficiently hydrophobicsegment. The GPGG motif insulates the centralhydrophobic segment from the surroundingsequence.19 The created glycosylation sites arethe most efficient sites based on the screening ofseveral candidates and are almost completelyglycosylated when the sites are translocated in thelumen. The constructs were expressed in a reticu-locyte lysate cell-free translation system supplemen-ted with dog pancreas rough microsomal membrane(RM). If the model sequence stops at the membrane,only the upstream glycosylation site (−102) isaccessible to OSTase and glycosylated (Fig. 1a). Ifthe polypeptide chain is fully translocated into thelumen, both glycosylation sites (−102 and +12) areglycosylated.The translocation behavior of the polypeptide

chain was examined under two contexts. In onecase, the mRNA possessed an in-frame terminationcodon and the nascent chain was normally releasedfrom the ribosome just after synthesis (Fig. 1d, Ter).In the other case, the mRNA possessed no in-frametermination codon and even the full-length nascentchain was retained in the ribosome as a peptidyl-tRNA to form the ribosome nascent chain complex(Fig. 1d, RNC). Because there are 65 amino acidresidues of the nascent chain between the ribosomepeptidyl transferase center and the OSTase in thelumen,27 the second glycosylation site could reachthe OSTase even when the C-terminus was retainedin the ribosome.The mRNAs were translated in the cell-free

system for 60 min. In the absence of the modelsequence, the [none] construct was diglycosylatedirrespective of the termination codon (Fig. 1b, lanes2 and 5), indicating that both glycosylation sites were

Fig. 1. Stop-translocation of mH-segments depends on whether the nascent chain is retained in the ribosome. (a) Theconstruct used here included the N-terminal signal peptide (S), the model sequence, and potential glycosylation sites(open circles). Numbers of amino acid residues are indicated. If the model sequence (filled rectangles) is fully translocatedinto the lumen, both glycosylation sites are glycosylated (forks on the right panel). If not, only the upstream site isglycosylated. Externally added ProK degrades only the cytoplasmic polypeptide. (b) Cell-free synthesis and membranetranslocation of the proteins at 60 min of translation. Synthesized mRNAs with the termination codon (Ter) or without thetermination codon (RNC) were translated in the absence or presence of RM for 60 min. Aliquots were treated with ProK onice. Radiolabeled products were analyzed by SDS-PAGE and visualized by image analysis. Diglycosylated (dots) andmonoglycosylated (triangles) forms are indicated. The membrane-protected fragments of the polypeptide spanning themembrane after ProK treatment are indicated (upward arrows). The partially trimmed ProK-resistant fragments (opencircles) were generated by degradation of the short C-terminal portion within the ribosome of RNC, as indicated in (d). (c)The percent translocation of the mH-segment was greatly affected by whether the C-terminus was in the ribosome (RNC)or released from the ribosome (Ter). (d) Geometry of nascent chain in the ribosome and the translocon. The mH-segment(filled rectangles) was statically stopped when the nascent chain was released from ribosome by termination codon, but itwas translocated when the chain retained in ribosome as RNC. In the RNC context, ProK could degrade the C-terminal30–40 residues in the ribosome to generate larger ProK-resistant fragments [open circles in (b)]. Numbers indicate aminoacid residue numbers in each region.

3207Movement of Hydrophobic Segment

in the lumen even though the nascent chain wasretained in the ribosome. When the product wastreated with proteinase K (ProK), which degrades the

polypeptide chain on the cytoplasmic side of themembrane, the diglycosylated form derived from theRNA with the termination codon was not degraded

3208 Movement of Hydrophobic Segment

(Fig. 1b, lane 3). The diglycosylated product in theRNC context gave significant amounts of thepartially degraded products in addition to theProK-resistant full-length product (Fig. 1b, lane 6,open circles). The C-terminal portion retained in theribosome could be degraded by a high concentrationof ProK (Fig. 1d, RNC).When the highly hydrophobic segment (10L9AC)

was included, the nascent chain was monoglycosy-lated (Fig. 1b, lanes 8 and 11, triangles). Themonoglycosylated form was degraded by ProK, anda ProK-resistant fragment of 22 kDa was observed(Fig. 1b, lanes 9 and 12). The 22-kDa fragment wasnot observed followingProK treatment in the presenceof amild nonionic detergent, Tx-100 (data not shown).The ProK-resistant fragment was protected by themembrane. Monoglycosylation and the appearanceof the 22-kDa membrane-protected fragment areindicators of the stop-translocation of the modelsequence. The same results were obtained for the10L9AC model sequence, whether or not the chainswere normally terminated or retained in the ribosome.In contrast, the stop-translocation of the mH-

segment (3L16AC and 4L15AC) was greatly affectedby the C-terminal context (Fig. 1b and c). When themRNA of the 3L16AC construct with the terminationcodon (Ter) was translated for 60 min, the majority ofthe product was stop-translocated and monoglycosy-lated, and littlewas diglycosylated (less than20%).Onthe other hand, the nascent chain retained in theribosome as RNC was mainly diglycosylated (78%).Similar results were obtained with the 4L15ACconstruct; the product released from the ribosomeby the termination codon was barely translocated,whereas the same nascent chain retained in the RNCwas translocated by nearly 50%. In the case of thenormally terminated chain, the faint diglycosylatedforms were resistant to ProK and the monoglyco-sylated form was converted into the membrane-protected 22-kDa fragment (Fig. 1b, lanes 15 and 21).In the cases of the chains retained in the ribosome, thediglycosylated forms were fully resistant or partiallydegraded by ProK, like the diglycosylated form of the[none] construct in the RNC context (Fig. 1b, lanes 18and 24, open circles), while the monoglycosylatedform was converted into the 22-kDa fragment, like the10L9AC construct (arrows). These results demon-strated that the extent of the stop-translocation of themH-segments depended on whether chain termina-tion is occurring or not (Fig. 1d). Thus, theC-terminallyassociated ribosomes seem to cause the transloca-tion of mH-segments.

The mH-segment first stops and then movesthrough the membrane depending on theC-terminal ribosome

To analyze the time course of the synthesis andglycosylation of the nascent chain, we synthesized a

constructed protein for a short period (Fig. 2). Thetranslation reaction was performed for only 15 minand the elongation was inhibited with cycloheximide,and then only the translocation was chased. Thenascent chains of the 3L16AC and 4L15AC con-structs were mainly monoglycosylated after the15-min translation (Fig. 2a and b, lane 1). In ourcell-free translation system, the full-length nascentchain becomes visible after the 10-min translation,and then the amount increases linearly (Fig. S2).The majority of the full-length nascent chains that aredetectable at the 15-min translation had beensynthesized within the last 5 min. Upon ProKtreatment, all the monoglycosylated products weredegraded and the 22-kDa ProK-resistant fragmentwas observed, like in the 10L9AC construct (Fig. 2aand b, lane 8), indicating that translocation of the3L16AC and 4L15AC segments clearly stopped atthe membrane at this stage. During the chasereaction, the monoglycosylated form was convertedinto the diglycosylated form (Fig. 2a and b, lanes2–7). In contrast, those normally released from theribosome by the termination codon were not con-verted (Fig. 2a and b, lanes 15–18). The diglycosy-lated forms were fully resistant or some populationswere converted into ProK-resistant C-terminallytrimmed fragments (Fig. 2a and b, open circles). Allthe monoglycosylated forms were sensitive and noProK-resistant monoglycosylated form was ob-served, indicating that the second glycosylation siteis an efficient substrate for OSTase and that themonoglycosylated product was actually stopped.Thus, the newly synthesized full-length polypeptidewith the mH-segment first stops at the membraneand then moves into the lumen only when theC-terminus of the nascent chain is retained in theribosome (Fig. 2c).

Ribosomes are critical for the movement

The forward movement of the mH-segment underthe RNC context suggested that the ribosome boundto the nascent chains is involved in the movement.We next examined the effects of ribosome disruptionby EDTA, ribosome degradation by RNase, andnascent chain release from the ribosome by puro-mycin (Fig. 3). The proteins were translated for15 min under the RNC conditions and the movementwas chased in the presence of the indicatedreagents. RNase and puromycin significantly inhib-ited the translocation. EDTA (2 mM) completelyhalted the movement. Under these conditions, themonoglycosylated forms were completely degradedby ProK, whereas the diglycosylated products werefully resistant. Thus, these treatments did not affectthe OSTase activity but suppressed the movementof the polypeptide chain into the lumen. In thecell-free system used here, the nascent chain isreleased by more than 75% from RNC within 2 min

Fig. 2. mH-segment first stops and then moves through the membrane. (a and b) The mRNAs were translated in thepresence of RM for 15 min and chain elongation was inhibited with cycloheximide. Translocation was then chased for theindicated time periods. An aliquot was removed at each incubation time, chilled on ice, treated with RNase (50 μg/ml), andsubjected to SDS-PAGE. Aliquots were treated with ProK on ice. Diglycosylated (dots) and monoglycosylated (triangles)forms were quantified and the percent translocation was calculated (right panels). (c) Translocation of the mH-segmentfirst stops, and then the nascent chain elongates up to the C-terminus. The mH-segment is then translocated into thelumen depending on the RNC conditions (RNC). When the nascent chain is released from the ribosome by the terminationcodon (Ter), the mH-segment does not move forward.

3209Movement of Hydrophobic Segment

(data not shown). The effect of puromycin lower thanthat of the other reagents might suggest that theribosome is still on the translocon and can be

effective for movement even after the release ofthe nascent chains. The partial effect of RNase mightreflect the rate or efficiency of the enzyme function.

3210 Movement of Hydrophobic Segment

These results strongly support the idea that theribosome associating with the nascent chain is thecritical determinant of the movement.

Movement is reversibly suppressed underhigh-salt conditions

The ribosome-translocon junction is sensitive tohigh-salt conditions23; the nascent chain betweenthe ribosome and translocon was disclosed by500 mM NaCl. When the chase reaction wasperformed in the presence of 500 mM NaCl,movement of the 3L16AC segment was completelyinhibited, as observed with EDTA (Fig. 4b, RNC).The titration experiment indicated that NaCl concen-trations higher than 200 mM caused completeinhibition (Fig. 5a). In contrast to this clear inhibitoryeffect of high-salt conditions on mH-segment move-ment, movement of the positive-charge cluster of thenascent chain is enhanced by a high NaClconcentration.22 The NaCl effect was examined ona cluster of 10 lysine residues (10K cluster; Figs. 4cand 5a, lower panel). The 10K cluster in the RNCcontext translocated slower than that on the normallyreleased nascent chain (Fig. 4c, compare Ter andRNC). The movement of the 10K cluster was greatly

Fig. 3. Movement of the mH-segment is inhibited by RNatranslated and chased as in Fig. 2. The translocation chase wa(1 mg/ml), or EDTA (2 mM).

enhanced by 500 mM NaCl to the same level,whether or not the chain was included in theribosome. Efficient movement of the 10K clusterindicated that the translocon function was notaffected by the high-salt environment. EDTA didnot inhibit movement of the 10K cluster, indicatingthat the function of the translocon was not affectedby EDTA. Movement of the mH-segment requiredthe high-salt sensitive step and an EDTA-sensitivemechanism that are different from translocon.The titration experiment indicated that 200 mM

NaCl completely inhibited mH-segment movement,whereas 50 mM NaCl had essentially no effect(Fig. 5a). When the chase reaction was first treatedwith 200 mM NaCl for 5 min and then the NaClconcentration was decreased to 50 mM by 4-folddilution, movement of the mH-segments clearlyrecovered (Fig. 5b). Thus, the effect of the high-saltcondition was completely reversible.We examined the reversibility of the 2-mM EDTA

treatment and found that the addition of 2 mM MgCl2could not recover the movement (Fig. 5c, EDTA thenMg2+). When MgCl2 was added to the chasereaction (up to 2 mM) prior to the EDTA treatment,translocation was partially inhibited, but significantmovement was observed. The addition of EDTA (up

se, puromycin, and EDTA. The truncated mRNAs weres performed in the presence of puromycin (1 mM), RNase

Fig. 4. Effect of high-salt conditions on movement of the mH-segment and positive-charge clusters. (a) Constructsincluded the 3L16AC model sequence or the 10K cluster. (b and c) The constructs were translated in the presence of RMfor 15 min and the translocation was chased in the presence or absence of 500 mM NaCl and/or 2 mM EDTA. Movementof the 3L16AC segment in the RNC context was completely suppressed by NaCl and EDTA, while movement of the 10Kcluster was greatly enhanced by NaCl and barely affected by EDTA.

3211Movement of Hydrophobic Segment

to 2 mM) after the 5-min chase enhanced themovement. The inhibition by NaCl was reversible,but that by the EDTA was irreversible. This findingsuggested that the ribosome is involved in themovement of the mH-segment. The complex qua-ternary structure of the ribosome could not be easilyreconstituted after the EDTA-mediated disruption.

Effect of downstream length on the movement

The sequences downstream of the mH-segmentwere partially deleted or extended to examine theeffect of the downstream length (Fig. 6). The proteinswere translated for 60 min and the glycosylationstatus was examined. The movement monitored bydiglycosylation strongly depended on the down-stream length. The 152-residue sequence, whichwas used throughout the study, caused the mostefficient movement. Although the shorter (112-resi-due) sequence is sufficient for glycosylation of the(+12) site (data not shown), nascent chains with ashorter sequence (112 and 132 residues) exertedlow level of translocation. Similarly, the longersequence had lower movement efficiency. It is likely

that the conformational properties of the downstreamregion are critical for mH-segment movement. Theconstructs used in this study were fortunatelyfavorable for movement of the mH-segment.

Discussion

Here, we demonstrated that the stop-transferfunction of mH-segment differs depending on wheth-er or not the chain termination is occurring andsuggested that there is a novel unstable stop-tran-slocation state prior to the stable state. During thestop-translocation, the mH-segments (3L16AC and4L15AC segments) first stop at the membrane butcan then move into the lumen as long as theC-terminus of the nascent chain is retained in theribosome.Large amount of the polypeptide chain with the

mH-segment could move into the lumen as long as itwas retained in RNC. The data on the mobility of themH-segment provide quantitative evidence that themH-segment is tentatively arrested in the mobilestate rather than in the stably stopped state. In other

Fig. 5 (legend on next page)

3212 Movement of Hydrophobic Segment

Fig. 6. Downstream 152-residuesequence is optimum for mH-seg-ment movement. The length of thesequence downstream of the modelsequence was shortened or extend-ed by the deletions and insertions,respectively. The proteins weretranslated for 60 min and subjectedto SDS-PAGE. Each band wasquantified and the translocation per-cent was calculated. The movementof themH-segments ismost efficientwith the 152-residue downstream.

3213Movement of Hydrophobic Segment

words, an unstable state of the stop-translocationoccurs before the stable state. The unstable state isconverted into the stable state by the release of thenascent chain from the ribosome. Cross-linkingexperiments have revealed various membranecomponents and stages that nascent chain experi-ences during insertion into the membrane. Littlequantitative information has been provided, howev-er, due to the limitation of the cross-link efficiency. Inthe case of the movement assay, the only limitationof the movement is the release of the nascent chainfrom the peptidyl transferase center of the ribosomecaused by spontaneous hydrolysis of the pepti-dyl-tRNA. In our system, the nascent chain in theRNC state obtained after the 15-min translation wasspontaneously released from the peptidyl transfer-ase center by 30% within the 30-min chase (Onishi,

Fig. 5. Inhibition of mH-segment movement by NaCl is revconcentration on movement of the mH-segment and 10K clustin the presence of the indicated concentration NaCl. (b) Dilutio3L16AC and 4L15AC constructs were translated for 15 min an200 mM NaCl. Where indicated, the chase mixtures were dilu(arrows). The movement inhibited by 200 mM NaCl was recoveby MgCl2 (2 mM). Movement of the 3L16AC segment was chaindicated, 2 mM MgCl2 or 2 mM EDTA was added after a 5-mdoes not recover the movement, whereas the addition of MgC

unpublished results). The movement efficiencyobserved here is therefore an underestimation.Our data indicate that the membrane insertion of

the mH-segment is not decided rapidly upon enteringthe translocon but remains undetermined by thepresence of the ribosome. It is widely accepted that asimple partitioning of the hydrophobic segment intothe lipid environment causes the stop-translocationand the subsequent membrane insertion.19,20 Actu-ally, the hydrophobic segment (10L9AC segment) isretained at the membrane irrespective of the C-ter-minal ribosome. If the stop-translocation of themH-segment is determined only by partitioning ofthe hydrophobic segment into the membrane, how-ever, the fate of the mH-segment would not beaffected by whether the polypeptide chain is retainedin the ribosome. Our data indicate that initial

ersible, but that by EDTA is irreversible. (a) Effect of NaCler. After the 15-min translation, the movement was chasedn of the high-salt concentration reversed the inhibition. Thed the movement was chased in the presence or absence ofted with the reaction buffer by 4-fold after a 5-min chasered by the dilution. (c) Inhibition by EDTA was not reversedsed in the presence of 2 mM EDTA or 2 mM MgCl2. Wherein chase (arrow). Addition of MgCl2 after EDTA treatmentl2 prior to the EDTA treatment suppressed the inhibition.

3214 Movement of Hydrophobic Segment

recognition of the mH-segment at the translocon isdifferent from such a stable state. The primaryrecognition would occur within the translocon priorto insertion into the membrane environment. Thepore ring region of the Sec61 molecule is involved inthe recognition and selection of the TM segment.28

Themutation in the pore ring region of Sec61 in yeastalters the recognition of the H-segment duringmembrane insertion. Ismail et al. examined forcegeneration by the hydrophobic TM segment on theelongating nascent chain during cotranslationalinsertion via the SecY complex and found that thereis an initial hydrophobic interaction between thehydrophobic segment and the translocon before theTM segment fully enters the translocon pore.29 Twoindependent processes are involved in membraneintegration at the translocon complex. Such atentative hydrophobic interaction would cause theinitial stop-translocation. Several reports haveaddressed membrane insertion of the TM segmentof the ribosome-associating nascent chain andindicated that the ribosome restricts the lateralrelease of the hydrophobic segment from thetranslocon. TM segments of the membrane proteinsare located adjacent to an ER membrane proteinthroughout an integration process.30 A TM segmentis tentatively retained around the Sec61α protein andTRAM as long as the chain is retained in theribosome.25 During the multi-spanning membraneprotein insertion, a TM segment is tentativelyretained near Sec61α until the next TM segment isinserted, and when the following TM segment isinserted into the translocon, the upstream TMsegment moves to the other position of thetranslocon.24 A recent report indicated that thehydrophobic TM segments of the multi-spanningmembrane protein are retained in the translocon andsegregated from the lipid environment as long as thenascent chain is retained in the ribosome.26 Inaddition to the release of TM segments intomembrane, the ribosome senses the sequence ofthe nascent chain within its tunnel and allostericallyregulates sealing of translocon pore.31,32 The ribo-somemight regulate the lateral gate of the transloconchannel to restrict the exposure of the mH-segmentand retain the mH-segments in an unstable state.During the unstable stop-translocation state of the

mH-segment, the nascent chain elongates up to theC-terminus. We acknowledge that the downstreamRSA sequence of 152 residues used in the presentstudy favors this movement and that the movement isdependent on the context (Fig. 6). The mH-segmentand RSA backbone allowed us to observe theunexpected movement after the tentative stop-translocation. Under the same situation, we alsoobserved the tentative arrest and movement of acluster of positive charges22 (Fig. 4). The RSAsequence might specifically favor a wide fluctua-tion range and allow the movement. In addition,

there should be a pulling force on the upstreamsequence from the ER lumen to cause themovement, even in the absence of the pushingforce of protein synthesis. Molecular chaperoneswould be able to cause chain movement. Utilizingthe ideal system, we found the unstable stage ofstop-translocation and its conversion into thestable state after release from the ribosome.It is possible that the ribosome on the ER translocon

simply tethers the mH-segment in the translocon tokeep it mobile. The following observation, however,rules out this possibility. First, the 10L9AC segmentstably stopped irrespective of the ribosome, indicatingthat the downstream 152-residue sequence cannottether the segment within the mobile environment.Second, when the downstream sequencewas shorterthan 152 residues (112 and 132 residues), movementdid not occur (Fig. 6). If only the tethering effect wasoperative, the mH-segment could also move in thesecases. Third, NaCl inhibition was completely revers-ible soon after by simple dilution. The ribosomereleased from the translocon can rebind and thensequesters the nascent chain from the cytoplasm.23 Ifribosome tethering causes the retention of themH-segment in the translocon, detachment of theribosome under high-salt conditions would allow themH-segment to be released into the membrane. Sucha membrane-inserted mH-segment would not beeasily reversed to the translocon. Rather thantethering, the ribosome has a positive action on thetranslocon to keep the mH-segment mobile.The mH-segment movement was completely inhib-

ited by the high-salt condition, while movement of thepositive-charge cluster was enhanced under thesame conditions. The high salt does not affect thetranslocon or OSTase but likely disrupts the associ-ation of the ribosome to the translocon. The high-saltconditions only cause the ribosome to detach from thetranslocon but do not affect the integrity of thetranslocon and ribosome, and thus when the NaClmixture was diluted, movement was reversed. On theother hand, inhibition of the mH-segment movementby EDTA was irreversible. EDTA does not inhibit themovement of the positive-charge cluster, indicatingthat it does not significantly inhibit the translocon.These observations indicate that mH-segment move-ment involves a complex structure requiring a divalentcation, likely the ribosome structure. Once collapsed,the ribosome cannot be easily reconstituted by simplyadding back the MgCl2.

Materials and Methods

Materials

RM33 and rabbit reticulocyte lysate34 were prepared aspreviously described. RM was treated with EDTA and then

3215Movement of Hydrophobic Segment

with the Staphylococcus aureus nuclease as describedpreviously.33 ProK (Merck), cycloheximide (Sigma), puro-mycin (Sigma), and theDNAmanipulating enzymes (Takaraand Toyobo) were obtained from the indicated sources.

Construction of proteins with model sequences

Constructs were based on RSA, as describedpreviously.21 The cDNA fragment encoding M1-H319 ofRSA (XbaI/ApaI), in which all the Cys codons werechanged to Ala codons in a previous study, was obtainedby polymerase chain reaction and subcloned into pRcCMV(XbaI/ApaI). The superscript numbers used here corre-spond to the original residue number of the RSApolypeptide. The cloned DNA fragment contained theKozak sequence at the 5′-region. The 3′-regions includedthe following. For normal termination, the H319 codon wasfollowed by the termination codon TAA. To generate thetruncated mRNA, we followed the H319 codon of RSA bythe sequenceGTGGATCC including the BamHI sequence,which was used for linearization of the template DNA (Fig.S1). In both cases, the ApaI site used for subcloning wasdestroyed to manipulate the site as follows. As indicated inFig. 1a and Fig. S1, the model sequences were insertedinto the middle portion. The Val residue in the EVASsequence corresponded to the Val166 of RSA and thecodons for the Ala-Ser sequence included the NheI site. Inthe GPGGSADDF sequence, the F corresponded to thePhe173 of RSA and the Gly-Pro codons included the ApaIsite. Oligonucleotides were synthesized and insertedbetween the NheI and ApaI sites. At each junction, thesix bases of the restriction enzyme site were designed toencode the two amino acid residues. For the glycosylationsites, V67QE was changed to NST (for the −102 site) andE177LLYYAEKYNEV188 to AQQNSTAAYIEI (for +12 site).All the constructed DNAs were confirmed by DNAsequencing. The entire DNA sequence and the codedamino acid sequence are shown in the supplement (Fig.S1). For deletion of the downstream portion, M271-K285 (forC-137), A269-T288 (for C-132), or G259-A298 (for C-112) ofthe 3L16AC construct was deleted. For extension of thedownstream portion, either the DNA fragment (possessingBglII/BamHI sites on each end) encoding theN319-D349 (forC-184), D289-D349 (for C-214), R249-D349 (for C-254), or theL217-D349 (for C-286) sequence of the 3L16AC constructwas introduced into the C-terminal BamHI site.

Cell-free transcription and translation

Cell-free transcription and translation were performedessentially as described previously.35,36 Plasmid DNAwaslinearized at the XhoI site within the vector for the mRNAwith the termination codon or at the BamHI site for thetruncated mRNA. The linearized DNA was then tran-scribed with T7-RNA polymerase.The RNA was translated in the reticulocyte lysate cell-free

system for the indicated time period at 30 °C, either in theabsenceor presenceofRM.The translation reaction included100 mM potassium acetate, 1.2 mM magnesium acetate,32% reticulocyte lysate, castanospermine (20 μg/ml), and15.5 kBq/μl [35S]EXPRESS protein-labeling mix (PerkinElmer). For the translocation chase experiment, translationwas performed for 15 min and inhibited with 1 mM cyclohex-

imide, and then the translation mixture was further incubatedfor the indicated time periods. Aliquots were sampled andkept on ice. For the ProK treatment, aliquots of the translationmixtureswere treatedwith ProK (200 μg/ml) for 30 min on iceand the reactionwas terminatedwith15%(w/v) trichloroaceticacid. The protein precipitates were washed with acetone andsolubilized with the SDS sample buffer for SDS-PAGE. Todigest the tRNA moiety bound to the nascent chain, thesample buffer included RNase (400 μg/ml). Radiolabeledpolypeptide chains were analyzed by SDS-PAGE, visualizedwith an imaging analyzer (BAS1800; Fuji Film), and quantifiedusing Image Gauge software (Fuji Film). Percent transloca-tion was estimated as the percentage of the diglycosylatedproduct among the monoglycosylated and diglycosylatedproducts.Supplementary data to this article can be found online at

http://dx.doi.org/10.1016/j.jmb.2013.05.023

Acknowledgements

This work was supported by Grants-in-Aid forScientific Research from the Ministry of Education,Culture, Sports, Science, and Technology of Japan.

Received 9 April 2013;Received in revised form 28 May 2013;

Accepted 30 May 2013Available online 6 June 2013

Keywords:protein topogenesis;membrane protein;

translocation;translocon;

glycosylation

† Y.O. and M.Y. contributed equally to this work.

Abbreviations used:EDTA, ethylenediaminetetraacetic acid; ER, endoplasmic

reticulum; OSTase, oligosaccharyl transferase; ProK,proteinase K; RM, rough microsomal membrane; RSA, rat

serum albumin; TM, transmembrane.

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