A stop transfer sequence recognizes receptors for nascent chain translocation across the endoplasmic reticulum membrane

Cell, Vol. 47, 711-719,

December

5, 1986, Copyright

0 1986 by Cell Press

Stop Transfer Sequence Recognizes Rece for Nascent Chain Translocation across the Endoplasmic Reticulum Membrane Nancy K. ize, David W. Andrews, and Vishwanath FL Lingappa Department of Physiology and Department of Medicine University of California, San Francisco San Francisco, California 94143 Kopogen San Francisco, California 94143

~~mrnary A stop transfer sequence derived from the extreme carboxyl terminus of membrane IgM heavy chain has been shown to confer predictable transmembrane orientation to secretory proteins by aborting translocation of subsequently synthesized protein domains. Mere we demonstrate that, in certain peptide sequence contexts, the same stop transfer sequence is also capable of initiating domain translocation across the endoplasmic reticulum (ER) membrane. Translocation directed by a stop transfer sequence is similar to, but distinguishable from, the action of a conventional signal sequence. Translocation is dependent on participation of the ribosome and protein receptors both in the cytoplasm and in the ER membrane. Moreover, both amino- and carboxy-terminal flanking protein domains can be translocated. Unlike a signal sequence, the stop transfer sequence is not itself translocated across the membrane. These results have implications for the nature of signal sequences, stop transfer sequences, and their receptor interactions.

How newly synthesized secretory and membrane proteins are transported into and across the membrane of the endoplasmic reticulum (ER) remains an unsolved problem (Walter and Lingappa, 1986). The evidence suggests that this problem can be resolved into three aspects. First a mechanism must exist to target the nascent chain to the ER membrane. Second, there must be a means of initiating translocation of protein domains. Finally, in the case of integral transmembrane proteins, there must be a means of terminating translocation such that the domains synthesized subsequent to the transmembrane region are localized to the cytosol. In the case of proteins spanning the membrane multiple times, the initiation and termination events of translocation must occur in a repeated, sequential, and programmed fashion to generate the particular transmembrane orientation of the polypeptide (Blobel, 1980). A large body of evidence has accumulated in favor of a receptor-mediated model by which nascent chains are targeted and translocation across the ER membrane is inilisted (Walter and Lingappa, 1986). In contrast, the underlying basis for termination of chain translocation, a phe-

nomenon also termed “‘stop transfer,” remains uncertain. An early suggestion was that stop transfer was a passive anchor phenomenon resulting from loss of a driving force for translocation upon completion of protein synthesis, which would operate only at the extreme carboxyl terminus of a transmembrane protein (Lodish et al., 1981). However, the demonstration that such carboxy-terminal anchors would nevertheless abort translocation even when engineered more than 100 codons from their native extreme carboxy-terminal position sugggested that stop transfer was directed by a discrete coding region, independent of its location in the chain (Yost et al., 1983). This result was consistent with, but did not prove, a receptor-mediated model for stop transfer. in fact, more recent work has suggested once again a specific role for hydrophobicity in termination of chain translocation (Davis and Model, 1985). Thus the issue of whether termination of translocation is active or passive, receptor-mediated or a simple consequence of extreme hydrophobicity, remains to be further elucidated. Molecular genetic manipulation of coding regions with subsequent expression in cell-free and whole cell expression systems has been a powerful tool for studying how translocation is initiated (Lingappaet al., 1984; Peraraand Lingappa, 1985; Perara et al., 1986). This approach has also provided an assay for termination of translocation (Gething and Sambrook, 1982; Rose and Bergmann, 1982; Boeke and Model, 1982; Yost et al., 1983). More recently, variations of this approach have been used to characterize topogenic sequences responsible for more complex transmembrane orientations using globin and other “passenger” coding regions as reporters of protein topology. Thus, it has been shown that carboxy- and aminoterminal sequences of hepatitis B virus surface antigen are translocated across the ER membrane (Eble et al., 1986; Eble et al., submitted), while in the cases of asialoglycoprotein receptor and transferrin receptor, sequences at the carboxyl but not the amino terminus are translocated (Spiess and Lodish, 1986; Perial et al., 1986). In the latter two cases the topology of the engineered proteins was as expected for topogenic elements intrinsically competent for carboxy- but not amino-terminal domain translocation, i.e., these topogenic elements appeared to contain both signal and stop transfer activities. However, it remained unclear as to whether signal and stop transfer activities within these sequences were separable or whether these topogenic elements were functionally a distinct class from conventional signal and stop transfer sequences as previously defined (Perara and Lingappa, 1985; Yost et al., 1983). It occurred to us that a further vartation of this approach might provide insight into the mechanism of stop transfer and termination of translocation. As a result of molecular genetic engineering of coding regions expressed in cellfree systems, our studies have unmasked a latent functional property of a stop transfer sequence. We demonstrate that a functionally defined stop transfer sequence,

Ceil 712

p§PSLMG SP6p *

“p1 S

NcoI EcoR,

I

L

WE,

I

jMj

I

G TAG

AUG

s”pr

pSPLMG SP6p

+

L

Nco, EcoR,

IMI

EWE,

G

AUG

TAG

Nco, BssH, BstE,

pSPgGSP 1 SP6p +

11

G

Ncq

Is

NC%

1

pvun

P TAG

A”G CA0 Ncor EssH,,

pSPgGMP I SP6p +

G

A”G CA0 Figure 1. Restriction

BstE,

II

1Mi

EcoR+(Pvuni

P TAG

Maps and Coding Regions of Plasmids

Coding regions are designated as follows: S, signal; L, lactamase; M, transmembrane segment of immtmoglobulin M; G, chimpanzee a-globin; P, mature bovine prolactin. SP6p indicates SP6 promoter; CHO indicates the site of insertion of the 7 codon glycosylation segment into globin. pSPgGSP was previously called pSPgGP (Perara and Lingappa, 1985). Its encoded products preGSP and Pl have been renamed GSP and P, respectively, in the text.

which normally acts only to terminate the translocation initiated by a preceding signal sequence, can itself initiate translocation when engineered in place of the signal sequence. Furthermore, we demonstrate that the signal-like behavior of this stop transfer sequence appears to be receptor-mediated and displays some, but not all, features of a conventional signal sequence. These results suggest that the mechanisms of initiation and termination of translocation may be related. Results A Stop Transfer Sequence Aborts Domain Translocation Initiated by a Preceding Signal Sequence To better understand the means by which multiple topogenie sequences can direct complex transmembrane orientations, we chose to study plasmid pSPSLMG (diagrammed in Figure 1 along with other constructions used in this study). This construction encodes, sequentially and in frame, a signal sequence (S) followed by a domain of lactamase (L), a stop transfer sequence derived from the carboxyl terminus of membrane b heavy chain (M segment), and, finally, globin (G). When pSPSLMG is expressed by cell-free, transcription-linked translation in the rabbit reticulocyte lysate, cell-free protein-synthesizing system in the absence of added microsomal membranes, the expected 40 kd protein was observed and demonstrated to be reactive with both lactamase (Figure 2, lane J) and giobin antisera (data not shown). In the presence of microsomal membranes, a new polypeptide was observed. Termed LMG, this polypeptide was also found to be reactive to lactamase (data not shown) and globin (Figure 2, lane K) antisera. LMG is approximately 2 kd smaller

than SLMG, presumably due to cleavage of the 23 amino acid residue lactamase signal sequence from its amino terminus. Digestion with proteinase K was carried out to determine the transmembrane orientation of inserted LMG. Proteolysis of LMG, synthesized in the presence of membranes, resulted in one major product of lower molecular weight, approximately 26 kd, that was reactive with iactamase, but not globin, antisera (Figure 2, lanes L and M) and was termed L. As can be seen in Figure 2, lane M, no globin-reactive protease digestion products were observed. Protection of the L fragment from proteinase K digestion was abolished when the protecting lipid bilayer was solubilized by nondenaturing detergents. From these results, LMG must span the bilayer asymmetrically with the lactamase domain within the vesicle lumen, the M segment spanning the bilayer, and the globin domain exposed on the cytoplasmic face. This result is consistent with previous work on a related coding region (Yost et ai., 1983). In the Absence of a Preceding Signal Sequence, a Stop Transfer Sequence Initiates Domain Translocation To study the behavior of the M segment alone without a preceding signal sequence, we constructed a deletion mutant of pSPSLMG, termed pSPLMG, in which the initial methionine was deleted and a new termination codon was introduced. Upon transcription-linked translation of this construction, initiation of protein synthesis would be expected to take place at the next (previously internal) AUG, formerly a methionine codon at residue 66 of authentic lactamase. Consistent with this expectation, the encoded product, termed LMG: was approximately 34 kd in size but was still reactive with both lactamase and globin antisera (Figure 2, lane 6). Since this product lacked the amino-terminal lactamase signal sequence and contained M segment as its only topogenic element, no shift in mobility was observed upon translation in the presence of microsomal membranes (Figure 2, lanes C and F) and it was not expected to be translocated across microsomal membranes. To our surprise, however, we found that both the lactamase domain and the globin domain of this protein appeared to be translocated across microsomal membranes when they were present during, but not when they were added after, translation. Translocation was inferred from the detection of both lactamase and globin immunoreactivity that was protected from proteinase Kin the form of two discrete fragments of approximately 19 and 15 kd termed L’ and G*, respectively, which were generated by cleavage of full-length LMG* (Figure 2, lanes D and G). It was observed that while both of the flanking domains could be translocated, the stop transfer sequence containing M segment itself could not be translocated, since proteolytic digestion of LMG* resulted in cleavage into the two protected products. Each of the protected domains was slightly larger than expected for the authentic lactamase and globin regions, presumably due to a small remnant of the M segment on each (Figure 2, lanes D and G). No protected fragments were observed upon proteolysis either in the absence of membranes (data not shown),

Topogenic 713

Sequences

for Protein Translocation

pSPSLMG

DSPLMG ab LbLL mb --++++++

LGGG

PK d@

+

+ - ----++-+

+

-

LLGLGL --tt++ t

+

ABCDEFGH .

68-

+------+ I J

+++ K

LMN

*:,

.

4% LMG*-

s.,

-15

Figure 2. Localization of lranslocated Protein Domains by Proteolysis of Products Encoded by pSPLMG and pSPSLMG SP6 polymerase transcription products were translated in reticulocyte lysate in the presence or absence of membranes. Products were subjected to posttranslationa! proteolysis with proteinase K and subsequent immunoprecipitation as described in Experimental Procedures. All lanes are immunoprecipitates. Designations are as follows: ab, antisera used for immunoprecipitation; L, anti-lactamase; G, anti-human hemoglobin. Additional components of the translation reaction are indicated by a + symbol above the lane. Components are coded as follows: mb, dog pancreas rough microsomes; PK, posttranslational digestion wiih proteinase K; det, Nikko1 detergent. Large arrows pointing down indicate full-length translation products. Arrows pointing up indicate proteolysis products. Products in lane A were synthesized in the absence of membranes but were incubated with membranes posttranslationally before proteinase K digestion. The unidentified bands slightly below LMG” in lanes B, C, F, J, and K, are presumed to result from internal initiation. Other unidentified bands result from nonspecific binding as demonstrated by nonimmune controls (data not shown). Positions of markers are identified by molecular weights in kd. The constructions and encoded products containing engineered deletions are denoted by an asterisk; the full-length parent molecules have no asterisk. Specificity of antisera was confirmed by immunocompetition of LMG precipitation using authentic human globin (data not shown).

in the presence of posttranslationally added membranes (Figure 2, lane A), in the presence of nonionic detergents added to solubilize the protecting lipid bilayer before proteolysis (Figure 2, lane H). Since L* was reactive with lactamase but not globin antisera and since G* was reactive to globin but not lactamase antisera (Figure 2, lanes D and G), it appeared that these fragments represented the two distinct domains flanking the M segment in LMG*. Previously it has been shown that these flanking domains lack intrinsic information for translocation, which can be conferred by engineering in a topogenic element such as an amino-terminal signal sequence (Lingappa et al., 1984). Thus, translocation of the lactamase and globin flanking domains in LMG* suggested to us that the stop transfer sequence within the M segment was the topogenie element responsible for directing translocation of both domains. To compare the signal-like function of M directly with that of a conventional signal sequence, we made use of two similar constructions, pSPgGMP and pSPgGSP pSPgGMP, diagrammed in Figure 1, contains the M coding region engineered between two flanking coding

regions (117 residues of globin including an artificially engineered glycosylation site and 150 residues of bovine prolactin). By comparison, pSPgGSP encodes a fusion protein containing the same 177 residues of chimpanzee globin followed by the signal sequence of bovine prolactin and the entire authentic prolactin coding region. Thus, pSPgGMP and pSPgGSP differ in that the former contains an internal stop transfer sequence while the latter contains an internal signal sequence. An additional difference, the lack of 50 authentic prolactin codons in pSPgGMP, is insignificant since expression of a similar deletion from pSPgGSP, a plasmid termed pSPgGSPK, demonstrates translocation behavior (Figure 38, lanes O-T) similar to that observed for pSP lanes K-N). We have shown previously that the former aminoterminal signal sequence of bovine reprolactin, when engineered to the internal position in pSPgGSR is nevertheless still capable of functional recognition of translocation receptors on the ER membrane in a cotranslationai fashion (Perara and Lingappa, 1985). The nascent protein encoded by pSPgGSP, termed GSP, was shown to interact productively with receptors for transfocation on the ER membrane. Cleavage of the signal sequence resulted in the generation of three fragments, GS, gGS, and P. The former two constitute the nonglycosylated and glycosyfated globin domains attached to the cleaved signal peptide; the latter fragment is mature bovine prolactin. All fragments were translocated across membranes as indicated by protection from proteinase K digestion (see also Figure 3, lanes K-N). The presence of similar flanking domains in pSPgGSP and pSPgGMP facilitates comparison of the signal-like behavior of the M segment stop transfer sequence with that of the prolactin signal sequence. Expression of pSPgGMP in the absence of membranes results in synthesis of a protein called GMP* (Figure 3, lanes C and D). When membranes are present cotranslationally, but not when added after completion of protein synthesis, a fraction of nascent GMP” is glycosylated, forming gGMP*. Glycosylation is particularly evident after proteolysis as the band gG in Figure 3 (lane G), as described below. Proteinase K digestion of translation products synthesized in the absence of membranes (Figure 3, lanes A and B) in the presence of membranes added posttranslationally (data not shown) results in no protease”protected fragments. However, when membranes were present during translation, subsequent proteolysis generated discrete protected fragments corresponding to the globin and prolactin domains. Two fragments immunoreactive with anti-globin serum were observed (Figure 3, lane G) and appeared to correspond to glycosylated (endoglycosidase H-sensitive, data not shown) and ~on~~ycosylated forms of globin containing a small remnant of M. One fragment was found to be immunoreactive with anti-prolactin and corresponds in size to that expected for the prolactin domain together with a part of the M segment (Figure 3, lane H). Thus, when expressed in the rabbit reticulocyte lysate cell-free system, pSPgGMP was found to translocate both the globin and the prolactin flanking domains, as well as

Cell 714

pSPgGMP

PGPGPGP

--

--

--++---

-

--

-I-+

+--

--+ --

ABC

DEFGHIJ

ab G P G mb PK &t

pSPgGSPK

pSPgGSP

++ ++++ --++++ ----++

KLMN

obP P G mb --f-+-k+-+ PK--det - - OPQ

G

P

P

++ - -

+ +

RS

T

60-

-60

45GMP*-

c-

-45

I 1

- GSPK -QG

JPK 7, Figure 3. Localization

of Translocated

Protein Domains

by Proteolysis

of Products Encoded

by pSPgGMP

pSPgGSP, and pSPGSPK

(A) SP6 transcription-linked translation and posttranslation procedures, as described in Figure 2. Lanes A-J, immunoprecipitates; lanes K-N, total products. Abbreviation are as follows: G, anti-human hemoglobin antisera; P anti-prolactin. Additional components of the translation reactions are indicated by a + symbol above the lane. Components are coded as follows: ab, antisera used for immunoprecipitation; mb, dog pancreas rough microsomes; PK, posttranslational digestion with proteinase K; det, Nikko1 detergent. Large arrows pointing down indicate full-length translation products. Arrows pointing up indicate proteolysis products. Relative mobilities for glycosyla?ed globin fragments are indicated as gG (pSPgGMP) and gGS (pSPgGSP). (6) Lanes O-T are immunoprecipitates. All lanes are from the same exposure, with lanes R-T printed darker. The positions of GSPK, gG, G, and PK are indicated. Specificity of antisera was confirmed by immunocompetition of GMP using ailthentic prolactin and globin (data not shown). Exposure time was 4 days for all lanes except G, which was 12 days.

to glycosylate the globin moiety (Figure 3, lane 6). However, in contrast to the signal sequence of pSPGSP the stop transfer sequence of pSPGMP was not itself translocated, as demonstrated by its accessibility to digestion by proteinase K (Figure 3, lanes G and H). It remains to be determined whether each domain is being translocated on the same, overlapping, or different populations of GMP chains.

Kmb - - + + NKmb f-b -PK-+-+ AB

CD

-=-I--+ ++--+-+ E F GH

b A -P Domain Translocation by a Stop Transfer Sequence Is Receptor-Mediated In view of the highly hydrophobic character of the stop transfer sequence, we wished to determine whether its signal-like function of initiating domain translocation proceeds in a protein- (receptor) independent or receptormediated fashion. We therefore compared the interaction of the signal sequence containing molecule GSP and the stop transfer containing molecules LMG and GMP’ with the ER membrane. As an assay for cytoplasmically exposed ER membrane proteins required for translocation, we used sensitivity to N-ethyl maleimide (NEM). NEM has been previously shown to render membranes translocation-incompetent, presumably by alkylating free sulfhydryl groups on membrane proteins required for translocation mediated by a conventional signal sequence (Gilmore et al., 1982; Hortsch et al., 1986). In Figure 4 NEM treatment of salt-washed membranes (KRMs) is shown to abolish domain translocation (as assayed by domain protection from proteinase K digestion) for both pSPgGSP (lane F) and pSPgGMP (lane 6) when translated in reticulocyte lysate and when compared to KRMs not treated with NEM (lanes D and H). Use of

P”-

A

Figure 4. Translocation of Nascent GSP and GMP’ is Abolished Pretreatment of Salt-Washed Microsomal Membranes with NEM

by

Transcription-linked translation of pSPGMP (lanes A-D) and pSPGSP (lanes E-H) was carried out in the reticulocyte lysate, cell-free translation system in the presence of salt-washed microsomal membranes either mock- (lanes C, D, G, and H) or NEM-treated (lanes A, 9, E, anti F). After completion of translation, aliquots of transcription-linked translation products of each construction in the presence of mock- or NEM-treated membranes were subjected to proteinase K digestion and proteolysis termination as described (Perara and Lingappa, 1965). Total products, 0.5 ~1, were separated by SDS-PAGE. Kmb: mock NEM-treated, salt-washed dog pancreas microsomal membranes. NKmb: NEM-treated Kmb. Large arrows pointing down refer to GMP’ (lanes A and C) or GSP (lanes E and G). Arrows pointing up refer to P’ (lane D) or P (lane G and H).

reticulocyte lysate containing signal recognition particle (SRP) and salt-washed membranes (SRP-depleted) ensures that any effect observed due to NEM alkylation does not result from inactivation of SRP Therefore the inactivation we observe with NEM treatment of membranes sug-

Topogenic 715

Sequences

for Protein Translocation

KRM --++---%+ SRP----++++ PK -i--+-+-+

ABCDEFGN

4--i---++9 --i---c I J

-t--k

KL

-GSP

l-

-+ LMG*

-+

-+

GSP

GMP*

Figure 5. Translation Arrest by SRP integrated optical density measured from autoradiograms of total translation products synthesized in wheat germ extract with (+) or without (-) added SW Translation products are as follows: PRL, prolactin; 6, globin; LMG: lactamase M segment globin; GSP globin signal prolactin; GMP*, globin M segment prolactin. To facilitate comparison, translation products for each molecule are arbitrarily normalized to 1.0 integrated QD unit for products synthesized in the absence of exogenous SRF. Quantitative comparisons were achieved by use of total products (rather than immunoprecipitates) analyzed without fluorography and with the exposure adjusted to the linear range of the X-ray film.

gests that translocation across the ER membrane, using either the signal sequence (pSPgGSP) or the intrinsic signal functions of the stop transfer sequence (pSPgGMP), is dependent on alkylation-sensitive membrane proteins. If both signal and stop transfer sequences share the requirement for alkylation-sensitive protein components in the ER membrane to translocate protein domains, might they not share more proximal binding sites in the pathway of chain translocation, i.e., functional interaction with SRP in the cytoplasm? SRP has two assayable functions in cell-free systems-arrest of nascent chain synthesis (Walter and Blobel, 1981b) and targeting to receptors on the ER membrane (Walter and Blobel, 1981a). The results of our investigation of these two roles for SRP in nascent GSP and GMP” translocation are summarized in Figures 5 and 6. Elongation of both molecules is arrested when compared with positive (bovine preprolactin and GSP) and negative (cytoplasmic chimpanzee globin) controls (Figure 5). Moreover, as shown in Figure 6, both molecules require exogenous SRP for translocation across microsomal membranes in the wheat germ cell-free system. In the absence of added membranes (Figure 6, lanes A and B) or in the presence of KRMs alone (lanes C, D, I, and ,t), no protease-protected products were observed. However, when both SRP and KRMs were provided (Figure 6, lanes G, i-l, K, and L), the prolactin domain of both GSP and GNP* was observed to be protected from digestion by proteinase K. Quantitative densitometry, corrected for the lower methionine content of the truncated prolactin domain of GMP’, revealed an efficiency of prolactin domain protection identical to that of GSP Also, addition of SRP and KRMs in an identical fashion has no effect on cytoplasmic globin (data not shown). From these results it appears that the pathway of the translocation process, including interaction with SRP and

Figure 6. SRP Dependence

of Translation of Gh?P and GSP

Transcription-linked translation of pSPgGMP (lanes A-H) and pSPgGSP (lanes I-L) in wheat germ extract at 24°C for 60 min. Addition to the translation reaction of various components is indicated by a + symbol above the lane. Components are coded as follows: KRM, salt-washed (SRP-depleted), dog pancreas microsomal membranes; SRP, signal recognition particles. PK indicates posttranslational digestion with proteinase K, final concentration 0.1 mglml, for 60 min at 20°C. All lanes were subjected to immunoprecipitation with anti-prolactin antiserum. Lanes G-H were exposed 7 times longer. Lanes I-J are the same exposure, with lanes J and L printed darker.

a secondary receptor in the ER membra~~e and possibly the ribosome binding site (discussed below), is the same for both signal-mediated and stop transfer sequencemediated domain translocation. Yet the translocation activity directed by these two topogenic sequences, while similar, is not identical. In GSP the signa! sequence is completely translocated (Perara and Lingappa, 1985). In contrast, we generally observe very few molecules of GMP* fully protected from proteinase K (see for example Figure 4, lane D). It seems likely that the occasional observation of a few chains of full-length GMP* after proteinase K digestion (see for example Figure 3, lanes G and H) results from incomplete proteolysis rather than translocation of the entire chain. Such a band is not observed in the more harsh proteolysis conditions used in the SRP experiments (Figure 6, lane H) and is virtually undetectable in experiments with LMG* (Figure 2, lanes D and G). Conversely, the proteolytic cleavage by exogenously added proteinase K observed only for the stop transfer sequence and not for the conventional signal sequence does not arise from the fact that the stop transfer sequence is not cleaved by signal peptidase. Evidence for this comes from the observation that some molecules of pSPgGPK are not cleaved by signal peptidase; yet, these uncleaved molecules, which contain the prolactin signal sequence rather than the M segment, can be detected completely translocated and protected from proteases (Figure 3, lanes R and S). In the course of the SRP experiments, an additional functional difference between conventional signal sequence-mediated and stop transfer sequence-mediated chain translocation was observed. Translocation of either flanking domain in GMP’ and LMG” appears to require

Cdl 716

nonmembrane-associated SRP lianslocation across microsomal membrane vesicles, either intact (data not shown) or salt-washed (Figure 6) and SRP-depleted, could be demonstrated in the wheat germ system (which lacks cytosolic SRP) only when purified SRP was added exogenously. Alternatively, translocation could be demonstrated across the same membranes in the reticulocyte lysate system (Figures 2, 3, and 4) known to provide endogenous cytosolic SRP (Meyer et al., 1982). This result contrasts with that of conventional signal sequence-mediated translocation in which the endogenous SRP pool associated with intact microsomal membranes is sufficient for translocation. This dependence on exogenous SRP may explain why translocation directed by a stop transfer sequence was not previously observed in the wheat germ system (Yost et al., 1983). The full physiologic significance of this phenomenon remains to be elucidated. Discussion We have shown that a stop transfer sequence previously defined by its ability to precisely abort chain translocation initiated by a preceding signal sequence can itself initiate translocation under the appropriate circumstances. The requirements for this behavior appear to include the expression of the stop transfer sequence in a cotranslational fashion, with no preceding signal sequence, and in the presence of receptor proteins in both the cytosol (SRP) and the ER membrane (NEM sensitivity). To understand the difference between the flanking domain translocation observed here and previously (Perara and Lingappa, 1985), it seems fruitful to distinguish conventional from nonconventional signal sequences. Conventional signal sequences are typically found on secretory proteins, are located at the amino terminus, are cleaved from the passenger polypeptide by signal peptidase, and display characteristic structural features (von Heijne, 1985). Nonconventional signal sequences are found in integral membrane proteins, are not cleaved by signal peptidase, and can be located at positions other than the extreme amino terminus (Bos et al., 1984; Eble et al., submitted; Friedlander and Blobel, 1985; Hay et al., unpublished data). In some but not all cases these nonconventional signal sequences are structurally distinct from the consensus features previously proposed (von Heijne, 1985). The signal-like activity described here for the M segment falls into the category of a nonconventional signal sequence. It is derived from a characteristic domain of an integral membrane protein and recognizes common receptors for the translocation of flanking domains. Yet, it is neither amino-terminal nor cleaved by signal peptidase. Moreover, aligning the amino acid sequence of the hydrophobic core of the M segment with consensus features for conventional signals as described (von Heijne, 1984, 1985) reveals that the M segment differs dramatically from conventional signals in both the N and C regions. The difference is most apparent in the N-terminus, which in a conventional signal would be expected to have a small net positive charge (mean of +1.7). In the M segment the analogous region has a net charge of -4.

The C-terminus lacks a characteristic cleavage site, as might be expected since the molecule is uncleaved. It seems possible that the similarities and differences between conventional and nonconventional signal sequences might reflect the existence of overlapping subsets of receptors for chain translocation-some recognized by all signal sequences and determining domain translocation, others recognized only by conventional signal sequences, thereby effecting complete translocation, cleavage, and release of those molecules. The results presented here are consistent with the signal-like activity of M segment representing a nonconventional signal sequence. It appears that stop transfer-mediated translocation is a specific receptor-mediated event that recognizes some, but not all, of the receptors engaged by a signal sequence and thus is able to effect flanking domain translocation but not translocation of itself. The relationship of signal and stop transfer sequences elucidated here provide a caveat for the interpretation of experiments attempting to dissect signal from stop transfer sequences by the engineering of reporter groups and passenger domains. It is possible that internal signal sequences defined by functional translocation of engineered flanking domains are, in fact, stop transfer sequences that, in their native location and sequence context, serve to abort rather than initiate translocation through closely related receptor-mediated mechanisms. Considerations for a Mechanism of Nascent Chain Translocation One possibility raised by these results and the distinction between conventional and nonconventional signal sequences is that translocation mediated by a signal sequence may be divided into two stages. The first stage, flanking domain translocation, taking place once binding of the putative secondary signal receptor in the ER membrane (Gilmore and Blobel, 1983; Hortsch et al., 1986) opens an aqueous tunnel (Gilmore and Blobel, 1985). The second stage, translocation of the signal sequence itself, becomes possible after opening of this tunnel and translocation of the domains has removed barrier and/or steric impediments to recognition of a (higher affinity) lumenally disposed signal receptor, perhaps signal peptidase itself. In this view, translocation mediated by the stop transfer sequence proceeds faithfully through the first stage but does not achieve the second, perhaps because it lacks features for recognition by the lumenally disposed receptor. It is possible that the stop transfer sequence does not resemble a conventional signal sequence for the same reason (von Heijne, 1985). On the Possible Receptor Mediation of Stop Transfer Activity Why should a stop transfer sequence be capable of recognizing receptors for transtocation? Our results using NEMtreated membranes suggests that such a recognition event is required. On initial consideration, this finding poses a paradox since the IgM & heavy chain stop transfer sequence is located at the extreme carboxyl terminus of the molecule, i.e., at a position where it cannot express

Topogenic 717

Sequences

for Protein Translocation

any cotranslational signal-like function because protein synthesis will have terminated before it even emerges from the ribosome (Bernabeu et al., 1983). Yet, this signallike activity appears to have been conserved through the course of evolution of p heavy chain. One appealing explanation is that the signal-like activity of the stop transfer sequence is required for its normal function of terminating translocation. That is, stop transfer may be achieved by recognition of the same receptor engaged by the signal sequence upon initiation of translocation. In such a view, the receptor in the ER membrane can be conceptualized functionally as a molecular switch. Binding of this receptor by a signal sequence initiates a process culminating in complete translocation. However, subsequent interaction with a stop transfer sequence results in termination of chain translocation. Whether receptor-mediated termination of chain translocation is in itself sufficient to consummate integration into the lipid bilayer remains to be established. As a result of the engineering and expression experiments presented here, a stop transfer sequence has been presented to the membrane in an inappropriate context, i.e., in the absence of a preceding signal sequence. The unexpected ability of the M segment to carry out some, but not all, signal sequence functions, may provide novel experimental approaches for distinguishing stages in translocation and characterizing early and late receptors.

Hydrophobicity and the Molecular Basis of Stop Transfer Function The precise role of hydrophobicity in membrane protein biogenesis remains to be elucidated. It is possible that receptor recognition by both signal and stop transfer sequences is mediated by hydrophobic interactions that are relatively degenerate, i.e., fulfilled by a variety of amino acid sequences within a set of conformational parameters as yet not clearly understood. It is also possible that two modes of stop transfer exist, one active, the other passive. The former mode would be receptor-mediated, and the latter mode would result from relatively nonspecific interactions of extremely hydrophobic sequences with the membrane. The former mode is most consistent with the data presented here, while the latter best explains earlier findings (Davis and Model, 1985). The bridge between the two concepts may lie in a better understanding of the hydrophobic basis for receptor recognition. Recent data demonstrate that some very hydrophobic sequences are capable of translocation (Davis and Hsu, 1986). The unexpected findings elucidated here further illustrate the power of a systematic approach to genetic engineering of functional domains from topogenic sequences. Establishing a minimal definition of signal (Lingappa et at., 1984) and stop transfer sequences (Yost et al., 1983) has set the stage for the generation of more complex probes (Perara and Lingappa, 1985). The concepts presented here provide a coherent framework for the use of such probes in studying the mechanisms of transmembrane integration. Further studies applying moEecular genetic approaches in cell-free systems should

continue to elucidate fundamental features of the processes of protein translocation, transport, and assembly. Experimental

Procedues

Materials All restriction endonucleases, n&ease Bal31, calf mtestinal alkaline phosphatase, SP6 RNA polymerase, T4 DNA ligase, and Klenow fragment of E. coli DNA polymerase were obtained from Boehringer Mannheim Diagnostics, Inc. (Houston, TX) or from New England BioLabs (Beverly, MA). RNAase inhibitor was from Promega Biotec (Madison, WI); Staphylococcal protein A-Sepharose, from Pharmacia, Inc. (Piscataway, NJ). Rabbit anti-E. coli lactamase antisera was a gift from Dr. Chung Nan Chang. Rabbit anti-human globin serum was from Cappet Laboratories (Cochranville, PA); rabbit anti-bovine prolactin, from ilnited States Biochemical Corp. (Cleveland, BH); proteinase K, from Merk (FRG); endoglycosidase H and ]35S]methionine (translation grade, >800 Cilmmol), from New England Nuclear (Boston, MA); Nikkol (octaethylene glyco-mono-n-dodecylether, a nonionic detergent), from Nikko Chemicals Co., Ltd. (Tokyo, Japan); Triton X-100, from Boehringer Mannheim (Indianapolis, IN); NEM, from Calbiochem Bering Corp. (CA). Constructions Recombinant DNA constructs were derived from existing plasmids, pG6 (Yost et al., 1983) and pSPgGSP (previously termed pSPgGP1; Perara and Lingappa, 1985). pG6 was modified by EcoRllBstEll elimination of 50 bp, followed by insertion of an Ncol linker (GCCATGGC), leaving 150 bp coding for the carboxyl terminus of membrane v heavy chain. The entire coding region was engineered behind the SP6 promoter. Thus, this pG6-derived construct, pSPSLMG, includes coding regions for lactamase, with its signal sequence (182 codons), for the transmembrane region of IgM (M segment, 50 codons), and for chimpanzee a-globin (143 codons). pSPLMG was constructed by truncation of the globin of pSPSLMG at BstEll and cleavage in theSP64 polylinker at Xbal with religation followed by partial digeslion of pSPSLMG with Sspl in the presence of ethidium bromide, Sal31 digestion, repair with Klenow fragment, and religation. DNA sequencing, using the dideoxy method (Sanger et al., 1977), confirmed deletion of the initial methionine and 14 subsequent codons of the signal sequence. Mobilities of the encoded, lactamase and globin immunoreactive, translation products on SDS-PAGE are consistent with initiation of synthesis at amino acid number 66 (methionine), which is the next A146 in the coding region of lactamase. Thus, pSPLMG encodes amino acids 66-182 of lactamase (without its signal sequence), the 50 amino acid M segment, and the first 110 codons of globin ending at BstEll. pSPgGMP was constructed from pSPgGSP by insertion of the M segment at the BstEII/Ncol site of pSPgGSP preceding the coding region for the prolactin signal. Subcloning was foilowed with deletion of the prolactin signal by cleavage at the EcoRl site at the distal end of the M segment and at the Pvull site 50 codons beyond the amino terminus of mature prolactin. Religation yielded pSPgGMP, which encodes the first 110 amino acids of chimpanzee a-gtobin, including a 7 amino acid glycosylation site at the BssHll site of globin. the 50 amino acid M segment, and the C-terminal 150 codons of mature prolactin. The parent plasmid, pSPgGSP encodes the same 117 amino acids of globin (with the glycosylation site), fused to the signal of prolactin (30 codons) and all 199 codons of mature prolactin. The coding regions of the four constructs are diagrammed in Figure 1. pSPgGSPK was constructed from pSPgGSP by cleavage with Pvull in the prolactin coding region and removal of approximately 50 codons of prolactin by digestion with Bal31, repair with Klenow fragment, insertion of a Kpnl linker, cleavage with Kpnl, and religation. Transcription-Linked Translation Transcription of SP6 plasmids was as described previously (Perara and Lingappa, 1985). Aliquots of the transcription reaction mixture were used directly in transcription-linked translations at a final concentration of 20%. Translation reactions of this kind in reticulocyte lysate have already been described (Perara and Lingappa, 1985). Translations in wheat germ extract were essentially as described (Erickson and Btobel, 1983); however, the final ion concentrations were maintained

Cdl 718

at 2.6 m M magnesium and 140 m M potassium and the pH was adjusted to 7.5 using TRIS base. Reaction mixtures were incubated at 24% for 60 min. Some translations were supplemented with either dog pancreas microsomes or salt-extracted (SRP-depleted) microsomes. Microsomal membranes were prepared as described (Walter and Blobel, 1983a), except that the column washing step was replaced by two consecutive washes of the membranes by centrifugation (30 min, 100,000 x g) and resuspension of the membranes in 50 m M triethanolamine, 1.5 m M Mg(OAc), 1 m M EDTA, 1 m M MT, and 0.5 m M PMSF. SRP was prepared from these membranes as described (Walter and Blobel, 1983b); however, the final sucrose gradient centrifugation step was omitted. Ion concentration for each column fraction was estimated from the measured conductance. Protein Processing and Translocation Assays In vitro translation products were immunoprecipitated and separated by SDS-PAGE, except for the NEM experiments presented in Figure 4 in which total products are shown. Bands were localized by autoradiography either with or without fluorography. Protease protection experiments were performed as described (Perara and Lingappa, 1985) except wheat germ products, in which digestion was allowed to proceed for 1 hr at 20aC, and microsomes were solubilized with Briton X-100 (0.1% final concentration) instead of Nikkol. To determine the extent of SRP arrest for each of the plasmid translation products, translations were carried out with and without SRP Total products were separated by SDS-PAGE and localized by autoradiography without fluorography. Bands were quantitated by densitometer scanning the autoradiogram using a LKB 2222 Ultrascan XL Laser Densitometer from LKB Instruments, Inc. (Gaithersburg, MD). To facilitate integration, peaks were smoothed by convolution with a Gaussian filter. In each case translation in the absence of SRP was normalized to 1.0 integrated OD units. NEY Treatment of Microsomal Membranes Salt washed microsomal membranes were prepared (Walter and Blobel, 1983b). A 9 ul aliquot at 50 Ass0 U/ml was adjusted to 5 m M NEM by addition of 1 nl of 50 m M NEM freshly prepared in 10 m M Tris (pH 8) and incubated at 22% for 15 min and at 4% for 30 min. The reaction was quenched by adjusting the aliquot to 10 m M DTT by addition of 1 pl of 0.1 M DTT in water followed by further incubation at 4% for 30 min. Mock NEM-treated membranes were prepared by premixing 1 ul of 50 m M NEM in 10 m M Tris (pH 8) with 1 ul of 0.1 M DTT in water and adding 9 ul of salt-washed membranes followed by sequential incubations for 15 min at 22% and 1 hr at 4%.

Davis, N. G., and Model, P. (1985). An artificial anchor hydrophobicity suffices to stop transfer. Cell 47, 607-614.

domain;

Davis, N. G., and Hsu, M.-C. (1986). The fusion-related hydrophobicdomain of Sendai F protein can be moved through the cytoplasmic membrane of Escherichia coli. Proc. Natl. Acad. Sci. USA 83, 5091-5095. Eble, B., Lingappa, V. R., and Ganem, D. (1986). Hepatitis B surface antigen: an unusual secreted protein initially synthesized as a transmembrane polypeptide. Mol. Cell. Biol. 6, 1454-1463. Erickson, A. H., and Blobel, G. (1983). Cell-free translation of messenger RNA in a wheat germ system. Meth. Enzymol. 96, 38-50. Friedlander, M., and Blobel, G. (1985). Bovine opsin has more than one signal sequence. Nature 378, 338-343. Gething, M. J., and Sambrook, J. (1982). Construction of influenza hemagluttinin genes that code for intracellular and secreted forms of the protein. Nature 300, 598-603. Gilmore, R., and Blobel, G. (1983). Transient involvement of signal recognition particle and its receptor in the microsomal membrane prior to protein translocation. Cell 35, 677-685. Gilmore, Ft., and Blobel, G. (1985). Danslocation of secretory proteins across the microsomal membrane occurs through an environment accessible to aqueous perturbants. Cell 42, 497-505. Gilmore, R., Elobel, G., and Walter, I? (1982). Protein translocation across the endoplasmic reticulum. I. Detection in the microsomal membrane of a receptor for the signal recognition particle. J. Cell Biol. 95, 463-469. Hortsch, M., Avossa, D., and Meyer, D. I. (1986). Characterization secretory protein translocation: ribosome-membrane interaction endoplasmic reticulum. J. Cell Biol. 103, 241-253.

of in

Lingappa, V. R., Chaidez, J., Yost, C. S., and Hedgpeth, J. (1984). Determinants for protein localization: beta-lactamase signal sequence directs globin across microsomal membranes. Proc. Natl. Acad. Sci. USA 87, 456-460. Lodish, H. F., Braell, W. A., Schwartz, A. L., Strous, G., and Zilberstein, A. (1981). Synthesis and assembly of membrane and organelle proteins. Int. Rev. Cytol. Supp. 72, 247-307. Meyer, D. I., Krause, E., and Dobberstein, B. (1982). Secretory protein translocation across membranes: the role of ‘docking protein.’ Nature 297, 647-650. Perara, E., and Lingappa, V. R. (1985). A former amino terminal signal sequence engineered to an internal location directs translocation of both flanking protein domains. J. Cell Biol. 707, 2292-2301. Perara, E., Rothman, Ft. E., and Lingappa, V. R. (1966). Uncoupling translocation from translation: implications for transport of proteins across membranes. Science 232, 348-352.

We wish to thank Eric Calhoun for technical assistance, Don Ganem and Peter Walter for helpful comments on the manuscript, and W. B. Hansen for useful discussions. N. K. M. is the recipient of a National Science Foundation Fellowship. D. W. A. is the recipient of a Medical Research Council of Canada Fellowship. This work was supported by National Institutes of Health grant GM31626. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

von Heijne, G. (1984). How signal sequences ficity. J. Mol. Biol. 773, 243-251.

Received August 20, 1986; revised October 3, 1986

von Heijne, G. (1985). Signal sequences: Biol. 784, 99-105.

References Bernabeu, C., Tobin, E. M., Fowler, A., Zabin, I., and Lake, J. A. (1983). Nascent polypeptide chains exit the ribosome in the same relative position in both eucaryotes and procaryotes. J. Cell Biol. 96, 1471-1474. Blobel, G. (1980). Intracellular Sci. USA 77, 1496-1500.

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Rose, J. K., and Bergmann, J. E. (1982). Expression from cloned cDNA of cell-surface and secreted forms of the glycoprotein of vesicular stomatitis virus in eukaryotic cells. Cell 30, 753-762. Sanger, F., Nicklen, S., and Coulson, A. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Nan. Acad. Sci. USA 74,5463-5468. Spiess, M., and Lodish, H. F. (1986). An internal signal sequence: the asialoglycoprotein receptor membrane anchor. Cell 44, 177-185. maintain cleavage speci-

the limits of variation. J. Mol.

Walter, P., and Blobel, G. (1981a). Translocation of proteins across the endoplasmic reticulum II. Signal recognition protein (SRP) mediates the selective binding to microsomal membranes of in-vitro-assembled oolysomes synthesizing secretory protein. J. Cell Biol. 97, 551-556. Walter, P., and Blobel, 6. (1981b). Translocation of proteins across the endoplasmic reticulum !II. Signal recognition protein (SRP) causes signal sequence-dependent and site-specific arrest of chain elongation that is released by microsomal membranes. J. Cell Biol. 97, 557-561.

Boeke, J., and Model, P (1982). A procaryotic membrane anchor sequence: carboxy terminus of bacteriophage fl gene III protein retains it in the membrane. Proc. Natl. Acad. Sci. USA 79, 5200-5204.

Walter, P., and Blobel, G. (1983a). Preparation of microsomal membranes for cotranslational protein translocation. Meth. Enzymol. 96, 84-93.

90% T., Davis, A. R., and Nayak, D. P. (1984). NH,-terminal hydrophobic region of influenza virus neuraminidase provides the signal function in translocation. Proc. Natl. Acad. Sci. USA 81, 2327-2331.

Walter, P., and Blobel, G. (1983b). Signal recognition particle. A ribonucleoprotein required for cotranslational translocation of proteins. Isolation and properties. Meth. Enzymol. 96, 682-691.

Topogenic 719

Sequences

for IProtein Translocation

Walter, f?, and Lingappa, V R. (1986). Mechanism of protein translocalion across the endoplasmic reticulum. Ann. Rev. Cell Biol., in press, Yost, C. S., Hedgpeth, J., and Lingappa, V. Ft. (1983). A stop transfer sequence confers predictable transmembrane orientation to a previously secreted protein in cell-free systems. Cell 34, 759-766. Zerial, M., Melancon, P., Schneider, C., and Garoff, H. (1986). The transmembrane segment of the human transferrin receptor functions as a signal peptide. EMBO J. 5, 1543-1550.