A nascent secretory protein may traverse the ribosome/endoplasmic reticulum translocase complex as an extended chain.

We have measured the minimum number of residues in a translocating polypeptide required to bridge the distance between the P-site in endoplasmic reticulum-bound ribosomes and the lumenally disposed active site of the oligosaccharyl transferase. The results suggest that a nascent chain may traverse the ribosome/translocase complex in a largely extended conformation, and that hydrophobic stop-transfer segments have a more compact, possibly alpha-helical conformation in the translocase.

Protein translocation through the membrane of the endoplasmic reticulum (ER) 1 is catalyzed by a complex multisubunit translocation machinery comprising cytoplasmic, integral membrane, and lumenal components (1,2). It has been proposed that the integral membrane components form a wateraccessible channel in the membrane through which nascent chains can pass (3). Experimental evidence based on a wide range of methods such as electrophysiology (4), urea extraction (5), photo-cross-linking (6), and fluorescence quenching (7,8) all support this proposal. Although the environment of nascent translocating polypeptide chains has thus been quite well characterized, their conformation during passage through the ER translocase is not known. In mitochondria, it has been shown that nascent chains traverse the two mitochondrial membranes in a largely extended conformation (9).
Previously, we have found that the lumenally oriented active site of the oligosaccharyl transferase (OST) enzyme (itself part of the translocation complex; Ref. 10), is positioned at a well defined distance above the surface of the ER membrane and can thus be used as a fixed point of reference against which the location of various parts of a nascent polypeptide in the translocase can be determined (11,12). We now report measurements of the minimum length of polypeptide chain required to bridge the distance between the ribosomal peptidyl transferase site (P-site) on the cytoplasmic side of the ER membrane and the OST active site on the lumenal side. The minimum distance has been measured both for nascent chains corresponding to a globular protein domain that normally becomes fully translocated to the lumen of the ER and for nascent chains containing a hydrophobic stop-transfer sequence which interrupts trans-location and ultimately becomes integrated into the lipid bilayer. Our results suggest that non-hydrophobic nascent chains may adopt a fully extended conformation during passage through the ribosome/translocase complex, whereas the hydrophobic stop transfer sequence appears to form a more compact, possibly helical, structure when located in the translocase. DNA Techniques-Insertion of the stop transfer sequence QQQL 17 VKKKK into the P2 domain of Lep was performed by introducing BclI and NdeI restriction sites in codons 214 and 220, respectively. Double-stranded oligonucleotides coding for the above amino acid sequence were then cloned between the BclI and NdeI restriction sites. Site-specific mutagenesis used to introduce an Asn-Ser-Thr glycosylation acceptor site in position 200 of Lep and to introduce restriction enzyme cleavage sites was performed according to the method of Kunkel (13), as modified by Geisselsoder et al. (14). For cloning into and expression from the pGEM1 plasmid, the 5Ј end of the lep gene was modified, first, by the introduction of an XbaI site, and second, by changing the context 5Ј to the initiator ATG codon to a "Kozak consensus" sequence (15). Thus the 5Ј region of the gene was modified to: . . . ATAACCCTCTAGAGCCACCATGGCGAATATG . . . (XbaI site and initiator codon underlined). Mutants of Lep made in phage M13 were cloned into pGEM1 behind the SP6 promoter as an XbaI-SmaI fragment. All constructs in pGEM1 were confirmed by sequencing of the plasmid DNA.

Enzymes and Chemicals-Unless
Templates for in vitro transcription of truncated mRNA were prepared using the polymerase chain reaction (PCR) to amplify fragments from pGEM1 plasmids containing the desired DNA constructs. The 5Ј primer was the same for all PCR reactions and had the sequence 5Ј-TTCGTCCAACCAAACCGACTC-3Ј. This primer is situated 210 bases upstream of the translational start, and all amplified fragments thus contained the SP6 transcriptional promoter from pGEM1. The 3Ј primers were chosen according to the positions of the truncations and were designed to have approximately the same annealing temperature as the 5Ј primer. None of the 3Ј primers contained translational stop codons. Amplification was performed with a total of 30 cycles using an annealing temperature of 59°C. The amplified DNA products were separated on a low melting-point agarose gel, excised, and purified using Wizard PCR purification resin (Promega) as described in the manufacturers protocol.
In Vitro Transcription-Truncated mRNAs were transcribed from the SP6 promoter. The reaction mixture was as follows: 10 l of 5 ϫ Promega transcription buffer, 5 l of 100 mM dithiothreitol, 2 l of 25 mM ribonucleotide triphosphates (A, U, C, and G), 5 l of 5 mM m7G(5Ј)ppp(5Ј)G, 3.5 l of H 2 O, 20 l of DNA template, 2.5 l (50 units) of RNasin, 2 l (40 units) of SP6 RNA polymerase. Transcriptions were carried out at 37°C for 1 h. The reactions were stopped by extracting with an equal volume of phenol/chloroform (1:1) followed by extraction with chloroform. Samples were precipitated by the addition of NaCl to 50 mM and 2 volumes of ethanol. The precipitates were pelleted by centrifugation for 10 min in a microcentrifuge at 4°C. The pellets were washed in 70% and then 99% ethanol and dried. The pellets were redissolved in 100 l of H 2 O, and 5 l were run on a 1% agarose gel to verify that the RNA band was at least 5-10 times more intense than the DNA template band.
In Vitro Translation-In vitro translation of [ 35 S]methionine-labeled proteins from the in vitro synthesized mRNA were carried out in 50-l reactions using wheat germ extract according to instructions supplied by the manufacturer. Translation reactions were performed at 25°C for 1 h. When relevant, the translation mixes were supplemented with 1 l (2 A 280 equivalents) of EDTA-washed dog pancreas microsomes (16,17) and 1 l of canine SRP (20 nM final concentration) prepared as described (18). For puromycin-treated samples, translation in the presence of microsomes and SRP was performed as above, followed by the addition of 2 l of 30 mM puromycin and incubation at 25°C for another 10 min. At the end of the reaction, samples were acid precipitated by the addition of an equal volume of 20% trichloroacetic acid and then processed for immunoprecipitation with a polyclonal antiserum against Lep. Gels were visualized on a Fuji BAS1000 phosphorimager and quantitated using the MacBas 2.1 software.
Translation in reticulocyte lysate in the presence of dog pancreas microsomes was performed as described (19). Translocation of polypeptides to the lumenal side of the microsomes was assayed by resistance to exogenously added proteinase K and by prevention of N-linked glycosylation through competitive inhibition by addition of a glycosylation acceptor tripeptide (N-benzoyl-Asn-Leu-Thr-N-methylamide) as described (11).

65
Residues Are Required to Bridge the Distance between the Ribosomal P-site and the OST Active Site-A well characterized model protein (Lep) (Fig. 1), normally located in the inner membrane of Escherichia coli, was used to measure the number of residues required to bridge the distance between the P-site and the OST active site (the "minimum glycosylation length"). When expressed in vitro in the presence of dog pancreas microsomes, Lep inserts in an SRP-dependent manner with the same orientation as in E. coli, i.e. with both the N-terminal tail and the C-terminal P2 domain in the lumen (Refs. 11 and 20; data not shown). Lep is efficiently glycosylated on Asn 214 when expressed as a full-length protein in the presence of microsomes (20).
To measure the minimum glycosylation length, mRNAs truncated in the P2 domain of Lep at various defined positions C-terminal to the potential glycosylation acceptor site (Asn 214 -Ser-Thr) were generated by PCR and in vitro transcription. When translated in a wheat germ lysate expression system in the presence of SRP, the resulting truncated proteins (which do not contain a translational stop codon) remain bound to the ribosomal P-site at their C-terminal end (21), while the more N-terminal parts of the P2 domain extend through the ribosome/translocase complex into the lumen of the ER.
As shown in Fig. 2 (panel B, lanes 1-3), when the truncation was 39 amino acid residues C-terminal to the acceptor site, no glycosylation was seen unless the nascent chain was released from the ribosome by treatment with puromycin, indicating that the protein had been correctly targeted to the ER membrane but did not extended sufficiently far from the P-site for the potential glycosylation site to reach the OST active site. Similar results were obtained for nascent chains truncated 51, 56, 61, 63, and 64 residues away from the potential glycosylation acceptor site (lanes 4 -18). Finally, for truncations 65 and 66 residues away from the potential glycosylation acceptor site, a sharp increase in the amount of glycosylation in the absence of puromycin was observed (lanes 19 -24). We conclude that a minimum of 65 amino acid residues is required to span the distance between the ribosomal P-site and the OST active site.
The Presence of a Stop-transfer Sequence Increases the Minimum Glycosylation Length-Based on the results of a previous study where the number of residues between the lumenal end of a hydrophobic transmembrane segment and the OST active site was measured, we suggested that transmembrane segments may be lipid-exposed and have a helical conformation when located in the ER translocase (12). Since the results reported above indicated that a nascent chain not containing any extensive hydrophobic stretches may traverse the translocase in an extended conformation (see "Discussion"), we considered the possibility that the minimum glycosylation length may be different if a hydrophobic segment is present in the nascent chain between the glycosylation acceptor site and the C terminus.
We thus constructed a protein, Lep-ST (Fig. 3A), that contains an engineered glycosylation site Asn 200 -Ser-Thr replacing amino acids 200 -202 and the stop-transfer sequence QQQL 17 VKKKK inserted between amino acid residues 215 and 220. When expressed in the presence of microsomes, Lep-ST was efficiently glycosylated on Asn 200 (Fig. 3B, lane 2) and Truncations were made at different sites downstream of the stop-transfer segment, and glycosylation in the presence of microsomes was assayed as above. As shown in Fig. 4, for truncations 52, 66, and 69 residues away from the potential glycosylation acceptor site no glycosylation was seen in the absence of puromycin (lanes 1-9). For truncations 70 and 71 residues away from the potential glycosylation site, there was a sharp increase in the amount of glycosylation observed in the absence of puromycin (lanes 10 -15), and complete glycosylation was observed when the truncation was 74 and 91 residues away (lanes 16 -21). It thus appears that the presence of a long hydrophobic stretch makes the nascent chain spanning the translocase complex more compact. DISCUSSION By measuring the minimum number of residues required to bridge the distance between the ribosomal P-site and the OST active site, we have sought to indirectly determine the conformation of a nascent chain in transit through the ER membrane. Our data show that a translocating nascent chain corresponding to a globular domain of a protein needs to be extended by a minimum of 65 amino acid residues from the P-site in the ribosome to be able to interact with the OST active site (Fig. 5). This length is increased to 71 residues when a 18-residue-long hydrophobic stop-transfer sequence is present in the nascent chain.
These results are broadly consistent with previously reported data, but have a significantly higher precision. An early study provided a rough estimate of the P-site/OST distance of 45-95 residues (22). More recently, it has been shown that protease digestion of nascent secretory polypeptides in detergent permeabilized microsomes results in protected fragments of around 70 amino acid residues (23), and that photochemically induced cross-linking between a nascent chain and Sec61␣ is possible at a maximum distance of ϳ70 residues away from the ribosomal P-site (6). Finally, it has been reported that disulfide bonds can form in a translocating nascent chain when it reaches a length of 50 -60 residues (24).
We have shown previously that at least 15 spacer amino acid residues are needed after the end of the hydrophobic core of a N lumen -C cyt -oriented transmembrane segment in order for a potential acceptor site for N-glycosylation to be glycosylated (11). Furthermore, around 40 amino acid residues of a nascent chain are protected by free ribosomes from protease digestion (23,25), and photochemical cross-links to Sec61␣ can be seen at positions in a nascent chain only 30 residues away from the P-site (6). Taken at face value, these results together imply the geometrical relationships depicted in Fig. 6. However, depending on the dimensions of the putative ribosome/translocase channel and the conformational flexibility of the nascent chain, a precise match between the results of the different assays may not be expected.
Electron microscopy studies of intact ribosomes suggest that nascent chains exit the large ribosomal subunit at a point ϳ150 Å from the P-site (26 -28). Assuming a typical 50-Å-thick membrane, this gives a first-order approximation to the physical P-site/OST distance of ϳ200 Å, suggesting that the nascent chain must be able to bridge the distance between the ribosomal P-site and the OST active site in a largely extended conformation (ϳ3.1 Å/residue, to be compared with 3.5 and 1.5 Å/residue in fully extended and ␣-helical conformations, respectively). Since these physical dimensions are only rough numbers, we cannot formally exclude that a short segment of the nascent chain is in fact helical, although we consider this unlikely.
In agreement with the suggestion of an extended conforma-tion, we find that the introduction of an 18-residue-long hydrophobic stop-transfer segment between the glycosylation acceptor site and the end of the nascent chain leads to an increase in the minimum glycosylation length of ϳ6 residues, suggesting a more compact conformation of the nascent chain. Since the hydrophobic segment is located only 19 residues downstream of the glycosylation acceptor site and since the acceptor site must be at least 17 residues away from the hydrophobic segment for glycosylation to be possible in this construct, 2 the stop-transfer segment is located within the translocase complex in the critical constructs (truncations 69 -74 residues away from the acceptor site). In the translocase, stop-transfer segments are believed to be in a partially lipid-exposed environment (12,29) and thus most likely in a helical conformation. Indeed, if 18 residues in a nascent chain were forced from a fully extended to an ␣-helical conformation, ϳ10 extra residues would need to be added to the chain in order to bridge a defined end-to-end distance. The 6-residue shift in the minimum glycosylation length that we observe is thus in reasonable agreement with an extended conformation for polar nascent chains and a helical conformation for hydrophobic segments.
In summary, a model where the nascent polypeptide chains passes through a water-accessible translocase channel in a largely extended conformation is consistent with all the available data. Further, hydrophobic stop-transfer sequences appear to have a more compact, possibly helical conformation when located within the translocase, suggesting a more lipidexposed environment.
FIG. 6. Geometry of the ER translocase complex. The OST active site is located ϳ15 residues away from the end of the hydrophobic transmembrane segment (Ref. 11, data not shown). 70 residues of the nascent chain are protease-protected in detergent-solubilized microsomes (23) and also represent the most distal position that can be cross-linked to Sec61␣ (6), 65 residues are required to bridge the P-site/ OST distance (this paper), 40 residues are protease-protected in isolated ribosomes (23), and cross-linking to Sec61␣ is possible from position 30 (6). From the minimum length of a nascent chain required for processing by signal peptidase (SPase), one can estimate that roughly 100 residues are needed to span the distance between the signal peptidase active site and the P-site in the ribosome (6,30,31), implying that the OST and signal peptidase active sites are not immediately adjacent in the complex.