Positive charges on the translocating polypeptide chain arrest movement through the translocon

Positively charged residues of membrane proteins have long been known to function as determinants of membrane topology, which is reflected in a statistical rule of membrane topology, i.e. the positive-inside rule of membrane proteins (Sipos and von Heijne, 1993; von Heijne and Gavel, 1988). Many membrane proteins in the secretory pathway in eukaryotic cells are integrated into the endoplasmic reticulum (ER) membrane by the protein translocation channel, the so-called translocon. Protein translocation and integration are initiated by a signal sequence on the nascent polypeptide chain. The signal sequences are primarily defined by a hydrophobic segment (H-segment). A signal recognition particle recognizes the H-segment emerging from the ribosome and induces targeting of the ribosome-nascent chain complex to the ER. The H-segment is transferred from the signal recognition particle to the translocon by the action of a signal recognition particle receptor, and the ribosome attaches to the translocon. The core part of the translocon comprises the Sec61 complex, which is composed of Sec61α, Sec61β and Sec61γ (Johnson and van Waes, 1999; Rapoport, 2007). In prokaryotic cells, a similar complex, the SecY complex, is involved in the protein translocation and membrane integration into the plasma membrane. On the basis of studies of crystal structures of the SecY complex (Tsukazaki et al., 2008; Van den Berg et al., 2004), 10 transmembrane (TM) segments of a single SecY molecule can form an aqueous pore through which a wide variety of hydrophilic polypeptide chains can move toward the lumen. The ten TM helices are arranged in pseudo symmetry and form a clam-shell-like structure that can open laterally to allow the TM segment to be released into the membrane lipid (Rapoport, 2007).

The positive charges determine the orientation of the signal sequences. The H-segment of the signal sequence penetrates the translocon, and then one side of the H-segment moves into the lumen to form the TM topology. During the insertion into the translocon, the orientation of the signal sequence is determined by the flanking positively charged amino acid residues (Goder and Spiess, 2001; Kida et al., 2006; Sakaguchi, 1997). If the N-terminal side of the H-segment is rich in positive charges, as observed in signal peptide and type II signal-anchor sequences, the N-terminus is retained on the cytoplasmic side and the segment forms the N_cytosol and C_lumen orientation (termed the type II orientation). The signal peptide is cleaved off by signal peptidase in the ER lumen, while the signal-anchor is retained in the final protein as a TM segment. Alternatively, if the C-terminal side is rich in positive charges, the N-terminus is translocated through the translocon and forms the N_lumen and C_cytosol orientation (the type I orientation) and the signal-anchor is retained as a TM segment. For example, the topology of synaptotagmin II is gradually converted depending on the number of positive charges downstream of the H-segment (Kida et al., 2006).

The positive charges also contribute to membrane spanning of the H-segment translocating through the translocon. The polypeptide chain movement through the translocon is halted at the hydrophobic TM segment and then the TM segment is released into the membrane lipid. These processes are called stop-translocation and membrane insertion, respectively. A simple partitioning into the lipid bilayer of the H-segment should be a major factor for stop-translocation and membrane insertion of the TM segment (Hessa et al., 2005; Hessa et al., 2007). The positive charges flanking the cytoplasm also have the essential function of enhancing the stop-translocation of marginal H-segments (Kuroiwa et al., 1990; Kuroiwa et al., 1991; Lerch-Bader et al., 2008). In special cases, a marginal H-segment that is insufficient for stop-translocation by itself can span the membrane only when it is accompanied by positively charged residues. Interestingly, the positive charges are effective even when they are separated from the H-segment by more than 60 residues (Fujita et al., 2010). In this case, the H-segment can be temporarily exposed to the lumen and then slides back into the translocon. The extent of the exposure in the lumen depends on the position of the positive-charge cluster. Such a marginal H-segment is not integrated into the membrane and remains in a water-accessible environment.

Here, we systematically examined the effect of positive charges of the nascent polypeptide chain on the translocation in the absence of an H-segment and found that the positive charges arrest the movement of the polypeptide chain through the translocon, independently of the H-segment. The polypeptide chain elongates up to the C-terminus during the arrest, and the full-length polypeptide is gradually translocated into the ER lumen. These findings demonstrate that the positive charge is an independent translocation regulator and provides the physicochemical basis underlying the fundamental function of membrane protein topogenesis.

To quantitatively assay the effect of charged amino acid residues on polypeptide chain translocation through the ER translocon, we constructed systematically designed model proteins consisting of rat serum albumin (RSA) as a backbone, a query sequence as the middle portion, potential glycosylation sites in the upstream and downstream regions and an N-terminal signal peptide (Fig. 1A). The signal peptide induces the co-translational translocation of the downstream polypeptide. Initially, the upstream portion moves into the lumen where the first potential glycosylation site is glycosylated by the oligosaccharyl transferase, the active site of which is located in the lumenal side (Fig. 1C). When the entire protein is translocated through the translocon, the second site is glycosylated. If the translocation is interrupted at the query segment, then only the upstream site is glycosylated. The fully translocated polypeptide is resistant to externally added proteinase K, whereas the membrane-spanning polypeptide is degraded (Fig. 1C).

We examined clusters of charged amino acid residues and hydrophobic residues as query sequences. The name of each query sequence was created using the single letter code of the amino acid and the number of each amino acid; for example, ‘14K’ stands for a cluster of 14 lysine residues, and ‘16L4A’ for 16 leucine and four alanine residues. When the models were synthesized in the reticulocyte lysate cell-free translation system for 1 hour in the absence of rough microsomal membranes (RM), prominent single bands were observed (Fig. 1A, squares). When synthesized in the presence of RM, 2-kDa- and 4-kDa-larger forms were observed (Fig. 1A,B, red triangles and open circles, respectively) and the nonglycosylated forms were rarely observed. In this system, the nascent chains were efficiently targeted to the RM so that the products were either monoglycosylated or diglycosylated. These larger forms were converted to the smaller form by endoglycosidase H treatment (data not shown), confirming that the larger forms comprised diglycosylated and monoglycosylated forms. The model protein containing the negatively charged cluster (e.g. 14D in Fig. 1A, and 14E in Fig. 1B) had lower mobility on the gel, for unknown reasons. As expected, the monoglycosylated forms (red triangles) were degraded by proteinase K, whereas the diglycosylated forms (open circles) were proteinase K resistant. The products were degraded by proteinase K in the presence of mild non-ionic detergent (Triton X-100), indicating that the proteinase K resistance of the diglycosylated forms was caused by the membrane.

We unexpectedly found that the 14K and 14R (arginine) models produced the monoglycosylated forms as much as the diglycosylated forms (Fig. 1A). More than 50% of their full-length products spanned the membrane and their C-terminal domains were on the cytoplasmic side even though there was no H-segment. The monoglycosylated forms were largely degraded by proteinase K, eliminating the possibility that the positive-charge cluster inhibited glycosylation in the lumen. The 0K model was completely translocated, diglycosylated in the lumen and resistant to proteinase K digestion; whereas the 16L4A model was membrane-integrated, monoglycosylated in the upstream region and sensitive to proteinase K. We estimated the translocation efficiency as the percentage of the diglycosylated form in the diglycosylated form plus the monoglycosylated form (Fig. 1D). The Lys and Arg residues showed essentially the same effect, whereas the negatively charged residues, aspartic acid (Asp) and glutamic acid (Glu), had no effect on the translocation. The data clearly indicated that the positive charges alone interrupted translocation in the absence of any H-segment and generated a full-length polypeptide spanning the membrane.

To examine the time course of the polypeptide chain synthesis and translocation, the translation was performed for 20 minutes and translation was inhibited with cycloheximide and then the translocations were chased (Fig. 2). The full-length polypeptide of the 0K model was not detectable after the 10 minutes translation but became visible at 15 minutes. The full-length chain synthesized within the 20-minute incubation was largely diglycosylated. From the estimation that there are 65–70 residues of nascent chain from the ribosome peptidyl transferase center to the oligosaccharyl transferase active site (Whitley et al., 1996), the second glycosylation site could be accessible to oligosaccharyl transferase after translation termination. The majority of the 0K model was thus diglycosylated shortly after being synthesized. In this experiment, the distance of 192 amino acid residues between the two potential glycosylation sites caused little, if any, difference in glycosylation timing (Fig. 2A,B, 0K).

When the K-cluster was included, the amount of the monoglycosylated forms observed at 20 minutes of translation varied depending on the charge numbers (Fig. 2A,B). In the case of the 12K and 10K models, less than 30% of the nascent chains were diglycosylated at 20 minutes of translation. The monoglycosylated full-length polypeptides were gradually converted to the diglycosylated form during the chase incubation. By contrast, little of the 20K model was translocated, even after a 100-minute chase. Proteinase K treatment of the 20-minute translation products completely degraded the monoglycosylated forms, whereas the diglycosylated forms were proteinase K resistant (Fig. 2C), indicating that the downstream portion of the monoglycosylated forms is exposed to the cytoplasmic side (Fig. 2D, h). In this experimental system, the full-length products of the 0K, 10K and 20K models became detectable after 14, 16 and 16–18 minutes of translation, respectively (supplementary material Fig. S1). The difference in the elongation rate does not explain the drastic differences in translocation. These data indicate that polypeptide chain movement is arrested by the positive-charge cluster trapped at the membrane. The downstream region was on the cytoplasmic side even after the polypeptide chain elongated up to the C-terminus, and then was translocated during the chase period (Fig. 2D).

To examine the effect of charge neutralization and charge density, Asp or serine (Ser) residues were introduced into the 10K cluster (Fig. 3). The 10K cluster caused the accumulation of the full-length monoglycosylated form after 20 minutes of translation. The monoglycosylated form was converted to the diglycosylated form. The 10K cluster used here was placed upstream of the SADD sequence. The effect of translocation arrest was more apparent than when the 10K cluster was placed downstream of SADD sequence (Figs 1, 2). When five Asp were inserted into the 10K cluster, the product was diglycosylated as efficiently as the 0K model. The effect of the 10K cluster was completely suppressed by the five Asp insertion. The 10 Asp insertion produced the same result. When 10 Ser were introduced, the effect of the 10K cluster was largely, but not completely, suppressed. The positive charge effect is neutralized by negative charges. The charge density is also crucial for the arrest of translocation.

The effect of the K-clusters on translocation is likely to be due to simple electrostatic interactions. If this is the case, movement of the full-length product should be accelerated under high salt conditions. The model proteins were translated in the presence of RM for 20 minutes, and the translocation was then chased in the presence of cycloheximide under various ionic conditions (Fig. 4). In the case of the 14K model, conversion of the monoglycosylated form to the diglycosylated form was accelerated by 500 and 1000 mM NaCl. Lower ionic solutions of 100 and 200 mM NaCl had no substantial effect on the rate of conversion to the diglycosylated form. Even in the case of the 20K model, a substantial increase of the diglycosylated form was observed with the higher concentrations of NaCl. These results also support the conclusion that electrostatic interaction caused the translocation arrest independent of the flanking H-segment.

To examine membrane anchoring of the translocation-arrested polypeptide chain, the membranes were extracted under high salt or alkaline conditions (Fig. 5). Under the high salt conditions, the products, irrespective of the glycosylation status, were recovered in the membrane precipitates, whereas the nonspecific 45-kDa product was in the soluble fraction. Under the alkaline conditions, in which the peripheral membrane proteins and soluble proteins in the lumen are extracted into the supernatant (Fujiki et al., 1982), the 14K and 20K models were recovered in the supernatant, whereas the monoglycosylated form of the 16L4A model was in the membrane precipitate. The 16L4A model was anchored to the membrane by hydrophobic interaction, whereas translocation-arrested monoglycosylated forms of the 14K and 20K models were not anchored to the membrane.

To address the geometry of the translocation-arrested polypeptide chain on the membrane, we examined the distance between the 14K cluster and oligosaccharyl transferase active site by glycosylation site scanning (Fig. 6). The glycosylation site located 106 residues upstream of the 14K cluster was silenced and a new one was created 33 residues upstream of the 14K cluster. When the model protein was translated for 60 minutes, a nonglycosylated form was not observed and the glycosylation status was similar to that of the model used above, indicating that the 33-residue upstream site was accessible to oligosaccharyl transferase. Thirty-five percent of the product was translocated into the lumen and 65% of the product was stalled at the membrane. As the distance between the glycosylation site and the 14K cluster was shortened by serial deletions, the amount of the monoglycosylated form decreased and the amount of the nonglycosylated form increased (Fig. 6B,C); the distance producing half maximum monoglycosylation was 29–30 residues from the 14K cluster. The distance of the oligosaccharyl transferase active site from the hydrophobic TM segment was estimated as a control. The monoglycosylation was decreased depending on the distance; the distance causing half maximum glycosylation was 18–19 residues for the 7L7A segment and 15–16 residues for the 16L4A segment. These values are consistent with the value of 15 residues between the oligosaccharyl transferase active site and the authentic TM segment (Nilsson and von Heijne, 1993; Popov et al., 1997). In the case of the short H-segment of 7L7A, an additional spacer should be required for glycosylation. We concluded that the positive charges were 30 residues away from the oligosaccharyl transferase. A hydrophilic segment of approximately 10 residues is likely to span the translocon in an extended conformation, whereas a hydrophobic TM segment of approximately 20 residues spans the membrane in a helix conformation. These data indicated that the 14K cluster was on the cytoplasmic side of the membrane (Fig. 6D).

To examine whether the translocation-arrested polypeptide chain is released from the ribosome tunnel, we performed a chemical modification experiment where the cysteine residue of the nascent chain was reacted with a high-molecular mass (2 kDa) reagent, polyethylene glycol maleimide (PEGmal), for a short period on ice (Fig. 7). A single cysteine (Cys) residue was added at the indicated position of the 12K model: 61, 121 and 145 residues downstream of the 12K cluster. To make a polypeptide chain that is retained in the ribosome as a ribosome-nascent chain complex, the RNA was truncated just after the Val320 that is 152 residues downstream of the 12K cluster. If the nascent chain was retained in the ribosome, the Cys(+145) should be included in the ribosome tunnel, the Cys(+121) near the exit site of the ribosome and the Cys(+61) out side the ribosome (Fig. 7B). When the RNAs with a termination codon were translated in the presence of RM, all the Cys residues were highly reactive; the reaction with PEGmal caused a shift up by 2 kDa (Fig. 7C, lanes 6, 12 and 18, and Fig. 7D). However, when the truncated RNA was translated, Cys(+145) and Cys(+121) showed low reactivity (Fig. 7C, lanes 8 and 14, and 7D), whereas the Cys(+61) was highly reactive (Fig. 7C, lane 2, and 7D). Immediately after the puromycin treatment, both of Cys(+145) and Cys(+121) became highly reactive. As a control we performed the modification reaction with the nascent chain synthesized in the absence of RM; the Cys(+61) fully-exposed from the ribosome, the Cys(+121) at the exit site of the ribosome and the Cys(+145) retained in the ribosome were highly, moderately and poorly reactive, respectively (Fig. 7C, lanes 20, 22 and 26, and 7D). After puromycin treatment, the Cys(+121) and Cys(+145) became fully reactive. All data indicated that the nascent chain was fully released from the ribosome tunnel during translocation arrest.

To examine the environment of the translocation-arrested polypeptide that spanned the membrane, we performed a chemical crosslinking experiment (Fig. 8). A single Cys residue was created at the fourth or fifth residue upstream of the 14K cluster using a Cys-less model protein [Cys(−4) and Cys(−5), respectively]. The models were translated in the presence of RM and the crosslinking reaction was performed with a homobifunctional crosslinker bismaleimidoethane (BMOE), the crosslinking distance of which is 8 Å. Crosslinked products were subjected to immunoprecipitation with anti-Sec61α antibody. A Cys(−5) gave a substantial crosslinked band of ~85 kDa that was immunoreactive with anti-Sec61α antibody, and the Cys(−4) also gave weak but distinct crosslinked form of the same molecular mass. Given that the nascent chain is ~40 kDa, the size of the crosslinking partner is consistent with that of Sec61α. In the absence of the 14K cluster, the crosslinking with Sec61α was not observed, indicating that the crosslinking was specific. These results demonstrated that the translocation-arrested polypeptide chain is adjacent to the translocon.

Positive charges are known to determine the transmembrane topology of membrane proteins. We demonstrated here that the positive charges of translocating nascent polypeptides temporarily arrest chain movement through the translocon even in the absence of the H-segment. The effect depends on the number of positive charges; in certain cases, the downstream portion of the positive charge is retained on the cytoplasmic side of the membrane, even after the nascent chain elongates up to the C-terminus and is released from the ribosome. The translocation-arrested full-length nascent chain is then gradually translocated into the lumen. In the case of the 10K cluster, translocation of the majority of the nascent chain was arrested 20 minutes after the start of translation, and it was then moved through the translocon during the chase incubation. With the 20K cluster, the translocation was almost completely arrested at 20 minutes of translation and little translocation was observed even after a chase of 100 minutes. The effect of the K-clusters was neutralized by negatively charged residues inserted into the cluster and was greatly suppressed by high salt concentrations. The positive charges stall on the cytoplasmic side of the membrane, probably at the ribosome–translocon junction.

Our data strongly suggested that a simple electrostatic interaction causes the translocation arrest independent of flanking hydrophobic sequences. The Coulomb force on the positive charges increases the energy barrier for the nascent chain to move into the translocon and consequently decreases frequency of the forward movement. The possible interaction partners are translocon subunits, ribosomal subunits, ribosomal RNA and charges of the membrane lipids. Goder et al. have indicated that charged residues of Sec61p of the yeast translocon contribute to the positive-inside rule (Goder et al., 2004). The charged amino acid residues within the Sec61α subunit might be involved in the interaction. In the ribosome, the positive charges of the nascent chain might slow down polypeptide chain elongation through electrostatic interaction with negative charges of the ribosome tunnel (Lu and Deutsch, 2008). In the present study, the effect of the positive charges on the protein synthesis rate was not drastic; similar amounts of the 0K model and the 20K model were observed after 20 minutes of translation (Fig. 2 and supplementary material Fig. S1). In contrast to the time range of the translation delay, the positive charge caused a much more drastic delay in translocation, which resulted in accumulation of translocation-arrested full-length polypeptide. For the accumulation of the monoglycosylated full-length polypeptide, the translocation must be arrested until the downstream polypeptide chain of 140 residues is synthesized. The 6K cluster induced substantial accumulation of arrested full-length product. We expect that such a translocation arrest also occurs with only a few positive charges, although they might not induce a much accumulation of the full-length polypeptide. Considering the time scales involved in polypeptide chain synthesis and topology determination of the nascent chain, short and temporary translocation arrest should be sufficient to establish the membrane topology of polypeptide chains. We found only two mammalian secretory proteins in a protein databases (human extracellular sulfatase Sulf-1 and human R-spondin-2) that have a cluster of 10 positive charges within a 12-residue window. Such a positive-charge cluster should be detrimental for translocation and thus excluded from secretory proteins during evolution.

The arrest of polypeptide chain translocation causes various topogenic events. If the N-terminal side of an H-segment of a signal sequence is arrested, then the opposite side moves into the lumen. In this case, the positive charges only have to retain the N-terminal side until its downstream sequence comprising, at most, 30 residues emerges from the ribosome. After that, the H-segment is settled as the TM α-helix with an N_cyto orientation. If the C-terminus of an H-segment of a signal sequence is arrested on the cytoplasmic side until the opposite N-terminal side is flipped into the lumenal side, the H-segment becomes a TM helix with an N_lumen orientation. For example, in the case of synaptotagmin II, there are eight positive charges just after the H-segment. When they are moved more than 20 residues downstream, they still affect the orientation of the H-segment, whereas those that are moved more than 30 residues downstream no longer affect the orientation (Kida et al., 2006). In many cases, the TM topology of a signal sequence is likely to be determined within a short range. The positive charges flanking an H-segment enhance stop translocation (Jaud et al., 2009; Kuroiwa et al., 1990; Kuroiwa et al., 1991; Lerch-Bader et al., 2008). The TM topology is achieved as soon as the positive charges reach the translocon. There is a considerable amount of data to support the contributions of positive charges to the membrane topology of H-segments, as indicated by the positive-inside rule (Sipos and von Heijne, 1993; von Heijne and Gavel, 1988). The positive charges have been discussed only in relation to the flanking H-segment and have been recognized as a topology modulator of H-segments. Recently, we found a longer range effect of positive charges than previously detected (Fujita et al., 2010). A positive-charge cluster located more than 60 residues downstream of a marginal H-segment can contribute to its membrane spanning (Fujita et al., 2010). After the upstream H-segment passes through the translocon, the positive charges at the 60 residues downstream arrests the translocation and the H-segment slides back into the translocon. In the present study, we found that the positive charges arrest polypeptide chain movement even in the absence of an H-segment. Tentative translocation arrest by positive charges provides the nascent chain in the translocon with a time interval that might allow the polypeptide segments to move back and forth, reorient and assemble with each other.

Our findings suggest that polypeptide chain movement is arrested during cotranslational translocation and that the forward movement resumes after completion of elongation. The positive charges stall at the translocon, even in the presence of the pushing force of the chain elongation, and can then continue to slowly advance in the absence of the pushing force. Even if the chain is not elongating, translocation tendency generated in the lumen should similarly contributes to the movement, e.g. binding of chaperones, folding of the polypeptide chain, glycosylation and disulfide bond formation, etc. In addition, there might be favorable conditions for translocation after termination of the translation. The cotranslational forward movement is driven by chain elongation on the ribosome but the rate is limited by the elongation rate. The degree of freedom of the polypeptide chain should be restricted during chain elongation. However, after the polypeptide chain is released from the peptidyl transferase center of ribosome, the nascent chain would be unrestricted and could thus fluctuate largely and freely. Such free motion might occasionally overcome the arrest caused by positive charges.

Rough microsomal membrane (RM) (Walter and Blobel, 1983) and rabbit reticulocyte lysate (Jackson and Hunt, 1983) were prepared as previously described. RM was treated with EDTA and then with Staphylococcus aureus nuclease as previously described (Walter and Blobel, 1983). Castanospermine (Merck, Tokyo, Japan), proteinase K (Merck, Tokyo, Japan), RNaseA (Wako, Osaka, Japan), DNA manipulating enzymes (Takara and Toyobo, Tokyo, Japan), 2 kDa PEGmal (Sunbright ME-020MA, NOF Corporation, Tokyo, Japan), bismaleimidoethane (BMOE, Thermo Scientific, Yokohama, Japan), puromycin (Sigma, Tokyo, Japan) and cycloheximide (Sigma, Tokyo, Japan) were obtained from the indicated sources.

In the following DNA construction procedure, DNA fragments were obtained by PCR using primers including the appropriate restriction enzyme site (indicated in parentheses). The fragments were subcloned into plasmid vectors that had been digested with the restriction enzymes. At each junction, the six bases of the restriction enzyme site were designed to encode two codons. Point mutations were introduced using the method of Kunkel (Kunkel, 1985) or the QuickChange procedure (Stratagene, La Jolla, CA). All the constructed DNAs were confirmed by DNA sequencing.

The model protein, based on RSA, was previously described (Fujita et al., 2010). Briefly, potential glycosylation sites were created at Asn67 and Asn259, each query sequence was introduced in the middle portion and the Val and termination codon were inserted after H319 (Fig. 1). Various query sequences, as indicated in the figures, were inserted into the middle portion of the model protein using the QuickChange procedure. The model proteins shown in Fig. 3 were created by inserting synthetic oligonucleotides encoding the indicated sequences between AS and SA (NheI–Aor51HI). For the glycosylation site scanning constructs (Fig. 6), the upstream regions of the query sequence were serially deleted or glycosylation sites were newly created by the mutations (T¹⁴⁹SF to N¹⁴⁹ST, S¹⁵⁰FQE to N¹⁵⁰STA, F¹⁵¹QE to N¹⁵¹ST, Q¹⁵²ENP to N¹⁵²STA, E¹⁵³NP to N¹⁵³ST, E¹⁵⁴NPT to T¹⁵⁴NST, N¹⁵⁵PTS to A¹⁵⁵NST and P¹⁵⁶TSF to A¹⁵⁶NST). The newly created potential sites were confirmed to be efficiently glycosylated in the absence of the query sequences. For chemical modification of the model proteins with PEGmal (Fig. 7), a single Cys residue was included at Cys229 Cys284 or Cys313 and DNA fragment encoding the 12K cluster was inserted between AS and SA (NheI–Aor51HI). For the construction, we exploited the Cys-less model protein which had glycosylation site 12 residues downstream of the 12K cluster. To make truncated mRNA, the restriction enzyme site (BamHI) was created just after the Val320 codon. The models for crosslinking (Fig. 8), a single Cys was included at Cys164 for Cys(−5) or Cys165 for Cys(−4) and DNA fragment encoding the 14K cluster was inserted between AS and SA (NheI–Aor51HI).

Cell-free transcription and translation were performed essentially as described previously (Sakaguchi et al., 1992), except that translation reactions with RM contained 20 μg/ml castanospermine to prevent trimming of the sugar chain, which results in heterogeneity of the products. The plasmids harboring RSA model proteins were linearized with XhoI or BamHI. The plasmids were then transcribed with T7-RNA polymerase. The RNA was translated in the reticulocyte lysate cell-free system for 1 hour at 30°C in either the absence or presence of RM. The translation reaction included 90 mM potassium acetate, 1.2 mM magnesium acetate, 32% reticulocyte lysate, 20 μg/ml castanospermine and 15.5 kBq/μl EXPRESS ³⁵S protein-labeling mix (Perkin Elmer, Waltham, MA). Where indicated, 2 mM cycloheximide was included at the indicated time points. After the translation reaction, aliquots were treated with proteinase K (200 μg/ml) for 1 hour on ice. Radiolabeled polypeptide chains were analyzed by SDS-PAGE, visualized with an imaging analyzer (BAS1800, Fuji Film) and quantified using Image Gauge software (Fuji Film). Where truncated mRNA was translated, the product was treated with RNase (42 μg/ml) before SDS-PAGE to completely remove tRNA from the nascent chain.

Where indicated, the translation reactions were terminated by the addition of cycloheximide (2 mM) and were then further incubated for the indicated times. At each time point, aliquots were removed and proteins were precipitated by TCA, and then analyzed by SDS-PAGE. Other aliquots were treated by proteinase K, in the absence or presence of detergent (Triton X-100) as previously describe (Sakaki et al., 1999). For chase reaction in the presence of NaCl, translation reaction mixture was diluted with an equal volume of appropriate NaCl buffer solution to adjust the final concentrations of salt and then incubated at 30°C for the indicated time. For alkaline or high salt extraction, the samples were treated as described previously (Ikeda et al., 2005); briefly, the translation mixtures were treated with high salt (500 mM NaCl) or alkali (100 mM NaOH) and subjected to ultracentrifugation for 5 minutes at 4°C (Hitachi S100AT4 Rotor, 50,000 r.p.m.), to separate membrane precipitates and supernatant.

For a PEGmal reactivity assay, the RNAs were translated in the presence of RM for 20 minutes at 30°C. To stop the translation, cycloheximide (2 mM) or puromycin (2 mM) was added and the reaction mixture immediately chilled on ice. Reaction with PEGmal was performed essentially as described (Kida et al., 2010). Then 1 μl 100 mM PEGmal dissolved in water was added to the mixture (9 μl) and incubated on ice for 5 minutes. The maleimide reaction was quenched with 1 μl 100 mM dithiothreitol (DTT), the mixture was treated with RNaseA and 20 μl SDS-PAGE sample buffer containing 100 mM DTT was added.

For chemical crosslinking, the RNAs were translated in the presence of RM for 60 minutes and the reaction was terminated with 2 mM cycloheximide. The chemical crosslinking was performed essentially as previously described (Kida et al., 2007). The products were treated on ice for 60 minutes with 10 mM BMOE or its solvent dimethyl sulfoxide. The crosslinking reaction was quenched for 10 minutes on ice with a sixfold volume of dilution buffer (30 mM HEPES, pH7.4, 150 mM potassium acetate, 2 mM magnesium acetate) containing 20 mM DTT. For immunoprecipitation, RM were isolated by centrifugation at 100,000 g for 5 minutes, solubilized with 1% SDS for 5 minutes at 95°C, and then diluted with a 30-fold volume of immunoprecipitation buffer [1% Triton X-100, 50 mM Tris-HCl, pH7.5, 150 mM NaCl]. After a centrifugation at 20,000 g for 5 minutes, the supernatant was incubated for 30 minutes with protein-A–Sepharose (GE Healthcare) alone to remove materials nonspecifically bound to resin. The unbound fractions were incubated for 2 hours with anti-Sec61α antiserum and then with protein-A–Sepharose for 14 hours. The resin was washed three times with immunoprecipitation buffer and the isolated proteins were eluted by boiling with SDS-PAGE sample buffer.

We thank Shigeki Mitaku and Runcong Ke (Nagoya University) for instruction on database searching.