4184
Research Article
Positive charges on the translocating polypeptide
chain arrest movement through the translocon
Hidenobu Fujita, Marifu Yamagishi, Yuichiro Kida and Masao Sakaguchi*
Graduate School of Life Science, University of Hyogo, Kouto Ako-gun, Hyogo 678-1297, Japan
*Author for correspondence (sakag@sci.u-hyogo.ac.jp)
Journal of Cell Science
Accepted 27 July 2011
Journal of Cell Science 124, 4184–4193
ß 2011. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.086850
Summary
Polypeptide chains synthesized by membrane-bound ribosomes are translocated through, and integrated into, the endoplasmic reticulum
(ER) membrane by means of the protein translocation channel, the translocon. Positive charges on the nascent chain determine the
orientation of the hydrophobic segment as it is inserted into the translocon and enhance the stop-translocation of translocating
hydrophobic segments. Here we show that positive charges temporarily arrested ongoing polypeptide chain movement through the ER
translocon by electrostatic interaction, even in the absence of a hydrophobic segment. The C-terminus of the polypeptide chain was
elongated during the arrest, and then the full-length polypeptide chain moved through the translocon. The translocation-arrested
polypeptide was not anchored to the membrane and the charges were on the cytoplasmic side of the membrane. The arrest effect was
prevented by negatively charged residues inserted into the positive-charge cluster, and it was also suppressed by high salt conditions. We
propose that positive charges are independent translocation regulators that are more active than previously believed.
Key words: Endoplasmic reticulum, Positive charge, Signal sequence, Translocation
Introduction
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 Sec61a, Sec61b and
Sec61c (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 Nterminal 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 Ncytosol and Clumen 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 Cterminal side is rich in positive charges, the N-terminus is
translocated through the translocon and forms the Nlumen and
Ccytosol 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
Positive charges arrest translocation
is an independent translocation regulator and provides the
physicochemical basis underlying the fundamental function of
membrane protein topogenesis.
Results
Effect of positive-charge clusters on protein translocation
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
Journal of Cell Science
essential function of enhancing the stop-translocation of marginal
H-segments (Kuroiwa et al., 1990; Kuroiwa et al., 1991; LerchBader 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 Hsegment 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
4185
Fig. 1. Positive-charge clusters arrest translocation in the absence of an H-segment. (A) Top panel: the rat serum albumin-based model protein included its
signal peptide (S) at the N-terminus, a query sequence of charged or hydrophobic residues in the middle portion, and glycosylation sites (circles) in the upstream
and downstream portions. The model proteins are named according to the included amino acids and their numbers, e.g. 10K stands for a cluster of 10 Lys residues.
16L4A has the following sequence: LLLLALLLLALLLLALLLLA. Bottom panel: the model proteins were translated in the absence and presence of RM for
1 hour. Aliquots were treated with proteinase K (ProK) in the absence or presence of nonionic detergent (Tx). The diglycosylated forms (open circles),
monoglycosylated forms (red triangles) and nonglycosylated forms (squares) are indicated. Nonspecific translation product is indicated by asterisks. (B) Model
proteins were translated in the presence of RM and half of each aliquot was treated with proteinase K. (C) Possible membrane topology of the model protein. If the
nascent chain is fully translocated into the lumen, both potential glycosylation sites are glycosylated (white circles) and the nascent chain is fully resistant to
proteinase K. If translocation is interrupted by the query sequence, only the upstream site is glycosylated, the C-terminal portion is exposed on the cytoplasmic
side of the membrane, and is degraded by proteinase K. The query sequence (black rectangles), glycosylated potential sites (white circles), nonglycosylated
potential sites (shaded dots) and ribosomes (shaded circle and oval) are indicated. (D) Quantification of translocated polypeptide chains. The diglycosylated form
and monoglycosylated form were quantified and translocation percentages were calculated using the following formula: diglycosylated form/[(diglycosylated
form)+(monoglycosylated form)]6100. The experiments were performed more than three times; values are means ± s.d. Lysine (K) and arginine (R) residues
produced almost the same effect, whereas aspartic acid (D) and glutamic acid (E) residues produced no effect.
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Journal of Cell Science
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 fulllength 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 positivecharge 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.
Positive charges temporarily arrest translocation
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
Fig. 2. Translocation is transiently arrested. (A) The mRNAs coding for
the model proteins were translated in the presence of RM for 20 minutes and
the chain elongations were inhibited with cycloheximide (CH). The reaction
mixtures were further incubated to chase the translocation for the indicated
time periods. An aliquot was removed at each incubation time and analyzed
by SDS-PAGE. Monoglycosylated forms (red triangles) and diglycosylated
forms (white circles) are indicated. 4L4A represents the following sequence:
LALALALA. (B) Translocation percentages at each time point were
calculated as described in Fig. 1. The experiments were performed twice and
the mean values are presented. (C) The monoglycosylated form is sensitive to
proteinase K. After 20 minutes of translation the products were treated with
proteinase K in the presence of CH. The monoglycosylated forms (red
triangles) were almost completely degraded, whereas the diglycosylated
forms were resistant. The monoglycosylated forms span the membrane and
their C-terminal region was sensitive to proteinase K. (D) In the absence of the
query sequence, the nascent chain was fully translocated and diglycosylated
(a–d), whereas the positive-charge cluster caused nascent chains to stall at the
translocon until the full-length chain was completed (e–h) and then gradually
moved through the translocon (h–j).
Journal of Cell Science
Positive charges arrest translocation
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).
The effect of charge neutralization and charge density
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 arrest by the K-cluster was suppressed in a
high-salt environment
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
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Fig. 3. Effect of charge neutralization and charge density. (A) Asp or Ser
residues were introduced into the 10K-cluster as indicated. The query
sequences were inserted upstream of the SADD sequence. The model proteins
were translated as described in Fig. 2 and chased for the indicated time
period. (B) Translocation percentages were calculated. The experiments were
performed twice and the mean values are presented. Insertion of five Asp
residues into the 10K cluster suppressed translocation arrest and resulted in
efficient translocation as in the 0K model. Insertion of Ser partially
suppressed the translocation arrest.
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.
The arrested K-cluster is on the cytoplasmic side of
the membrane
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
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Fig. 4. Effect of ionic strength on translocation rate. The 14K and 20K model proteins were translated in the presence of RM for 20 minutes. An equal volume
of NaCl solution was added to produce the indicated salt concentration. The mixtures were further incubated and aliquots were removed at the indicated times and
subjected to SDS-PAGE. Translocation percentages were calculated. The experiments were performed more than three times; values are means ± s.d.
Fig. 5. Alkaline extraction of the translocation-arrested polypeptide.
(A,B) The model proteins with the indicated sequences were translated for
60 minutes and the reaction mixtures were treated with high-salt solution (A)
or alkaline buffer (B). The mixture was subjected to ultracentrifugation to
separate membrane precipitates (P) and supernatant (S). These fractions and
the total translation product (T) were analyzed by SDS-PAGE. Diglycosylated
forms (white circles) and monoglycosylated forms (black triangles) are
indicated. Nonspecific products are indicated by asterisks. The
monoglycosylated forms of the 14K and 20K models were not anchored to the
membrane, whereas the 16L4A model was anchored in the membrane. The
trace amount of an apparently diglycosylated form of the 16L4A-model in the
P fraction was not examined further.
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
Journal of Cell Science
Positive charges arrest translocation
4189
Fig. 6. Geometry of the translocation-arrested polypeptide chain. (A) To assess membrane topology of the lysine cluster (14K), a potential glycosylation site
was introduced 33 residues upstream of the 14K cluster. The distance from the 14K cluster was altered by serial deletion, as indicated. As a control, the
glycosylation site was placed at various points upstream of the H-segments (7L7A and 16L4A), as indicated. (B) The models were translated in vitro in the
presence of RM for 60 minutes and analyzed by SDS-PAGE. Diglycosylated forms (white circles), monoglycosylated forms (black triangles) and nonglycosylated
forms (white squares) are indicated. The distance between the glycosylation site and the query sequence is indicated at the top of the gel. The 14K cluster did not
completely stop the translocation. Under the condition used, 40% of the nascent chain was fully translocated after the 60 minutes translation and consequently
diglycosylated irrespective of the positions of glycosylation site. (C) Percentage of the monoglycosylated form in the nonglycosylated plus the monoglycosylated
form was calculated. The experiments were performed twice and the mean values are presented. (D) The 14K cluster is more than 30 residues away from
oligosaccharyl transferase (OSTase), whereas the authentic TM segment is 15 residues away from the enzyme. The hydrophilic 14K cluster is on the cytoplasmic
side of the membrane.
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).
The arrested polypeptide chain is released from
the ribosome
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 ribosomenascent 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) fullyexposed 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.
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Fig. 7. The nascent polypeptide chain was fully released from the ribosome during translocation arrest. (A) To assess reactivity with PEGmal, a single
Cys residue was created at the indicated position of the Cys-less model protein. The positions relative to the 12K cluster of the Cys residue, glycosylation
sites and C-terminus are indicated in parentheses. The 12K cluster was inserted between AS and SA, as in Fig. 3. (B) Geometry of Cys residues of the
nascent chain. In the ribosome-nascent chain complex (RNC), Cys(+145) is in the ribosome and not reactive with PEGmal, whereas Cys(+121) is near the
exit site and partially reactive. Cys(+61) is outside the ribosome and freely accessible to PEGmal. In the presence of a termination codon, the nascent chain
was released from the ribosome immediately after synthesis and even the Cys(+145) reacted with PEGmal. (C) The RNAs, including those with a
termination codon (Ter) or truncated after the Val codon (RNC and Puro) were translated in the presence or absence of RM for 20 minutes and subjected to
PEGmal treatment on ice for 5 minutes. Where indicated, the nascent chain was released from the ribosome with puromycin (Puro) before the PEGmal
reaction. The PEGylated form (red squares), monoglycosylated form (red triangles) and nonglycosylated form (open squares) are indicated. Nonspecific
products are indicated by asterisks. (D) The efficiency of the PEGmal reaction. The experiments were repeated twice with each model protein and the mean
PEGylation efficiency was plotted.
Translocation-arrested polypeptide was crosslinked with
the Sec61a subunit
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-Sec61a antibody. A
Cys(–5) gave a substantial crosslinked band of ,85 kDa that was
immunoreactive with anti-Sec61a 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 Sec61a. In the
absence of the 14K cluster, the crosslinking with Sec61a was not
observed, indicating that the crosslinking was specific. These
results demonstrated that the translocation-arrested polypeptide
chain is adjacent to the translocon.
Discussion
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 Kclusters 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.
Journal of Cell Science
Positive charges arrest translocation
Fig. 8. Crosslinking of translocation-arrested polypeptide chains with
Sec61a. A single Cys residue was added at the indicated position of
Cys-less 14K or 0K model proteins. The position relative to the 14K-cluster
was indicated in the parenthesis. Supposed geometry of the Cys residues is
also shown. The model proteins were translated in the presence of RM for
60 minutes, and subjected to BMOE treatment on ice for 1 hour (BMOE +
lanes). Aliquots of the products were subjected to immunoprecipitation with
anti-Sec61a antiserum (IP lanes). The bands immunoreactive with Sec61a
antibody (arrowheads) and the nascent chains (triangle) are indicated.
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 Sec61a 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 fulllength 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
4191
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 Rspondin-2) that have a cluster of 10 positive charges within a 12residue 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 Nterminal side until its downstream sequence comprising, at most,
30 residues emerges from the ribosome. After that, the Hsegment is settled as the TM a-helix with an Ncyto 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 Nlumen 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 Hsegments, 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 Hsegments. Recently, we found a longer range effect of positive
charges than previously detected (Fujita et al., 2010). A positivecharge 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
4192
Journal of Cell Science 124 (24)
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.
Materials and Methods
Materials
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.
Journal of Cell Science
Construction of model proteins
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).
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 ml 100 mM PEGmal dissolved in water was added to the mixture (9 ml)
and incubated on ice for 5 minutes. The maleimide reaction was quenched with
1 ml 100 mM dithiothreitol (DTT), the mixture was treated with RNaseA and
20 ml 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 TrisHCl, 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-Sec61a 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.
Acknowledgements
We thank Shigeki Mitaku and Runcong Ke (Nagoya University) for
instruction on database searching.
Funding
This work was supported by Grants-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and
Technology of Japan, and Japan Society for the Promotion of
Science [19058013, 20370041 and 23370055 to M.S.; 21770123 and
23770151 to Y.K.; 23-6629 to H.F.]; the Global COE program; the
Sumitomo Foundation [grant number 080178 to Y.K.]; and the
Hyogo Science and Technology Association [grant number 229035
to Y.K.].
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.086850/-/DC1
In vitro synthesis and membrane translocation
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