From the Laboratory of Cellular Biophysics, The
Rockefeller University, New York, New York 10021 and
§ The Department of Cell Biology, The Scripps Research
Institute, La Jolla, California 92037
Received for publication, July 25, 2002, and in revised form, December 12, 2002
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ABSTRACT |
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The tight coupling between ongoing translation
and translocation across the mammalian endoplasmic reticulum has made
it difficult to determine the requirements that are specific for
translocation. We have developed an in vitro assay that
faithfully mimics the co-translational targeting and translocation of
the amino terminus of opsin without ongoing translation. Using this
system we demonstrate that this post-translational targeting and
translocation requires nucleotide triphosphates but not cytosolic
proteins. The addition of GTP alone was sufficient to fully restore
targeting. The addition of ATP was not specifically required, and
non-hydrolyzable analogs of ATP that blocked 90% of the ATPase
activity also had no inhibitory effect on translocation.
Targeting to the mammalian endoplasmic reticulum
(ER)1 membrane has been
extensively studied and is a model system for other targeting events.
Proteins with the appropriate signal sequence (1) bind to the signal
recognition particle (SRP), which pauses translation (2-4). SRP then
binds to the SRP receptor, which brings the ribosome and nascent
peptide to the ER and releases the translational pause (5-7). The
nascent peptide is then translocated through protein-conducting
channels (8). Most studies on targeting to the ER membrane have used
secreted proteins with cleaved amino-terminal signal sequences for
which there is tight coupling between ongoing translation and
translocation (9-13). This tight coupling between translation and
translocation has complicated the analysis of the requirements for
translocation in mammalian systems, as distinct from the requirements
for translation.
The analysis of translocation in yeast has been more straightforward
since a completely post-translational pathway has been demonstrated.
Some yeast proteins can be targeted to the ER after complete synthesis
and release from the ribosome (14, 15). This has allowed the in
vitro analysis of the process in isolation of the requirements for
ongoing translation, which has provided a number of insights into
mechanisms of translocation. ATP is required for post-translational
translocation across the yeast ER (15), as a consequence of the
involvement of both cytosolic and ER-lumenal ATPase chaperones (14, 16,
17). The lumenal chaperone BiP (also known as Kar2 and Grp78) plays a
critical role in yeast translocation. Both temperature-sensitive
mutations in BiP and decreased expression of BiP cause translocation
defects in vivo (18, 19). Proteoliposomes reconstituted with
purified yeast ER proteins required BiP for translocation as well
(20).
The role of ATP and BiP in mammalian translocation is unresolved. ER
microsomes that had been depleted of lumenal contents (including BiP)
were competent to target proteins to the membrane but failed to
translocate them across the microsomal membrane (21). In other
experiments, proteoliposomes reconstituted with purified ER membrane
components did not require BiP (22, 23). Because ongoing translation
was required in these assays, the requirement for ATP could not be
independently evaluated. Other in vitro systems have been
used to partially dissociate the coupling between translation and
translocation across the mammalian ER membrane. However, the targeting
and translocation efficiencies were very low (24-27), suggesting that
efficient translocation may only occur coupled to translation.
To investigate translocation in a mammalian system, we developed an
assay using translation intermediates of opsin. Like most members of
the G-protein-coupled receptor family, opsin does not have cleaved
amino-terminal signal sequence, but nevertheless, the amino terminus is
translocated across the ER membrane. The first transmembrane segment
functions as the signal sequence, and it must emerge from the ribosome
to bind SRP and target to the membrane (28). This implies that the
segment of opsin that is on the amino side of the first membrane
segment can only translocate uncoupled from translation. Indeed, for
nascent polypeptides paused as the first transmembrane segment emerges
from the ribosome, the nascent opsin can efficiently target to the ER
membrane and translocate uncoupled from ongoing protein synthesis
("post-translationally") (29).
Thus, a transient step in the biogenesis of opsin can be investigated
in vitro, isolated from the requirements for translation. Using this system, we have observed that GTP is necessary for translocation and that there is no specific requirement for ATP or
cytosolic proteins.
General chemicals were from Sigma or Fisher unless otherwise
noted. Puromycin stock was 20 mM in 500 mM
Hepes-KOH (pH 7.4). Cycloheximide stock was 20 mg/ml. Emetine stock was
20 mM. ATP, GTP, and UTP stocks (100 mM) were
from Amersham Biosciences. Restriction enzymes were from New England
Biolabs (Beverly, MA). Translations were labeled with translation grade
[35S]methionine from Amersham Biosciences or PerkinElmer
Life Sciences.
Constructs--
Opsin truncation mutants have been previously
described (29). The nomenclature we use for this shortened nascent
opsin construct is tOP××, for truncated
opsin, with the superscript number indicating the number of
codons after the end of the first transmembrane domain (which ends at
amino acid 61). The first transmembrane segment is used as a point of
reference because it is the first information targeting the nascent
opsin to the ER membrane (28). Throughout this study we use
tOP30, which contains 91 amino acids total, 30 after the
first transmembrane domain.
Transcriptions--
DNA plasmids were digested with
BamHI and purified with a QIAQuick PCR purification kit
(Qiagen, Chatsworth, CA). The cut DNA was subsequently transcribed with
a Message Machine SP6 kit (Ambion, Austin, TX) according to the
manufacturer's specifications.
Translations--
The nuclease-treated rabbit reticulocyte
lysate extract and RNasin were from Promega (Madison, WI). Canine
pancreatic membranes (ER membranes) were purchased from Promega or
prepared as previously described (30). Translations were performed
according to the manufacturer's specifications, except as described in
the text.
Post-translational Reactions--
After translation with no ER
membranes present, further translation was inhibited with cycloheximide
(2 mg/ml final) or emetine (2 mM final) for 10 min at
25 °C. ER membranes (0.75 µl/12.5-µl reaction) were added after
treatment. The reaction was incubated at 25 °C for an additional 30 min. Some samples were explicitly released from the ribosome with
puromycin (2 mM final) and incubated for 5 min at 25 °C
then 5 min at 37 °C.
Purification of Ribosome-bound Nascent Opsin Polypeptides (RBOps)
by Desalting--
Translation reactions (prepared as above for
post-translational reactions) were desalted using Microspin® G-25
columns (Amersham Biosciences). The column was equilibrated with
translation/translocation buffer (TB:120 mM potassium
acetate (pH 7.5), 50 mM Hepes-KOH (pH 7.5), 2 mM magnesium acetate) supplemented with 1 mM
dithiothreitol just before use. The column was centrifuged at 735 × gav for 25-30 s. The translation was diluted
with buffer to a volume of 50 µl final and then added to the column.
The sample was centrifuged into the column at 735 × gav for 25-30 s, and the flow-through was
discarded. The desalted translation mix was eluted by centrifugation for 2 min at 735 × gav and diluted with
TB. Supplemented reactions were typically incubated at 25 °C for 30 min.
Purification of RBOp by Centrifugation--
Translations
(prepared as above for post-translational reactions) were diluted in TB
plus dithiothreitol, overlaid on standard harvesting buffer (1 M sucrose, 150 mM potassium acetate (pH 7.5), 50 mM HEPES-KOH (pH 7.5), 2.5 mM magnesium
acetate), and centrifuged in TLA 100 rotor (Beckman Instruments) at
62,000 rpm (~148,000 × gav) for 30 min
at 4 °C. The resulting ribosome nascent opsin complex pellet was
resuspended in TB plus dithiothreitol.
Post-translational Reactions with RBOp--
For reactions,
10-µl aliquots were supplemented with 1 µl of a 10× energy stock
or reticulocyte-lysate (cytosol). ER membranes (0.5 µl) were added to
each 10-µl reaction.
ER Membrane Pretreatment with Inhibitors--
ER membranes were
diluted in TB containing 1 mM AMPPNP or GMPPNP and
incubated for 10 min at 25 °C. Mock-treated samples were diluted in
buffer alone under the same conditions. ER membrane aliquots were then
added to purified RBOp for post-translational reactions as above. The
final concentration of inhibitor was less than 200 µM in
the reaction.
ATPase assay--
ATPase activity was measured by determining
the concentration of NADH using a Genesys 2PC spectrophotometer
(Spectronic Instruments, Rochester, NY) with a coupled system (that
converted 1 molecule of NADH to NAD+ for each molecule of
ADP generated) as previously described
(31).2 Briefly, 200-µl
reactions containing 1 mM MgATP, 3 mM
phosphoenolpyruvate, 0.21 mM NADH, 65 mM
Hepes-KOH (pH 7.5), 1.3 mM MgCl2, and 1.3 mM dithiothreitol were supplemented with 3.5 µl of
protein kinase/lactate dehydrogenase enzymes (Sigma) and 15 µl
of ER membranes (prepared from canine pancreas). The
A340 was monitored for 10 min at 1-min intervals to determine the rate constant. As a control, ADP was added
to some samples to directly stimulate degradation of NADH. AMPPNP did
not inhibit the coupled system (data not shown).
We previously showed that nascent intermediates of opsin could
target to the ER membrane and even translocate the amino terminus across without ongoing translation (29). In this work we examined the
specific requirements for this process by removing components from the
translation mix before the addition of ER membranes. As before, we
generated RBOps by in vitro translation of mRNA that had
been truncated at specific positions within the coding region without a
stop codon. Then the RBOp were isolated from other components of the
translation mix using a G-25 column or ultracentrifugation. These
purified RBOp could be incubated with ER membranes and other
supplements to determine the requirements for targeting and
translocation. RBOp were purified over a G-25 column after treatment
with emetine to block any further translation (Fig.
1). The indicated band represents the
nascent polypeptide still covalently bound to the tRNA as previously
shown (29). The nascent polypeptides were still bound to functional
ribosomes, since 93% could be released from the tRNA by puromycin
(lane 7). Release of nascent polypeptides by puromycin
requires the labile aminoacyl-tRNA bond to be positioned near the
functional peptidyltransferase site (33). Some components required for
targeting and/or translation were removed by the G-25 column, because
adding ER membranes to depleted RBOp did not result in appreciable
glycosylation of the nascent peptides (lane 1). Without ER
membranes added, supplementation of RBOp with cytosol alone did not
stimulate glycosylation (lane 2). However, supplementation
with cytosol and ER membranes together did result in efficient
glycosylation (62%, lane 3). This was comparable with the
efficiency of glycosylation of samples that were not run over the G-25
column (Untreated Translation, 83%, lane 9).
Because the majority of the nascent polypeptides could be released by
puromycin and the efficiency of glycosylation was comparable with the
untreated samples, we can conclude that the treatment did not damage
the ribosome-bound nascent chains.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Desalted translations require the addition of
nucleotide triphosphate energy sources for efficient targeting and
translocation. A translation of tOP30 was incubated at
25 °C for 30 min and treated with emetine (2 mM final)
for 10 min at 25 °C. Aliquots of the translation (lanes 8 and 9) were incubated in the presence (lane 8) or
absence (lane 9) of ER membranes. The rest of the
translation was depleted of small molecules with a G-25 column. The
resulting eluted volume was diluted to 100 µl final. An aliquot was
treated with puromycin (2 mM final) for 5 min at 25 °C
and 5 min at 37 °C (lane 7). Aliquots were supplemented
with ER membranes only (lane 1), cytosol only
(lane 2), or both cytosol and ER membranes (lane
3). Other aliquots were supplemented with ER membranes and the
indicated nucleotides (lanes 4-6). Samples were then
incubated for 30 min at 25 °C (except lane 7). The
numbers indicate the percent glycosylation (except lane
7).
To determine the specific energetic requirements for targeting and translocation, individual nucleotide energy sources were added to RBOp, with ER membranes present (lanes 4-6). The addition of GTP supported glycosylation as efficiently as cytosol (compare lanes 3 and 5). The addition of ATP and GTP was no more efficient than GTP alone (compare lane 5 to lane 6). The addition of ATP alone (lane 4) supported moderate glycosylation of the tRNA-nascent opsins (48%). Because the addition of GTP was as efficient as supplementation with cytosol, the only significant/relevant factors removed by the desalting treatment were small energy molecules.
In yeast, post-translational translocation depends on cytosolic
proteins to maintain the nascent polypeptide in a translocation competent state (17, 34). To investigate which, if any, cytosolic proteins are required for translocation of the amino terminus of opsin,
RBOp were harvested by centrifugation in physiological (150 nM) or high salt (500 mM potassium acetate)
conditions (Fig. 2). A high salt wash has
been used previously to remove the nascent polypeptide-associated
complex from ribosome-nascent peptide complexes (35). The translation
was diluted with buffer (physiologic or high salt) and centrifuged
through a similarly buffered sucrose cushion. Both the physiological as
well as the salt-washed RBOp were then incubated with ER membranes and
supplemented as indicated.
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RBOp that had been harvested in high salt were as efficient in targeting and translocation as those harvested under physiological conditions (Fig. 2). Incubation with cytosol and ER membranes together resulted in glycosylation of 67% of the nascent opsin polypeptides in physiological (lane 3) and 66% in high salt (lane 9). The ability of nucleotide triphosphates to substitute for cytosol was tested in the presence of ER membranes (lanes 4-6, 10-12). The addition of ATP stimulated glycosylation of 29% of the tRNA-nascent opsin in physiological salt (lane 4) and 30% in high salt (lane 10). Consistent with the previous observations (Fig. 1), the addition of GTP was more efficient than ATP and supported glycosylation of 71% of the tRNA-nascent opsin in physiological salt (lane 5) and 70% in high salt (lane 11). Once again the GTP was as efficient as the addition of cytosol to the RBOp purified with physiological salt (lane 3) or with high salt (lane 9) and nearly as efficient as the untreated sample (lane 14). The combination of ATP and GTP was no more efficient than GTP alone (lane 6 and 12). The addition of ER membranes alone showed only barely detectable levels of glycosylation (lanes 1 and 7), indicating that this RBOp purification procedure also removed the majority of cytosol. As expected, cytosol in the absence of ER membranes did not support glycosylation (lanes 2 and 8). The ability of puromycin to efficiently release tRNA-nascent polypeptide demonstrated that the nascent opsin was bound to a functional ribosome throughout the salt wash and purification. RBOps were incubated with (lanes 16 and 18) or without puromycin (lanes 15 and 17), and about 90% of the nascent polypeptides were released by puromycin.
Lumenal chaperones have been implicated in both co-and
post-translational translocation of proteins across the ER membrane (18, 19, 21, 36-38). Because these chaperones are ATPases (39) we had
expected a role for ATP in the translocation of opsin. The endoplasmic
reticulum has transporters for ATP and other nucleotides (40). Our
results indicated only a modest effect of ATP that was less efficient
than that of GTP alone (Figs. 1 and 2). We examined whether the ATP
would have a more robust effect in the presence of reduced levels of
GTP. RBOps purified by centrifugation were incubated with membranes and
supplemented with a range of GTP concentrations either alone (Fig.
3, closed circles) or in the
presence of 1 mM ATP (open circles).
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Consistent with the results in Figs. 1 and 2, 1 mM ATP (open circle, zero point on GTP axis) alone stimulated translocation but was less efficient than even 10 µM GTP alone (closed circles). However, when ATP was supplemented with even low concentrations of GTP, translocation was greatly improved, approximating that observed in samples supplemented with high levels of GTP.
There are three possible explanations for the observation that ATP augmented the effects of low concentrations of GTP on the efficiency of targeting and translocation. First, GTP is necessary and sufficient for targeting and translocation, and ATP is only being used to regenerate GTP from GDP (dinucleotide exchange). Second, there are distinct ATP- and GTP-dependent steps. From previous work, these steps are potentially targeting (GTP-dependent) and translocation (ATP-dependent). In this model, high GTP concentrations could allow RBOp to target multiple times to the ER membrane, eventually overcoming the inefficient ATP-dependent step. Lower GTP concentrations require a more efficient translocation step and more ATP. Third, there are distinct ATP- and GTP-dependent steps (as described above), but the ER membranes or purified RBOps contain residual adenine nucleotides (but not residual guanine nucleotides). With only GTP added, sufficient ATP can be regenerated. However, since there is no residual guanine nucleotides, GTP cannot be regenerated. Further experiments were done to differentiate these possibilities.
In cells there are abundant enzymes to generate the other nucleotide
triphosphates from the large pool of ATP. One well characterized enzyme
is nucleotide diphosphate kinase. Nucleotide diphosphate kinase
non-specifically catalyzes the transfer of the -phosphate from a
nucleotide triphosphate to another nucleotide diphosphate. Nucleotide
diphosphate kinase is a homohexamer of 17-kDa subunits. Thus, it is
expected to be too small to co-purify with ribosomes under the
centrifugation conditions used. Nevertheless, it could be present with
the purified RBOps or the ER membranes. To test for triphosphate
exchange, UTP was used instead of ATP to supplement low levels of GTP
(Fig. 3, triangles). UTP was as effective as ATP in
increasing the efficiency of translocation at low levels of added GTP.
This is still consistent with the first and third models. The
specificity seen in the preceding experiments still may be due to
specific requirement or differences in the levels of residual
nucleotides present. To help distinguish these possibilities, more
specific tests for the nucleotide requirements were used, namely the
ability of analogs to inhibit the reactions.
GDP has been shown to inhibit GTP-dependent enzymes by
favoring the inactive GDP-bound form. GDP addition alone had very
little effect on stimulating translocation (Fig.
4). When ATP was present with lower
levels of added GDP, translocation was stimulated, but at high levels
of GDP there was no effect. We interpret this to mean that high levels
of GDP specifically inhibit the GTP-dependent steps. At
lower levels of added GDP, however, there is rapid conversion of the
majority of the GDP to GTP, which can be used to support targeting and
translocation. These data provide strong evidence that there is
nucleotide triphosphate transfer and that GTP (or another species such
as GDP that can be converted to GTP) is required. Still, a specific
role for ATP may exist, since sufficient residual ATP/ADP may be
present and regenerated itself in the presence of sufficient GTP.
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To further explore the energetic requirements for targeting and
translocation, ER membranes were treated with non-hydrolyzable analogs
of GTP, 1 mM GMPPNP, and ATP, 1 mM AMPPNP (Fig.
5). The top panel shows ER
membranes treated with inhibitors or mock-treated used with unpurified
translations. There was efficient glycosylation of nascent opsin (60%)
with the addition of untreated control ER membranes (lane
5), which was comparable with mock-treated membranes (lane
2, 49%) and AMPPNP-treated membranes (lane 3, 57%).
In contrast, the glycosylation of nascent opsin was reduced to 19% in
the presence of ER membranes pretreated with GMPPNP (lane
4). With no membranes added, background glycosylation was not
detectable (lane 1). Similar results were observed with the inhibitors used at 10 mM (data not shown).
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The treated ER membranes were then tested with purified RBOp and specific nucleotide supplementation. Mock-treated ER membranes (lanes 6-11) gave results very similar to those seen earlier with untreated ER membranes. Glycosylation was efficient with the addition of membranes and cytosol (lane 7). The addition of 10 µM GTP stimulated translocation above background (lane 8), whereas 1 mM GTP was nearly as efficient as cytosol (lane 9). The addition of ATP, even at 1 mM, was less efficient than 10 µM GTP (compare lanes 10 and 11 to 8 and 9). Without any added energy source (lane 6), translocation was barely detectable.
When AMPPNP pretreated membranes were supplemented with the same energy sources, the results were very similar to the mock-treated membranes (compare the bottom panel to the middle panel). Cytosol stimulated translocation efficiently (lane 13), low GTP was less efficient (lane 14), and high GTP was as efficient as cytosol (lane 15). Low and high ATP were still inefficient (lanes 16 and 17). With no added energy there is only barely detectable glycosylation (lane 12).
With the GMPPNP pretreated membranes (bottom panel) there were two differences from the mock- and AMPPNP-treated membranes. First there was a higher background level of glycosylation with only membranes added (lane 18), which is consistent with previous work showing that GMPPNP can have a positive effect on targeting (25-27, 41) but still less efficiently than a similar concentration of GTP (data not shown). Second, GMPPNP inhibited stimulation of translocation by other energy sources. The addition of cytosol (lane 19) did not stimulate translocation detectably above background (compare with lane 18). Low GTP (lane 20) had no stimulatory effect, but high GTP (lane 21) moderately increased the targeting and glycosylation. The addition of ATP had no significant effect (lanes 22 and 23).
Because AMPPNP did not inhibit translocation it was important to
independently confirm that ATPase activity in the treated ER membranes
had indeed been inhibited by the compound. The ER has been shown to be
able to transport ATP and other nucleotides (40). However, we tested
for effects of GMPPNP and AMPPNP on the lumenal ATPase activity of ER
membranes (Fig. 6). Membranes were
pretreated with the non-hydrolyzable nucleotide triphosphate analogues
as before, and ATP hydrolysis was assayed by coupling release of ADP to
the oxidation of NADH, which was monitored by A340. Membranes pretreated with AMPPNP showed an
inhibition of 89% of the ATPase activity. There was only a slight loss
of activity with mock treatment and a moderate loss of activity with 1 mM GMPPNP treatment.
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DISCUSSION |
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Here we examine the requirements for targeting and translocation across the mammalian ER. This has proven difficult due to the tight coupling between targeting/translocation and ongoing translation with the commonly studied model proteins (9, 42). We therefore sought other proteins with a stage of biogenesis that could potentially be uncoupled; that is, "tail-anchored" proteins (43) and membrane proteins with translocated amino-terminal extensions but no cleaved signal sequence (44). The tail-anchored proteins target and translocate after release from the ribosome (45), independently of SRP (46) and the Sec61 complex (47). A representative protein from the second group is opsin, whose first transmembrane segment is the signal sequence (28). The amino-terminal domain of opsin has 36 amino acids that are synthesized before the beginning of the first transmembrane segment, which is not sufficient for targeting (48). In addition, there are two N-linked glycosylation sites in this domain that can be used as a "reporter" to follow the translocation of this segment of the protein.
We have established an assay for efficient targeting and translocation of nascent opsin polypeptides without ongoing translation (29). The efficiency of post-translational translocation in this in vitro assay was nearly identical to the efficiency of co-translational translocation. SRP is required, and the nascent polypeptide must remain bound to an active ribosome (29). Thus, the in vitro translation-independent assay likely mimics a physiologic step in the in vivo targeting and translocation. Because ongoing translation is not required, the system can be manipulated in ways that were previously not possible. In the present study we examined the requirements for energy sources and cytosolic factors by first removing small molecules from the translation and then by purifying RBOp from the bulk translation by centrifugation. Glycosylation of the amino terminus of opsin was used to as a marker of translocation due to the ease of the assay and the relative efficiency of the reaction with optimized ER membranes. This assay then has an end point that reflects only those nascent chains that have both targeted to and translocated the amino terminus across the ER membrane. The intermediate step of targeting before translocation is much more difficult to examine experimentally, since detection requires physical separation of the targeted and untargeted material. Using a quick centrifugation method, this was done previously, and the majority of targeted nascent chains chased into a glycosylated form within 5-10 min of targeting.3 In addition, in all experiments that assayed targeting directly (using physical separation methods) glycosylated nascent polypeptides were not present in the untargeted material (29).
The removal of small molecules from translation reactions by desalting resulted in ribosome-bound nascent opsin complexes that were functional intermediates in biogenesis. This was assessed in two ways. First, nascent opsin polypeptides could still be released from the tRNA by puromycin. This requires that nascent opsin be bound to the ribosome (33, 49, 50). Second, targeting and translocation of the amino terminus, as assayed by glycosylation, could be reconstituted with the addition of the appropriate nucleotide energy source. This indicated that only small molecules were removed by the treatment. Reconstitution was as efficient as the addition of cytosol and 75% as efficient as the reaction with ribosome nascent opsin complexes that had not been desalted.
The efficiency of both the regular and the reconstituted post-translational translocation reaction was somewhat variable. When comparisons were made between experiments, the results were normalized to the maximum translocation efficiency of the unpurified samples (as in Figs. 3 and 4). When normalized this way, the error bars were very small. This suggests the variability as in the isolation of the labile translation intermediate (29).3
With pure nucleotides added back, GTP was more efficient (62%) in supporting targeting and translocation than ATP, which in the same experiment was 48%. However, this is quite different from the 1% observed in the absence of any energy source. Because cytosolic enzymes might not be completely removed by desalting, it is possible that there was some enzyme-mediated transfer of triphosphates between the nucleotide pools. Previously published work has shown that there is an energy requirement for targeting and translocation (24). Specifically, GTP has been found to be required for targeting nascent opsin to the ER membrane (25-27, 41, 51). Examination of the role of ATP for mammalian translocation has not been possible due to the requirements for ongoing translation. To more specifically examine the role of ATP in translocation, centrifugation was used to further purify RBOp from cytosolic components. These more highly purified RBOps could efficiently target and translocate opsin to the ER when supplemented only with nucleotide triphosphate energy sources. The efficiency of targeting and translocation was unaffected by a high salt wash of the RBOp. Only SRP has ever been shown to stay bound to ribosomes under these conditions (52). This indicates that cytosolic proteins were not required for the efficient targeting and translocation of ribosome nascent opsin complexes. The lack of a cytosolic protein requirement distinguishes this mammalian post-translational reaction from the yeast post-translational reaction, which requires two ATP-dependent chaperones, one in the cytosol (14-17) and one in the lumen of the ER (18, 19, 39, 53, 54).
Based on several criteria, we conclude that GTP is specifically required for the targeting and translocation of ribosome nascent opsin complexes. First, the addition of only GTP to RBOp was as efficient as the addition of complete cytosol and nearly as efficient as the unfractionated translation reaction. Second, the level of targeting and translocation correlated in a dose-dependent fashion with the amount of GTP added. Third, although low levels of GDP (with ATP present) are stimulatory, high levels of GDP inhibited the targeting and translocation reaction. Fourth, pretreatment with non-hydrolyzable GTP analogs inhibited the targeting and translocation reaction, whereas pretreatment with ATP analogs did not.
Several steps of targeting are known to require GTP. It is required for SRP binding to the signal sequence of nascent proteins (3, 55, 56) and for the SRP receptor to bind SRP (57-59). GTP hydrolysis is required in each case for proper recycling. GMPPNP has been shown to cause SRP to remain associated with the ER membrane, presumably bound to the SRP receptor, and to block further targeting to those membranes (60). Although pretreatment of the ER membranes with GMPPNP was inhibitory, the addition of this non-hydrolyzable analog to a targeting and translocation reaction had a mildly stimulatory effect (data not shown). One explanation may be that GMPPNP can support a single SRP cycle by displacing GDP. In previously published work, GMPPNP was fully or partially effective at replacing GTP (25-27, 41, 51).
ATP is thought to be important for translocation (61-63). In yeast systems, there are two ATP requirements. First there are cytosolic chaperones that presumably keep the proteins in a translocation-competent state (17, 34). The second ATP requirement is for the lumenal chaperone BiP, which is an ATPase and required for translocation (14, 39). We have examined the requirements for ATP in this mammalian-dissociated system. When the level of added GTP was reduced, ATP could stimulate translocation but only to the level seen with high GTP alone. This effect could be entirely explained by triphosphate energy transfer. First, the same effect of ATP was observed with low levels of added GDP, and second, UTP could substitute for ATP in similar assays. Because it is clear there is triphosphate exchange, this did not eliminate the possibility that there was residual ATP/ADP in the system, which would account for our observed lack of an ATP requirement (since it could be continuously regenerated).
To eliminate the possibility that there was an ATP-dependent step that we failed to uncover due to residual ATP/ADP, ER membranes were pretreated with non-hydrolyzable inhibitors before the addition to purified RBOp. Non-hydrolyzable inhibitors have been shown to prevent BiP from releasing bound peptide even in the presence of ATP (64). AMPPNP has also been used at high concentrations to compete tightly bound ADP from purified mammalian BiP (65). Pretreatment of ER membranes with 1 mM AMPPNP inhibited 89% of the ATPase activity (Fig. 6). However, ER membranes pretreated with AMPPNP (1 mM) showed no targeting or translocation defect (Fig. 5). Membranes that were pretreated with even higher concentrations of AMPPNP (10 mM) were also fully functional for targeting and translocation (data not shown).
We can, therefore, conclude that there is no specific requirement for ATP in this in vitro targeting and translocation system based on several criteria. First, ATP alone had only a modest effect on targeting and translocation, which was much lower than the effect of cytosol or of GTP alone (Figs. 1, 2, and 3). Second, the effect of ATP was much more pronounced when even very small amounts of GTP or GDP were present (Figs. 3 and 4), suggesting that the ATP is only acting by regenerating usable GTP. Third, the effect of ATP could be completely mimicked by other nucleotide triphosphates (Fig. 3) or by a nucleotide-regenerating system (data not shown), which further suggests that ATP only serves as a source of high energy triphosphates. Fourth, non-hydrolyzable ATP analogs do not affect the targeting or translocation reactions, which suggests that there is no specific ATPase that is required.
The post-translational targeting and translocation assay that we have
developed for short nascent opsin polypeptides permits the uncoupling
of these steps from ongoing translation. Thus, it is now possible to
investigate the energetic requirements for targeting and translocation
as distinct from the requirements for the translation process itself.
Although GTP was required for targeting and translocation, ATP was
dispensable. Because GTP is known to be important for targeting to the
ER membrane, we can assign the GTP requirement to the targeting step.
We detected no further energy requirements for the translocation step.
The ATPase BiP is thought to have a role in the translocation of
proteins across the ER. We were unable to detect any specific
requirement for ATP hydrolysis. The amino terminus of opsin is only 36 amino acids and may not be large enough to require any additional
proteins to cross the membrane. There are other members of the
G-protein-coupled receptor family with considerably longer
amino-terminal extensions. It is possible that such proteins have
additional requirements. Because G-protein-coupled receptor family
members with amino termini longer than 200 amino acids encode cleaved
signal sequences, it is possible that there are upper limits on the
size of the peptides that can be translocated post-translationally.
This can now be studied using in vitro systems similar to
the one described here in which targeting/translocation steps can be
dissociated from translation itself.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant R01EY12346-01 (to S. M. S. and E. M. K.), the Tri-Institutional MD-PhD program (to E. M. K.), and The Robert Mealey Program for the Study of Macular Degenerations (to M. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Rockefeller University, 1230 York Ave., #304, New York, NY 10021. Tel.: 212-327-8130; Fax: 212-327-8022; E-mail: simon@mail.rockefeller.edu.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M207462200
2 L. Greene and S. Henikoff, Kinesin Home Page, www.proweb.org/kinesin/methods/ATPase_assay.html.
3 E. M. Kanner and S. M. Simon, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
ER, endoplasmic reticulum;
RBOp, ribosome-bound opsin polypeptide;
tOP, truncated opsin;
SRP, signal recognition particle;
TB, translation/translocation buffer;
AMPPNP, adenosine
5'-(,
-imino)triphosphate;
GMPPNP, guanylyl
imidodiphosphate.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Blobel, G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 1496-1500[Abstract] |
2. | Walter, P., and Blobel, G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7112-7116[Abstract] |
3. | Walter, P., Ibrahimi, I., and Blobel, G. (1981) J. Cell Biol. 91, 545-550[Abstract] |
4. | Walter, P., and Blobel, G. (1981) J. Cell Biol. 91, 557-561[Abstract] |
5. | Walter, P., and Blobel, G. (1981) J. Cell Biol. 91, 551-556[Abstract] |
6. | Gilmore, R., Walter, P., and Blobel, G. (1982) J. Cell Biol. 95, 470-477[Abstract] |
7. | Gilmore, R., Blobel, G., and Walter, P. (1982) J. Cell Biol. 95, 463-469[Abstract] |
8. | Simon, S. M., and Blobel, G. (1991) Cell 65, 371-380[Medline] [Order article via Infotrieve] |
9. | Blobel, G., and Dobberstein, B. (1975) J. Cell Biol. 67, 852-862[Abstract] |
10. | Katz, F. N., Rothman, J. E., Lingappa, V. R., Blobel, G., and Lodish, H. F. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 3278-3282[Abstract] |
11. | Lingappa, V. R., Devillers-Thiery, A., and Blobel, G. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2432-2436[Abstract] |
12. | Sabatini, D. D., Kreibich, G., Morimoto, T., and Adesnik, M. (1982) J. Cell Biol. 92, 1-22[Medline] [Order article via Infotrieve] |
13. | Brown, P. A., Halvorson, H. O., Raney, P., and Perlman, D. (1984) Mol. Gen. Genet. 197, 351-357[Medline] [Order article via Infotrieve] |
14. | Rothblatt, J. A., and Meyer, D. I. (1986) Cell 44, 619-628[Medline] [Order article via Infotrieve] |
15. | Waters, M. G., and Blobel, G. (1986) J. Cell Biol. 102, 1543-1550[Abstract] |
16. | Rothblatt, J. A., Webb, J. R., Ammerer, G., and Meyer, D. I. (1987) EMBO J. 6, 3455-3463[Abstract] |
17. | Waters, M. G., Chirico, W. J., and Blobel, G. (1986) J. Cell Biol. 103, 2629-2636[Abstract] |
18. | Vogel, J. P., Misra, L. M., and Rose, M. D. (1990) J. Cell Biol. 110, 1885-1895[Abstract] |
19. | Nguyen, T. H., Law, D. T., and Williams, D. B. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1565-1569[Abstract] |
20. | Panzner, S., Dreier, L., Hartmann, E., Kostka, S., and Rapoport, T. A. (1995) Cell 81, 561-570[Medline] [Order article via Infotrieve] |
21. | Nicchitta, C. V., and Blobel, G. (1993) Cell 73, 989-998[Medline] [Order article via Infotrieve] |
22. | Yu, Y., Zhang, Y., Sabatini, D. D., and Kreibich, G. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9931-9935[Abstract] |
23. | Görlich, D., and Rapoport, T. A. (1993) Cell 75, 615-630[Medline] [Order article via Infotrieve] |
24. | Perara, E., Rothman, R. E., and Lingappa, V. R. (1986) Science 232, 348-352[Medline] [Order article via Infotrieve] |
25. | Connolly, T., and Gilmore, R. (1986) J. Cell Biol. 103, 2253-2261[Abstract] |
26. | Wilson, C., Connolly, T., Morrison, T., and Gilmore, R. (1988) J. Cell Biol. 107, 69-77[Abstract] |
27. |
Hoffman, K. E.,
and Gilmore, R.
(1988)
J. Biol. Chem.
263,
4381-4385 |
28. | Friedlander, M., and Blobel, G. (1985) Nature 318, 338-343[Medline] [Order article via Infotrieve] |
29. | Kanner, E. M., Klein, I. K., Friedlander, M., and Simon, S. M. (2002) Biochemistry 41, 7707-7715[CrossRef][Medline] [Order article via Infotrieve] |
30. | Walter, P., and Blobel, G. (1983) Methods Enzymol. 96, 84-93[Medline] [Order article via Infotrieve] |
31. |
Huang, T. G.,
and Hackney, D. D.
(1994)
J. Biol. Chem.
269,
16493-16501 |
32. | Deleted in proof |
33. | Traut, R. R., and Monro, R. E. (1964) J. Mol. Biol. 10, 63-72 |
34. | Chirico, W. J., Waters, M. G., and Blobel, G. (1988) Nature 332, 805-810[CrossRef][Medline] [Order article via Infotrieve] |
35. | Wiedmann, B., Sakai, H., Davis, T. A., and Wiedmann, M. (1994) Nature 370, 434-440[CrossRef][Medline] [Order article via Infotrieve] |
36. | Simon, S. M., Peskin, C. S., and Oster, G. F. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3770-3774[Abstract] |
37. | Brodsky, J. L., Goeckeler, J., and Schekman, R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9643-9646[Abstract] |
38. | Matlack, K. E., Misselwitz, B., Plath, K., and Rapoport, T. A. (1999) Cell 97, 553-564[Medline] [Order article via Infotrieve] |
39. | Kassenbrock, C. K., and Kelly, R. B. (1989) EMBO J. 8, 1461-1467[Abstract] |
40. | Hirschberg, C. B., Robbins, P. W., and Abeijon, C. (1998) Annu. Rev. Biochem. 67, 49-69[CrossRef][Medline] [Order article via Infotrieve] |
41. | High, S., Flint, N., and Dobberstein, B. (1991) J. Cell Biol. 113, 25-34[Abstract] |
42. | Palade, G. E. (1975) Science 189, 347-358[Medline] [Order article via Infotrieve] |
43. | Kutay, U., Hartmann, E., and Rapoport, T. A. (1993) Trends Cell Biol. 3, 72-75[CrossRef] |
44. | Schechter, I., Burstein, Y., Zemell, R., Ziv, E., Kantor, F., and Papermaster, D. S. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2654-2658[Abstract] |
45. | Borgese, N., and Gaetani, S. (1983) EMBO J. 2, 1263-1269[Medline] [Order article via Infotrieve] |
46. | Anderson, D. J., Mostov, K. E., and Blobel, G. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7249-7253[Abstract] |
47. | Kutay, U., Ahnert-Hilger, G., Hartmann, E., Wiedenmann, B., and Rapoport, T. A. (1995) EMBO J. 14, 217-223[Abstract] |
48. | Audigier, Y., Friedlander, M., and Blobel, G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5783-5787[Abstract] |
49. | Skogerson, L., and Moldave, K. (1968) Arch. Biochem. Biophys. 125, 497-505[Medline] [Order article via Infotrieve] |
50. | Pestka, S. (1974) Methods Enzymol. 30, 261-283[Medline] [Order article via Infotrieve] |
51. | Lauring, B., Sakai, H., Kreibich, G., and Wiedmann, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5411-5415[Abstract] |
52. | Powers, T., and Walter, P. (1996) Curr. Biol. 6, 331-338[Medline] [Order article via Infotrieve] |
53. |
Tokunaga, M.,
Kawamura, A.,
and Kohno, K.
(1992)
J. Biol. Chem.
267,
17553-17559 |
54. |
Corsi, A. K.,
and Schekman, R.
(1997)
J. Cell Biol.
137,
1483-1493 |
55. | Miller, J. D., Wilhelm, H., Gierasch, L., Gilmore, R., and Walter, P. (1993) Nature 366, 351-354[CrossRef][Medline] [Order article via Infotrieve] |
56. | Rapiejko, P. J., and Gilmore, R. (1992) J. Cell Biol. 117, 493-503[Abstract] |
57. | Connolly, T., and Gilmore, R. (1993) J. Cell Biol. 123, 799-807[Abstract] |
58. | Rapiejko, P. J., and Gilmore, R. (1994) Mol. Biol. Cell 5, 887-897[Abstract] |
59. | Rapiejko, P. J., and Gilmore, R. (1997) Cell 89, 703-713[Medline] [Order article via Infotrieve] |
60. | Connolly, T., Rapiejko, P. J., and Gilmore, R. (1991) Science 252, 1171-1173[Medline] [Order article via Infotrieve] |
61. | Dierks, T., Volkmer, J., Schlenstedt, G., Jung, C., Sandholzer, U., Zachmann, K., Schlotterhose, P., Neifer, K., Schmidt, B., and Zimmermann, R. (1996) EMBO J. 15, 6931-6942[Abstract] |
62. | Mayinger, P., and Meyer, D. I. (1993) EMBO J. 12, 659-666[Abstract] |
63. |
Clairmont, C. A.,
De Maio, A.,
and Hirschberg, C. B.
(1992)
J. Biol. Chem.
267,
3983-3990 |
64. | Flynn, G. C., Chappell, T. G., and Rothman, J. E. (1989) Science 245, 385-390[Medline] [Order article via Infotrieve] |
65. |
Wei, J.,
and Hendershot, L. M.
(1995)
J. Biol. Chem.
270,
26670-26676 |