Signal Recognition Particle Binds to Ribosome-bound Signal
Sequences with Fluorescence-detected Subnanomolar Affinity That
Does Not Diminish as the Nascent Chain Lengthens*
John J.
Flanagan
§,
Jui-Chang
Chen§¶
,
Yiwei
Miao¶,
Yuanlong
Shao¶,
Jialing
Lin**,
Paul E.
Bock
, and
Arthur E.
Johnson
¶§§¶¶
From the ¶ Department of Medical Biochemistry and Genetics,
Health Science Center, and the Departments of
Biochemistry and Biophysics and
§§ Chemistry, Texas A&M University, College
Station, Texas 77843, the ** Department of Biochemistry and
Molecular Biology, University of Oklahoma Health Sciences Center,
Oklahoma City, Oklahoma 73104, and the

Department of Pathology, Vanderbilt
University School of Medicine, Nashville, Tennessee 37232
Received for publication, January 8, 2003, and in revised form, March 4, 2003
 |
ABSTRACT |
The binding of signal recognition particle
(SRP) to ribosome-bound signal sequences has been characterized
directly and quantitatively using fluorescence spectroscopy. A
fluorescent probe was incorporated cotranslationally into the signal
sequence of a ribosome·nascent chain complex (RNC), and upon
titration with SRP, a large and saturable increase in fluorescence
intensity was observed. Spectral analyses of SRP and RNC association as
a function of concentration allowed us to measure, at
equilibrium, Kd values of 0.05-0.38 nM for SRP·RNC complexes with different signal sequences.
Competitive binding experiments with nonfluorescent RNC species
revealed that the nascent chain probe did not alter SRP affinity and
that SRP has significant affinity for both nontranslating ribosomes
(Kd = 71 nM) and RNCs that lack an
exposed signal sequence (Kd = 8 nM). SRP can therefore distinguish between translating and
nontranslating ribosomes. The very high signal
sequence-dependent SRP·RNC affinity did not decrease as
the nascent chain lengthened. Thus, the inhibition of
SRP-dependent targeting of RNCs to the endoplasmic
reticulum membrane observed with long nascent chains does not result
from reduced SRP binding to the signal sequence, as widely thought, but
rather from a subsequent step, presumably nascent chain interference of
SRP·RNC association with the SRP receptor and/or translocon.
 |
INTRODUCTION |
In mammalian cells, ribosomes are found both in the cytoplasm and
at the membrane of the endoplasmic reticulum
(ER).1 These two classes of
ribosome differ in the nature of their translation products, with
membrane-bound ribosomes synthesizing secretory or membrane proteins
that are being translocated across or integrated into the ER membrane
cotranslationally. The structural feature that distinguishes the
cytoplasmic ribosomes from the membrane-bound ribosomes is the presence
in the latter of a nascent chain that contains a signal sequence.
When a nascent chain signal sequence emerges from the ribosome, it is
recognized and bound by a ribonucleoprotein termed the signal
recognition particle (SRP) (for review, see Ref. 1). The binding of SRP
to the signal sequence-containing ribosome-nascent chain complex
(RNC) temporarily prevents it from synthesizing protein. The resulting
"elongation-arrested complex" then diffuses to the ER membrane
where a GTP-dependent interaction with the SRP receptor
initiates a series of events which includes the binding of the RNC to
the site of cotranslational translocation and integration at the ER
membrane (the translocon), the release of the signal sequence from the
SRP, the release of SRP and the SRP receptor from the translocon, and
the resumption of protein synthesis by the ribosome (for review, see
Refs. 1-3). After targeting is complete, nascent chain translocation
or integration then proceeds at the translocon (for review, see Ref.
4).
SRP therefore has a critical regulatory role in the cell because it is
responsible both for the proper trafficking of newly synthesized
proteins and for the conversion of a cytoplasmic ribosome to a
membrane-bound ribosome. In doing so, SRP functions in multiple ways to
effect RNC targeting to the ER membrane, including the selection of
appropriate RNCs, the regulation of RNC translation, and specific
interactions with the SRP receptor and translocon at the membrane.
Although much progress has been made, and models are abundant, the
molecular mechanisms that accomplish each of these functions are still
largely undefined experimentally. The necessity to discriminate
accurately between ribosomes with and without signal sequences is
especially interesting because there is no consensus for the sequences
that can serve as signal sequences (5). The signal sequence binding
site on SRP is therefore promiscuous, yet it must simultaneously be
exceptionally accurate to avoid improper trafficking of the nascent
chains. These conflicting demands are most likely resolved
thermodynamically by discrimination between satisfactory and
unsatisfactory sequences which is based on the binding affinity between
the SRP and the putative signal sequence. This model would predict that
legitimate signal sequences in RNCs would bind much more tightly to SRP
than would other nascent chain sequences.
Soon after the discovery of SRP, Walter and colleagues (6) examined the
binding of SRP to ribosomes with nascent chains that either contained
or lacked signal sequences. Their experiments yielded an estimate of
Kd less than 8 nM for SRP bound
to an RNC with a preprolactin (pPL) nascent chain, and a
Kd less than 50 µM for
nontranslating ribosomes. Because these binding affinities were
estimated using a nonequilibrium sedimentation velocity technique, the
authors noted that the above values represent the minimal binding
affinities. Thus, these early experiments demonstrated a high affinity
interaction between SRP and RNCs which was signal sequence-dependent, but no rigorous thermodynamic analysis
of these interactions has been done.
Elongation arrest was also identified early as a property of SRP when
experiments revealed that SRP inhibits translation of secretory
proteins, but not cytoplasmic proteins, in the absence of ER microsomes
(1, 6). Because some nascent chains become translocation-incompetent
when they become too long, elongation arrest was proposed as a
mechanism for slowing translation and thereby prolonging the time
during which an RNC could be targeted successfully to the ER membrane.
For example, the targeting of RNCs with pPL nascent chains to ER
microsomes was shown to be abolished when the nascent chain exceeded
140 residues in length, presumably because SRP affinity for the RNC had
been reduced by the lengthening and folding of the nascent chain which
interfered with SRP access to the signal sequence (7).
We have here employed fluorescence techniques to monitor directly the
association of SRP with various RNC complexes. This approach has
allowed us to quantify for the first time the interactions of SRP with
the signal sequence, the nascent chain, and the ribosome at
equilibrium, and hence to obtain accurate measurements of the Kd values for complexes containing SRP. The
resulting data reveal that some long standing views about the
mechanisms involved in SRP-dependent ribosome selection and
targeting need to be revised. The following new conclusions are among
those revealed by the fluorescence experiments. The RNC·SRP
Kd is more than 100-fold smaller than estimated
previously; SRP has different affinities for different signal
sequences; SRP binds more tightly to translating ribosomes than to
nontranslating ribosomes because of a conformational change in the
ribosome that is recognized by the SRP; and SRP binds with very similar
affinity to signal sequence-containing RNCs with short and long nascent
chains. Hence, in contrast to current dogma, the inability of RNCs with
long nascent chains to target to the ER membrane is not the result of a
failure of SRP to bind to the signal sequence.
 |
EXPERIMENTAL PROCEDURES |
Plasmids and mRNA--
Plasmids coding for golden hamster
BiP, rat preproinsulin I (pPI), and chimpanzee
-globin were
generous gifts from Dr. Linda Hendershot (8), Dr. Sandra Wolin (9), and
Dr. David Andrews (10), respectively. The pPLss coding sequence was
prepared by Dr. Veronica Worrell by fusing the signal sequence of
bovine pPL (11) to a long stretch of Bcl-2 which lacks a lysine codon
(12). An amber stop codon was introduced into the coding sequences of pPL, pBiP, pPI, and globin using the QuikChange protocol (Stratagene, La Jolla, CA). The primary sequence of each protein and derivative was
confirmed by the DNA sequencing of its plasmid. Truncated mRNAs
coding for peptides of defined length were transcribed in vitro by SP6 RNA polymerase as described previously (13) using either plasmid DNA cut in the coding region with a particular restriction endonuclease or PCR-produced DNA fragments of the desired length.
SRP and SRP Proteins--
SRP was purified as described
previously (14), except that the sucrose density sedimentation was
omitted. SRP proteins and SRP RNA were purified and reconstituted as
before (15, 16). SRP concentration was determined using
280
nm = 1.0 × 106 M
1
cm
1
tRNA--
Yeast Lys-tRNALys and
N
-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminohexanoyl]-Lys-tRNALys
(
NBD-Lys-tRNALys) were purified and prepared as detailed
earlier (17). Escherichia coli tRNALys and a
derivative with a single base change in the anticodon which converted
the tRNALys into a tRNA that recognizes the amber stop
codon (generously provided by Dr. Greg Beckler, Promega Corp.; here
termed tRNAamb) were synthesized in vitro using
T7 RNA polymerase as described earlier for SRP RNA (16). The resulting
RNA samples were purified by anion exchange chromatography using a
Pharmacia FPLC equipped with a Mono Q HR 10/10 column. The RNA was
eluted in 10 mM NaOAc (pH 4.5), 5 mM
MgCl2 with a 115-ml linear gradient of NaCl from 0.48 to
1.0 M. The fractions containing functional
tRNAamb were detected by aminoacylation assays (18), except
that MgCl2 was at 6 mM, and no KCl was added;
most of the tRNAamb eluted near 0.55 M NaCl.
The fractions with the highest tRNAamb content were
aminoacylated with [14C]Lys, chemically modified with
NBD, purified, and stored as described previously with the above
changes (17). The resulting amber suppressor tRNAs have exhibited
excellent suppression efficiencies with a variety of mRNAs, often
exceeding 50% even for amber codons located far into the coding
sequence (data not shown). As seen previously (17), the NBD
modification has no detectable effect on the ability of
Lys-tRNAamb to function in protein synthesis.
Translation Intermediates--
Truncated mRNAs were
translated for 25 min at 26 °C in a wheat germ extract as described
previously (17, 19, 20). Translations (250 or 500 µl total volume)
contained 0.60 pmol/µl of either
NBD-[14C]Lys-tRNALys/amb or unmodified
[14C]Lys-tRNALys/amb. Following translation,
RNCs were purified by gel filtration at 4 °C using a Sepharose CL-6B
column (1.5-cm inner diameter × 20 cm) and an elution buffer
containing 50 mM HEPES (pH 7.5), 140 mM KOAc, 5 mM Mg(OAc)2, and 1 mM
dithiothreitol (Buffer A). A slow flow rate was used during gel
filtration to ensure the removal of noncovalently bound fluorophores
(see Ref. 17). The radioactivity and A260 of a
70-µl aliquot of each 550-µl fraction were used to identify those
fractions containing the RNCs that elute in the void volume, and only
the leading half of the void volume peak was pooled.
For competitive binding experiments, nontranslating ribosomes were
prepared by incubating wheat germ extract with 2 mM
puromycin for 15 min at 26 °C and then purifying the ribosomes by
gel filtration as above. Nonfluorescent RNCs were prepared as above,
except that translations included unmodified
[14C]Lys-tRNALys/amb instead of NBD-labeled
Lys-tRNA. Prior to addition to NBD-RNC samples, nontranslating
ribosomes and nonfluorescent RNCs were concentrated using a Centricon
YM-30 (Millipore, Bedford, MA).
Targeting of RNCs to the ER Membrane--
Translations (25 µl)
of various RNCs were performed as before without added tRNA and with
[35S]Met for nascent chain detection. After 25 min at
26 °C, cycloheximide and then SRP were added to 1 mM and
40 nM, respectively. Following another 10-min incubation at
26 °C, 3-4 eq of high salt- and EDTA-washed microsomal
membranes were added (14). After incubating for 5 min at
26 °C in the presence of membranes, each sample was layered on a
50-µl cushion (0.5 M sucrose in Buffer A) and sedimented for 3 min at 4 °C and 20 p.s.i. in a Beckman Airfuge (30 °
rotor). The pellets were resuspended in SDS-sample buffer,
whereas the supernatants were precipitated in acid before resuspension
in sample buffer. The protein contents of the membrane and supernatant pellets were analyzed on a 10-15% SDS-polyacrylamide gradient gel,
and radioactivity was detected and quantified using a Bio-Rad FX PhosphorImager.
Fluorescence Spectroscopy--
Steady-state fluorescence
intensity was monitored using an SLM 8100 photon-counting
spectrofluorometer with a two-grating excitation monochromator, a
single grating emission monochromator, and a 450-watt xenon lamp.
Samples were excited at 468 nm (4-nm bandpass), and emission was
detected at 530 nm (4-nm bandpass). All spectral measurements were done
at 4 °C in Buffer A using 4 × 4-mm quartz microcuvettes.
Sample mixing in these cuvettes was accomplished as described
previously (21). The sample compartment of the fluorometer was flushed
with a stream of N2 to minimize condensation on the cold
cuvettes. After each addition of titrant and mixing, no spectral
measurements were made for at least 10 min. This delay was sufficient
to ensure that the samples had reached equilibrium in terms of both
ligand binding and temperature and that all condensation had
evaporated. To prevent RNC adsorption to the walls of the cuvette
during the titration, the interiors of the cuvettes were coated with
purified phosphatidylcholine (22, 23).
For spectroscopic analysis, 250-µl samples of purified NBD-labeled
RNCs (usually ~1 nM) in Buffer A were titrated at 4 °C by the sequential addition of known amounts of SRP in small volumes. After each addition, the emission intensities of the NBD and blank samples were measured after reaching equilibrium. After blank subtraction and then dilution correction, the net NBD emission intensity (F) at each point in the titration was compared
with the initial intensity (F0) of the sample in
the absence of SRP. At the end of each titration, the amount of
[14C]Lys in each cuvette was measured to determine the
actual concentration of nascent chains.
To correct for the significant background signal (mostly light
scattering) observed with samples lacking NBD, we initially performed
two translations in parallel, one with
NBD-Lys-tRNA and one with
Lys-tRNA to serve as a blank. NBD and blank samples with equivalent
background signals were then created from the separate RNC pools by
equating (by dilution) either their absorbances at 260 nm (because
ribosomes are responsible for the observed light scattering in these
samples) or their emissions at 485 nm (
ex = 468 nm)
(17). The signal of the blank was then routinely subtracted from the
signal of the fluorescent sample to yield the net NBD emission
intensity. In later experiments, blank samples contained only ribosomes
that had been purified by the gel filtration of wheat germ extract (the
same amount used in the NBD translation) because the presence of
residual tRNA, mRNA, nascent chains, and SRP after purification and
titration did not detectably alter the amount of light scattering.
Data Analysis--
The total concentration of NBD-RNCs in a
purified sample was determined directly by counting the
[14C]Lys present in the solution using a scintillation
counter and experimentally determined counting efficiencies. The number
of RNCs equaled the number of [14C]Lys in all but the
pPLss samples because there is one
NBD-[14C]Lys/ribosome-bound nascent chain when the
probe is incorporated using the amber suppressor. In the case of pPLss,
the number of nascent chains was twice the number of
[14C]Lys because each pPLss nascent chain contains two
lysines, and only one-fourth of the incorporated lysines are
NBD-[14C]Lys because of the competition between
NBD-[14C]Lys-tRNALys and endogenous
Lys-tRNALys (13). The ribosome concentration was determined
by absorbance at 260 nm (1 A260 unit = 23.1 pmol (24)).
We have previously derived equations to analyze the competitive binding
of a ligand to two alternate receptors, one of which is labeled with a
fluorescent probe. The dissociation constant for the unlabeled
competing receptor is obtained by analysis of the dependence of a
spectral change on the concentrations of ligand and receptors (25-28).
Here, because every sample contains more than one ribosomal species
that can bind to SRP, the titration data have been analyzed using
either the cubic equation (Equation 2), which relates the
spectral change to two simultaneous receptor binding equilibria, or the
quartic equation (Equation 10), which was derived for three competing
equilibria (see Fig. 1B). In both cases, fluorescence data
were expressed as the fractional change in the initial fluorescence
|
(Eq. 1)
|
where F0 is the net initial emission
intensity of NBD-RNCs and F is the net dilution-corrected
intensity observed at a given SRP concentration.
Fluorescence studies of SRP binding to NBD-RNCs with nontranslating
ribosomes as a competitor were performed by measuring the fluorescence
change in the presence of excess nontranslating ribosomes. NBD-RNCs at
a total concentration of [N]0 in the sample and a large
excess of nontranslating ribosomes at a total concentration of
[R]0 were titrated with SRP, and the change in NBD-RNC
emission was measured as a function of the total SRP concentration
([S]0). Data from these titrations and titrations with no
excess ribosomes were analyzed using the cubic equation (Equation 2) to
obtain the maximum fluorescence change
(
Fmax/F0) and the
dissociation constants for the SRP·NBD-RNC
(Kn) and the SRP·ribosome
(Kr) complexes that best fit the data. For the
nonlinear least squares analyses, the parameters
(Kn, Kr, and
Fmax/F0) were fit with
the stoichiometric factors n and r, the number of SRP
binding sites/NBD-RNC or nontranslating ribosome, respectively, set to 1, an assumption that fits the titration data very well.
Although the cubic equation has been published previously using
different nomenclature (28), the equation with our nomenclature is
shown below (Equation 2), where the fraction of sites on NBD-RNC occupied by SRP is
F/
Fmax.
|
(Eq. 2)
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|
(Eq. 3)
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|
(Eq. 4)
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|
(Eq. 5)
|
|
(Eq. 6)
|
For competitive ligand binding to three receptors, one of which
is fluorescent, a quartic equation was derived from the equilibrium constants and conservation equations where the SRP·ribosome
concentration is termed [RS], the SRP·nonfluorescent RNC
concentration is designated [US], the SRP·NBD-RNC concentration is
termed [NS], and
F/
Fmax = [NS]/n[N]0. In addition to the parameters in
the cubic equation, the quartic includes Ku
(Kd for SRP·nonfluorescent RNC) and
u (number of SRP binding sites/nonfluorescent RNC).
|
(Eq. 7)
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(Eq. 8)
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(Eq. 9)
|
Substitution of Equations 8 and 9 into Equation 7 and
rearrangement gave Equation 10 for
F/
Fmax as a function of the
total concentrations of all of the interacting species and binding
parameters. For the nonlinear least squares analyses, the dissociation
constants for the various SRP·ribosomal species and the maximum
change in emission intensity
(
Fmax/F0) were fit
simultaneously by the quartic equation (Equation 10) with the
stoichiometric factors (n, r, and u)
fixed at 1.
|
(Eq. 10)
|
|
(Eq. 11)
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(Eq. 12)
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|
(Eq. 13)
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|
(Eq. 14)
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(Eq. 15)
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(Eq. 16)
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(Eq. 17)
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(Eq. 18)
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(Eq. 19)
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The validity of the quartic equation was confirmed by finding
indistinguishable fits to the data by a model in which the expressions for the equilibrium constants and conservation equations were solved
simultaneously by SCIENTIST software (Micromath Software, Salt Lake
City, UT).
The above equations provide exact solutions for the various
Kd values and
Fmax/F0 that are valid
for all concentrations of the interacting species. These exact
equations are necessary for the analysis of tight binding interactions
such as those studied here, where simplifying assumptions regarding
equivalence of free and total concentrations cannot be made. The
equations are generally applicable to the analysis of interactions at
equilibrium where one ligand binds independently (noncooperatively) to
two (Equation 2) or three receptors (Equation 10), one of which is
fluorescent. All nonlinear least squares analyses were performed with
the SCIENTIST program. All reported estimates of error represent ± 2 S.E.
The Kd values for SRP complexes with
NBD-pPL86-RNC and nontranslating ribosomes were determined
by combining all of the individual titration data sets in the presence
and absence of excess free ribosomes and analyzing them simultaneously
by nonlinear least squares fitting of the cubic equation using the
SCIENTIST fitting program (28). Similarly, the data from multiple
independent experiments were combined and analyzed simultaneously to
determine the Kd values using the quartic
equation. Many different preparations of purified SRP and translation
components have been used in our experiments over several years, and we
observed little dependence of the Kd values on
sample origin.
 |
RESULTS |
Experimental Approach
Equilibrium Kd Values Determined Using Fluorescence
Spectroscopy--
To determine most accurately the affinity of SRP for
ribosomes with various nascent chains, it is necessary to measure the Kd of each complex at equilibrium.
Nonequilibrium techniques estimate the extent of complex formation in a
sample by first separating the complex from the unbound species and
then measuring the amount of complex. Kd values
calculated from such data are typically much higher than the true
dissociation constants because the complex dissociates during the
separation process. For example, we found that
Kd values for aminoacyl-tRNA·EF-Tu·GTP
ternary complexes determined using nonequilibrium techniques were
10-1,000-fold higher than the actual equilibrium dissociation constant
(26, 27). Because nonequilibrium methods may substantially
underestimate SRP affinity for signal sequences and ribosomes, we have
chosen to measure Kd values at equilibrium.
The optimal approach for quantifying the amounts of bound and unbound
species in a sample at equilibrium is to use a spectroscopic technique
that can distinguish between the bound and free species without
separating them. Here we positioned a fluorescent probe in the signal
sequence with the expectation that SRP association with the signal
sequence would alter the environment of the fluorophore and hence its
fluorescence signal. As shown below, this expectation was realized, and
we were able to measure directly the fraction of signal sequences bound
to SRP in a sample. Another advantage of using a nondestructive
spectroscopic technique to monitor binding is that the sample can be
examined over time to ensure that the spectral signal has reached a
constant value and hence that the sample is at equilibrium.
Kd values are determined experimentally from the
concentration dependence of complex formation, and hence it is
necessary to examine samples with measurable amounts of both free and
bound RNCs and SRP. Because Walter et al. (6) showed that
the Kd for SRP binding to signal
sequence-containing RNCs was less than 8 nM, significant
amounts of both free and bound species would be observed only in
samples containing nanomolar concentrations of RNCs and SRP.
Fluorescence is the only acceptable choice for this study because it is
the only spectroscopic technique that can detect and measure probe
concentrations that are nanomolar or lower.
Selective Labeling of the Nascent Chain--
To position a
fluorescent probe solely in nascent chains (which constitute much less
than 1% of the total protein in our samples), a fluorescent labeled
amino acid must be incorporated into the nascent chain as it is being
synthesized by the ribosome. This requires a functional aminoacyl-tRNA
analog whose amino acid has been chemically modified, an approach that
was originated by us (29). Here we have used the well characterized
NBD-Lys-tRNALys to incorporate NBD probes into nascent
chains (17).
NBD-Lys is incorporated into polypeptide at the same
rate and to the same extent as unmodified Lys, so the translation
machinery does not discriminate against the abnormally large amino
acid. Yet
NBD-Lys-tRNALys must compete with endogenous
Lys-tRNA for incorporation at Lys codons in the mRNA, and hence
only about 25% of the lysines incorporated in an in vitro
translation are
NBD-Lys (13). This competition complicates the
determination of the number of nascent chains in a sample because only
the
NBD-[14C]Lys are radioactive. Hence, we have also
used a tRNA that can be aminoacylated with Lys and modified with NBD
but that recognizes an amber stop codon during translation. This amber
suppressor tRNA,
NBD-Lys-tRNAamb, incorporates its
NBD-Lys wherever an amber stop codon has been introduced into the
mRNA, and only termination factors compete for decoding the stop
codon. Because termination factor action releases the nascent chain
from the ribosome, the total number of ribosome-bound nascent chains in
an RNC sample programmed with an amber stop codon-containing mRNA
is equal to the number of incorporated
NBD-[14C]Lys
residues, and every RNC contains an NBD dye. This approach allows us to
examine SRP-signal sequence interactions in the context of the ribosome
using ribosome-bound nascent chains rather than free signal
sequence-containing peptides. Also, because only functional ribosomes
will be able to synthesize a nascent chain, every
Kd measurement involves only functional RNCs.
Homogeneous RNCs--
Homogeneous RNC samples were created using
mRNAs that were truncated at a specific site within the coding
sequence. Translation of such an mRNA proceeds normally until the
ribosome reaches the end of the truncated mRNA. Because there is no
stop codon, the ribosome does not dissociate from the mRNA but
instead remains associated with the peptidyl-tRNA and its nascent chain
to create a translation intermediate (RNC) with a nascent chain length
that is dictated by the length of the truncated mRNA
(e.g. (13)). To confirm that every RNC in our samples had
the same length of nascent chain, samples were examined by SDS-PAGE
after gel filtration, and a single band containing 90-100% of the
NBD-[14C]Lys-labeled nascent chains was observed with
the expected molecular mass (data not shown).
RNCs were prepared using plasmids coding for the proteins shown in Fig.
1A. In one, the pPL signal
sequence was fused to a 109-residue stretch of polypeptide which did
not contain any lysines; this construct was designated pPLss. When
pPLss mRNAs were translated in the presence of
NBD-Lys-tRNALys,
NBD-Lys was incorporated only into
positions 4 and 9 of the nascent chain where lysines are located in the
native pPL sequence. In a different construct, here termed pPL, a codon
in the middle of the hydrophobic core of the pPL signal sequence was
converted into an amber stop codon. An uncharged and nonpolar
NBD-Lys was then incorporated into position 18 of the pPL signal
sequence when mRNAs of this pPL derivative were translated in the
presence of
NBD-Lys-tRNAamb.

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Fig. 1.
Protein constructs and competitive binding
experiments. A, the protein constructs used to form
RNCs in this study are shown schematically with signal sequences in
gray. Solid lines indicate the position of either
lysine codons (pPLss) or amber stop codons (pPL, pPI, pBiP, globin)
used to incorporate NBD-Lys. B, competitive binding
equilibria. When SRP (light gray ovals) is titrated into a
sample containing three different ribosomal species, the extent of SRP
binding to each reflects the affinity of SRP for each ribosomal
species. At equilibrium, the free SRP concentration will be the same
for each of the three equilibrium equations that hold simultaneously,
and the distribution of SRP between the ribosomal species is determined
by their relative Kd values. NBD-labeled RNCs
are depicted with a fluorescent probe (small black circles)
incorporated in the signal sequence (black sawtooth lines)
of the nascent chain (black lines), whereas unlabeled RNCs
are shown without the fluorescent dye. Nontranslating ribosomes are
shown without a nascent chain. Although unlabeled signal
sequence-containing RNC is shown in the figure as the competitor, the
same competitive binding approach may be used to determine the
dissociation constant for SRP binding to other ribosomal species
(e.g. an unlabeled globin RNC that lacks a signal
sequence).
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Competitive Binding Experiments--
Kd
values for complexes containing SRP and an unmodified,
nonfluorescent RNC can be determined spectroscopically using
competitive binding experiments. In this approach, the fluorescent RNCs
serve solely to quantify the distribution of SRP within the sample. When SRP is added to a sample (a cuvette) containing both unmodified RNC and NBD-labeled RNC (NBD-RNC) complexes, two competing binding equilibria are established which reflect the relative affinities of SRP
for RNC and NBD-RNC. Because the amount of SRP·NBD-RNC in a sample is
given directly by the magnitude of the observed spectral change, the
extent of competition by the nonfluorescent RNC for binding to SRP is
given by the extent to which the RNC reduces SRP·NBD-RNC complex
formation (i.e. lowers the magnitude of the spectral
change). After calculating the distribution of SRP in the sample (bound
to NBD-RNC, bound to RNC, or free), the Kd value
for the nonfluorescent SRP·RNC complex is determined from this
distribution and the spectroscopically determined
Kd for SRP·NBD-RNC. The equations representing
the two equilibria for competitive SRP binding to RNC and to NBD-RNC
can be solved simultaneously because the free SRP concentration is the
same for each equation. This yields a cubic binding equation as an exact solution, relating the observed fluorescence change to the known
total concentrations of the components and the binding parameters. This
equation is required to analyze high affinity interactions where
assumptions about the equivalence of free and total component concentrations cannot be made.
In the present case, the weak binding of SRP to nontranslating
ribosomes (6) further complicates matters. Because many ribosomes
(50-75%) in in vitro translations do not synthesize protein and because our purification procedures do not separate RNCs
from nontranslating ribosomes, any binding of SRP to nontranslating ribosomes would reduce the amount of SRP available for binding to RNCs.
Thus, there may actually be three species vying for SRP binding at
equilibrium, each with its own binding affinity. This is illustrated in
Fig. 1B, where the Kd values for the
NBD-RNC, unlabeled (nonfluorescent) RNC, and nontranslating ribosomes
are given by Kn, Ku, and
Kr, respectively. Because the free SRP
concentration is the same for each interaction at equilibrium, the
binding equations can be solved simultaneously to determine the
individual dissociation constants. The resulting quartic binding
equation expresses the change in observed fluorescence as a function of
the total concentrations of the interacting species, with the
individual dissociation constants, stoichiometric factors, and maximum
fluorescence change as parameters. This is an exact solution applicable
to any concentration of the various components, independent of
assumptions regarding free and total concentrations of ligand.
Fluorescence-detected Binding of SRP to RNCs
To ascertain whether the binding of SRP to a signal sequence can
be detected spectroscopically, fluorescent probes were positioned at
three different locations within the pPL signal sequence.
NBD-Lys was introduced in place of the Lys residues normally found at positions
4 and 9 of the 30-residue pPL signal sequence in
NBD-pPLss65-RNC complexes (the subscript indicates the
length of the nascent chain), and in the middle of the hydrophobic core
(position 18) of the pPL signal sequence in NBD-pPL86-RNC
complexes. When each of these complexes was titrated with purified SRP,
a substantial increase in NBD emission intensity was observed (Fig.
2). The SRP-dependent increase in fluorescence intensity averaged 65% for
NBD-pPLss65-RNC and 60% for NBD-pPL86-RNC.
Thus, binding of SRP to a signal sequence in an RNC can be detected
spectroscopically. Furthermore, SRP binding is detected equally well by
fluorescent probes located at different sites within the signal
sequence.

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Fig. 2.
Fluorescence-detected SRP binding to signal
sequence-containing RNCs by direct titration. A, 5 nM NBD-pPLss65-RNC was titrated as described
under "Experimental Procedures" with the indicated total
concentrations of SRP ( ) or SRP(-SRP54) ( ), SRP that was
reconstituted from purified SRP RNA and SRP proteins lacking SRP54.
B, binding of SRP to RNCs containing 1.1 nM
NBD-pPL86 ( ), NBD-pPL35 ( ), or
NBD-globin84 ( ). The observed change in emission
intensity is F, and the initial fluorescence intensity of
the sample is designated F0. Data shown are
representative of multiple independent titrations performed for each
RNC.
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A number of controls were done to eliminate the possibility that the
observed fluorescence change was an artifact. First, the mammalian SRP
contains one RNA of 300 nucleotides and 6 polypeptides designated SRP9,
SRP14, SRP19, SRP54, SRP68, and SRP72, where the number refers to the
molecular mass of the protein in kDa (1). Because photocross-linking
experiments showed that the signal sequence binds to SRP54 (11, 30), we
prepared an SRP lacking SRP54 by reconstituting purified SRP and the
other five SRP proteins as described previously (16). When
NBD-pPLss65-RNC was titrated with SRP(-SRP54), no spectral
change was observed (Fig. 2A). This result is fully
consistent with SRP54 being the signal sequence-binding component of
SRP. Second, no change in fluorescence occurred when
NBD-pPL35-RNC was titrated with SRP (Fig. 2B).
Because this nascent chain is too short for the signal sequence to have
emerged from the ribosome, no SRP-dependent spectral change
was expected. Third, no fluorescence change was detected when SRP was
titrated into RNCs containing an 84-residue nascent chain of globin
which lacked a signal sequence but had an NBD probe positioned in the
nascent chain at nearly the same location as in NBD-pPL86
(Figs. 1A and 2B). Fourth, no
SRP-dependent increase in emission intensity was observed
when NBD-pPL86 nascent chains were released from their RNCs
using puromycin prior to the addition of SRP (data not shown). Thus,
the SRP-dependent increase in fluorescence shown in Fig. 2,
A and B, required a fully assembled and
functional SRP as well as a nascent chain that had an exposed signal
sequence and was bound to a ribosome.
Photocross-linking was also used as an independent assay of SRP
association with RNCs. Photoreactive probes were incorporated into the
same sites occupied by fluorophores in the various nascent chains, and
the resulting RNCs were photolyzed after the addition of SRP as
before (11). Photocross-linking to SRP54 was observed only in samples
containing nascent chains with an exposed signal sequence; no
cross-linking to globin or pPL35 nascent chains was observed (data not shown).
SRP Binds to Nontranslating Ribosomes
The extent of SRP·NBD-RNC formation can be determined directly
from the magnitude of the fluorescence changes shown in Fig. 2,
A and B, at different SRP concentrations. In
principle, we can use these data to determine the dissociation
constants for each SRP·NBD-RNC complex. However, as noted above, such
an analysis is complicated by the presence of another potential SRP
binding partner in the sample, the nontranslating ribosome. This
potential competition must be taken into account in any calculations of Kd values.
Samples containing a 30-60-fold excess of nontranslating ribosomes
over NBD-pPL86-RNCs were titrated with SRP. If
SRP·ribosome complexes form, fewer SRPs would be available to form
SRP·NBD-pPL86-RNC complexes, and the
SRP-dependent fluorescence change at low SRP concentrations
would be lower for samples with the large excess of ribosomes than for
samples without the added ribosomes. Fig. 3 shows parallel SRP titrations of
NBD-pPL86-RNC with or without a 47-fold molar excess of
nontranslating ribosomes over RNCs, and it is clear from these data
that the nontranslating ribosomes do compete, albeit poorly, with RNCs
for binding to SRP. Thus, SRP does bind to nontranslating ribosomes,
but weakly.

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Fig. 3.
Competitive binding of ribosomal species to a
limited amount of SRP. Competitive binding experiments show SRP
titrations of 1.3 nM NBD-pPL86-RNCs
supplemented with no added ribosomes ( ), a 47-fold molar excess of
nontranslating ribosomes ( ), or a 2.1-fold molar excess of
nonfluorescent pPL86-RNCs ( ). For the three titrations
shown, nonlinear least squares fitting of the titration data, using
either the cubic equation or the quartic equation and assuming
n = r = u = 1 , yielded
the best fit lines shown. For these particular titrations, the best fit
parameters were: Kn = 0.18 nM,
Kr = 71 nM, and
Fmax/F0 = 0.61 for the
cubic equation with no added ribosomes; Kn = 0.23 nM, Kr = 71 nM, and
Fmax/F0 = 0.62 for the
cubic equation with added nontranslating ribosomes; and
Kn = 0.20 nM,
Kr = 71 nM,
Ku = 0.13 nM, and
Fmax/F0 = 0.59 for the
quartic equation with added unlabeled pPL86-RNCs. Data
shown are representative of multiple independent titrations performed
for each RNC.
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The Kd values for SRP complexes with both
NBD-pPL86-RNC and nontranslating ribosomes were determined
by simultaneous nonlinear least squares fitting of the combined data of
many titrations by the cubic binding equation (Equation 2) to yield a
Kd of 0.21 nM for
SRP·NBD-pPL86-RNC and a Kd of 71 nM for SRP·ribosome complexes (Table
I). SRP therefore binds with very high
affinity to RNCs with an exposed signal sequence, and the SRP-signal
sequence interaction is responsible for a 350-fold reduction in
Kd. As expected, this interaction is largely or
solely hydrophobic because increasing the ionic strength by 0.5 M KOAc did not alter the Kd (data
not shown).
Although SRP binds with significant affinity to nontranslating
ribosomes, their ability to compete with RNCs for SRP is limited. For
example, when titration data were simulated assuming
Kd = 0.21 nM for NBD-pPL-RNCs,
Kd = 71 nM for nontranslating
ribosomes, and
F/F0 = 0.60, a
5-fold excess of ribosomes over NBD-pPL-RNCs (the maximum expected in
the usual in vitro translation; we typically observed a
ratio of ribosomes to RNCs between 2:1 and 1:1 in our experiments (data
not shown)) had almost no effect on the predicted SRP-dependent fluorescence change. Thus, the concentration
of nontranslating ribosomes present in a typical in vitro
translation does not detectably reduce SRP binding to NBD-RNCs with
exposed signal sequences.
In fact, the affinity of SRP for nontranslating ribosomes is so much
less than it is for RNCs with signal sequences that it is difficult to
determine the Kd value for empty ribosomes
accurately even in equilibrium experiments. This is evidenced by the
fact that the Kd calculated for the
SRP·ribosome complex can be altered by even small variations in the
value for the maximum
F/F0, which explains the large uncertainty in the 71 nM
Kd value.
In early experiments, the Kd for SRP binding to
ribosomes lacking signal sequences was estimated to be less than 50 µM (6). This value differs greatly from the above
Kd of 71 nM because the latter was
determined at equilibrium, and the former was determined by a
nonequilibrium technique. Similarly, the dissociation constant determined here for SRP binding to RNC (0.21 nM) is 40-fold
lower than the previous estimate (8 nM). Because the
dissociation rate is typically greater for low affinity complexes than
for high affinity complexes and because a high dissociation rate will
reduce the amount of complex detected in a nonequilibrium approach, it is not surprising that the difference between the equilibrium and
nonequilibrium Kd values is greater for the
weaker SRP·ribosome complex.
The Fluorescent Probe Does Not Interfere with SRP
Binding
To determine whether the fluorescent probe in the signal sequence
influenced SRP binding either positively or negatively, we compared the
binding of SRP to NBD-pPL86-RNC and to
pPL86-RNC. The parallel titrations in Fig. 3 show that the
2.1-fold excess of nonfluorescent RNCs competes much more strongly with
NBD-RNCs than does the 47-fold excess of nontranslating ribosomes.
These experiments involved three competing equilibria representing SRP binding to NBD-pPL86-RNCs, pPL86-RNCs, and
nontranslating ribosomes (Fig. 1B). These results were
analyzed by simultaneous nonlinear least squares fitting of the
combined data with the quartic binding equation (Equation 10) that
provides an exact solution for this binding model. All of the titration
data shown in Fig. 3 were combined with the data from several
independent titrations of the same ribosomal species for this global
analysis. These experiments revealed that the presence of the NBD probe
had little effect on the binding of SRP to pPL86-RNC (Table
I). The Kd values for SRP·NBD-pPLss65-RNC and SRP·pPLss65-RNC
were also nearly identical (data not shown). Hence, SRP binding to the
pPL signal sequence in RNCs was altered only slightly by a probe in one
of the three probe positions. The near invisibility of the probe during
SRP-RNC association probably results both from the long, flexible side chain of
NBD-Lys, which allows it to adjust its location to minimize steric interference of SRP binding to the signal sequence, and also
from the NBD probe being too small to provide either significant binding energy or steric interference.
SRP Affinity for RNCs Lacking an Exposed Signal
Sequence
When RNCs containing NBD-globin84 or
NBD-pPL35 nascent chains were titrated with SRP, no change
in NBD emission intensity was observed (Fig. 2B). Although
these results indicate that SRP does not interact directly with these
nascent chains, this type of experiment does not address whether the
presence of such nascent chains either promotes or inhibits SRP
association with ribosomes. To determine the affinity of SRP for
translating ribosomes that do not have signal sequences exposed to the
cytosol, we performed competitive binding experiments. SRP was titrated
into a sample that contained RNCs with an NBD-pPL86 nascent
chain and a 7-fold excess of RNCs with globin84 nascent
chains that lacked any signal sequence. When the data from three
independent titrations were analyzed together, the SRP affinity for the
globin-containing RNCs was found to be significantly higher than it was
for nontranslating ribosomes (8.0 versus 71 nM,
respectively; Table I). Thus, SRP binds to translating ribosomes with
an exposed nascent chain that lacks a signal sequence. Furthermore, SRP
can also distinguish between ribosomes that are translating and
ribosomes that are not translating.
This conclusion was confirmed by competitive binding experiments using
RNCs that were translating a secretory protein with a signal sequence
but whose nascent chains were not long enough for the signal sequence
to have emerged from the ribosome. When the data from five titrations
containing a 7-12-fold excess of pPL35-RNC over
NBD-pPL86-RNCs were combined and analyzed simultaneously by
nonlinear least squares fitting of the data with the quartic binding
equation, the SRP·pPL35-RNC complex was found to have a
Kd of 8.6 nM (Table I). Thus, SRP
binds more tightly to translating ribosomes than to nontranslating
ribosomes. But the additional binding energy does not
originate from a nonspecific interaction between SRP and the nascent
chain because the nascent chain is still inside the ribosome in the
pPL35-RNC.
SRP Affinity for Signal Sequences Is Not Uniform
To determine whether SRP binds every signal sequence with the same
affinity, RNCs were prepared with either of two other nascent chains
containing a signal sequence, pPI or pBiP. An
NBD-Lys was
incorporated into the middle of the signal sequence of the pPI and pBiP
derivatives shown in Fig. 1A, and the association of SRP
with either NBD-pPI86-RNC or NBD-pBiP87-RNC was
monitored using fluorescence. The data shown in Fig.
4 and summarized in Table I reveal that
SRP binding to signal sequences other than pPL can be detected
spectroscopically. The magnitude of the SRP-dependent increase in NBD emission intensity is not the same for each signal sequence, but this spectral difference is unimportant for
Kd measurements because the fraction of NBD-RNC
bound to SRP in a sample is given by
F/
Fmax, and the
Kd is then determined from the SRP concentration dependence of the normalized spectral value
F/
Fmax. Because the
Kd values for the pPI, pPL, and pBiP signal
sequences differ significantly, these titrations demonstrate that SRP
affinity for an RNC is dictated by its signal sequence.

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Fig. 4.
SRP binding to RNCs with different signal
sequences. SRP titrations of 1.6 nM
NBD-pBiP87-RNCs ( ) or 1.0 nM
NBD-pPI86-RNCs ( ) are shown. For these two titrations,
nonlinear least squares fitting of the titration data using the cubic
equation and assuming n = r = 1 yielded
the best fit lines shown. For these particular titrations, the best fit
parameters were Kn = 0.05 nM,
Kr = 71 nM, and
Fmax/F0 = 0.46 for the
pBiP RNCs, and Kn = 0.35 nM,
Kr = 71 nM, and
Fmax/F0 = 0.28 for the
pPI RNCs. Data shown are representative of multiple independent
titrations performed for each RNC.
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SRP Affinity for Signal Sequences Is Unaffected by Nascent Chain
Length
The effect of nascent chain elongation on SRP binding to the
nascent chain signal sequence was assessed by creating a set of
translation intermediates with nascent chains of different length, each
with an NBD probe in the signal sequence. We were particularly
interested in determining whether SRP was unable to bind to pPL-RNCs
with nascent chains longer than 140 amino acids. As was true in all of
the experiments reported here, the NBD-RNCs were prepared, purified,
and characterized spectroscopically before any SRP was added to the
sample. Thus, there was plenty of time for the nascent chains to fold
before they were exposed to SRP.
As shown in Table II, we observed no
reduction in the affinity of SRP for the pPL signal sequence even when
the nascent chain was nearly full-length (220 versus 229 amino acids). Thus, the length of the nascent chain has little
influence on the ability of SRP to bind to the signal sequence, even
when SRP is added subsequent to nascent chain emergence from the
ribosome rather than the more physiological SRP scanning of the nascent
chain as it leaves the ribosome. Although partial nascent chain folding may alter the kinetics of SRP accessibility to a particular signal sequence, our data indicate that SRP can bind to the signal sequence even after more than 180 nascent chain residues are exposed to the
cytosol.
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Table II
Kd values for SRP·RNC complexes with NBD-pPL nascent chains
of various lengths
All Kd values and
Fmax/F0 were determined by
fitting data with Equation 2 while setting n = r = 1 and Kr = 71 nM.
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The above results led us to examine whether these long nascent chains
were able to target SRP·RNCs to microsomes, and targeting was
evaluated using the same procedures described earlier (7). As reported,
we found that targeting efficiency decreased dramatically as the length
of nascent chain increased (Fig. 5). In
particular, even though the NBD-pPL220-RNCs bound tightly
and completely to SRP (Table II), very few pPL220-RNCs were
targeted successfully to the ER membrane. Hence, it is clear from the
combined fluorescence and targeting data that the long nascent chain
interferes with targeting at some step subsequent to the binding of SRP
to the signal sequence.

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Fig. 5.
Targeting of pPL-RNCs to ER membranes is
dependent on nascent chain length. [35S]Met-labeled
RNCs were incubated with SRP and high salt- and EDTA-washed microsomal
membranes (EKRMs) as indicated and as described under "Experimental
Procedures." Targeting of RNCs to EKRMs was assessed by sedimentation
of the microsomes to separate membrane-associated RNCs in the pellet
(P) from untargeted RNCs in the supernatant (S).
Locations of molecular mass markers are shown on the
right.
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 |
DISCUSSION |
The affinities of SRP for various ribosomal species were
determined directly and at equilibrium. This is very
important because, as noted above, only equilibrium
Kd values provide an accurate measure of
affinities and thermodynamic free energies. The fluorescence approach
used here reveals that SRP binds to signal sequences in RNCs with
exceptionally high affinity (0.05-0.38 nM; Table I). The
physiological requirement for such tight binding of SRP to RNCs with
signal sequences is likely 2-fold: (i) to ensure that only those RNCs
with legitimate signal sequences are selected by SRP and targeted to
the ER membrane, and (ii) to maximize the time of elongation arrest,
thereby maximizing the chances of the RNC targeting successfully to the
ER membrane. Because RNCs with long nascent chains are less likely to
target successfully to translocons (7), a high affinity interaction
between SRP and RNCs with signal sequences would slow the dissociation
rate of SRP from the SRP·RNC complex and cause nascent chain
elongation to be inhibited for a longer time, thereby increasing the
time available for successful targeting. Thus, the high affinity
SRP·RNC interaction appears to be required to select RNCs accurately
for targeting to the ER membrane and also to target efficiently those RNCs to the membrane.
However, RNCs with different signal sequences bind to SRP with
different affinities. The measured Kd values for
the three SRP·RNC complexes examined here differ by 8-fold (Table I),
and these differences are presumably physiologically significant. Because SRP will bind preferentially to the RNC signal sequences for
which it has the highest affinity, the SRP-signal sequence affinity may
correlate with the priority of the nascent chain for translocation
within the spectrum of nascent chains that are vying for an empty
translocon. The extremely high affinity of SRP for the pBiP signal
sequence appears to be consistent with this idea because when the cell
is under stress, BiP must take precedence and be transported quickly
into the ER lumen as part of the unfolded protein response (31). Of
course, the rate of release of the signal sequence from the SRP at the
translocon during targeting will also be affected by the strength of
the SRP-signal sequence interaction, so other considerations will undoubtedly influence the optimal SRP affinity for a particular signal
sequence and nascent chain. Thus, the variation in SRP-signal sequence
affinities presumably evolved through a balancing of different
physiological or mechanistic requirements within the cell. More
SRP·RNC Kd values will have to be determined
and correlated with targeting efficiency, among other things, to
appreciate fully the functional significance of different SRP·RNC affinities.
Early on, SRP was proposed to scan the nascent chain as it emerged from
the ribosome to ascertain whether or not the nascent chain had an
appropriate signal sequence (32). This mechanism of signal sequence
detection requires SRP to bind to ribosomes that lack an exposed signal
sequence, and Walter et al. (6) did observe weak binding of
SRP to ribosomes that lacked a signal sequence
(Kd < 50 µM). The competitive
binding experiments reported here confirm that observation by
demonstrating that SRP binds to ribosomes that lack either a signal
sequence or a nascent chain. However, the equilibrium dissociation
constants determined here show that SRP affinity for nontranslating
ribosomes is actually quite high (Kd = 71 nM). Although this dissociation constant was determined
with canine SRP and wheat germ ribosomes because of the difficulty of
purifying mammalian ribosomes free of the relatively high concentration
of endogenous SRP, the well documented compatibility of the wheat germ
and canine molecules involved in targeting and translocation suggest
that the dissociation constant for a mammalian SRP and ribosome would
be similar. The spectroscopic data are therefore consistent with the
view that SRP is typically bound to a ribosome and not free in the
cytosol. This would allow the SRP to scan the emerging nascent chain
for a signal sequence and detect its presence as quickly as possible,
thereby minimizing the chances that the nascent chain would become too
long to target to the translocon efficiently.
At first glance, the affinity of SRP for nontranslating ribosomes
reported here may appear to be too high. However, the
Kd for the SRP·ribosome complex is reasonable
given the concentrations of these species in the cell. The SRP
concentration in the mammalian cytoplasm has been estimated to be about
10 nM (7), and the total ribosome concentration has been
estimated to be about 10-100-fold higher than the SRP concentration in
different organisms (33). Because a significant fraction of the
ribosomes in a mammalian cell (up to ~50%) will be bound to the ER
membrane at translocons and hence unavailable for SRP binding, the
actual ribosome concentration will be lower than 100-1,000
nM and may be lower than 50-500 nM in
pancreatic cells. Although these estimates are very rough, the low
cytosolic concentrations of SRP and free (not bound to the ER membrane)
ribosomes reveal that a Kd below 100 nM is required to ensure that most of the SRP is bound to a
ribosome in the cytosol. Because the actual SRP and ribosome
concentrations will vary with cell type, it seems likely that the
Kd value has evolved to balance and best satisfy
the needs of a variety of different cells and circumstances.
The competitive binding experiments also revealed an unexpected
property of the ribosome, that of a conformational difference between
translating and nontranslating ribosomes which is detected by SRP
(Table I). The discovery that SRP can distinguish between translating
and nontranslating ribosomes provides even more compelling evidence for
the cotranslational scanning and detection of nascent chain signal
sequences by SRP because the 9-fold difference in Kd values will lead to a preferential binding of
SRP to ribosomes that are engaged in protein synthesis. It is also
important to note that this preference exists even before the nascent
chain emerges from the ribosome. This result shows that the increased affinity of SRP for an RNC over a nontranslating ribosome is caused by
a change in ribosome conformation at the SRP binding site on the
ribosome, not by an interaction between the SRP and nascent chain.
The inability of RNCs to target to the ER membrane when their nascent
chains become too long was originally attributed to an inability of the
SRP to bind to the signal sequence of RNCs with a long nascent chain,
presumably because folding of the longer nascent chains interfered with
SRP accessibility to the signal sequence (7). Yet by placing a
fluorescent probe in the signal sequence to monitor SRP binding
directly, we found that SRP can recognize and bind to a signal sequence
in a long nascent chain that had plenty of time to fold into a stable
conformation before SRP was added (Table II). Furthermore, the affinity
of the SRP for the signal sequence in an RNC was essentially
independent of nascent chain length once the signal sequence had fully
emerged from the ribosome and was able to contact its binding site on the SRP optimally (Table II). We therefore conclude that a long nascent
chain interferes with RNC targeting to the ER membrane at some point
after the formation of the SRP·RNC complex. Because the SRP·RNC
complex next interacts with the SRP receptor and the translocon, it
appears that the extra polypeptide of an elongated nascent chain
interferes sterically with either the interaction of SRP with the SRP
receptor and/or the interaction of the RNC with the translocon. The
latter possibility seems particularly reasonable because the cytosol
contains many ribosomes that are synthesizing cytoplasmic proteins, and
such ribosomes will undoubtedly diffuse to and collide with empty
(ribosome-free) translocons at some frequency. If long nascent chains
were to interfere with the binding of such ribosomes to free
translocons, the cell would reduce the chances of improperly binding a
ribosome to the translocon. The discrimination against an RNC with a
long nascent chain may therefore constitute a significant safety
mechanism to minimize improper trafficking.
A very important and controversial issue is the effect that GTP binding
to SRP has on its ability to bind to a signal sequence. Because the
RNCs examined here were purified by gel filtration, the spectral data
reported above were obtained in the absence of nucleotides. We have
since examined the nucleotide dependence of RNC selection by SRP
spectroscopically and quantitatively, but those results will be
reported elsewhere because they are outside the scope of this paper.
In summary, the spectroscopic detection of SRP binding to a RNC signal
sequence has allowed us to characterize this interaction quantitatively, and this in turn has allowed us to reexamine the mechanism of SRP selection of RNCs for targeting to the ER membrane. Our data reveal, among other things, that SRP affinity for RNC signal
sequences is both variable and exceedingly high and is also unaffected
by the length of the nascent chain. Furthermore, SRP binds with
nanomolar affinity to ribosomes that lack signal sequences and can
distinguish between translating and nontranslating ribosomes.
 |
ACKNOWLEDGEMENTS |
We are grateful to Drs. David Andrews,
Greg Beckler, Linda Hendershot, Sandra Wolin, and
Veronica Worrell for providing plasmids, to Dr. Hung Do for
photocross-linking experiments, and to current and former members of
the Johnson laboratory for helpful discussions and advice.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants GM 26494 (to A. E. J.) and HL 38779 (to P. E. B.), by Training Grant T32 GM08523 (to J. J. F.), and by
the Robert A. Welch Foundation (to A. E. J.).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.
§
These authors contributed equally to this work.
Present address: Division of Molecular Medicine, Dept. of
Medical Research, China Medical College Hospital, Taichung, Taiwan.
¶¶
To whom correspondence should be addressed: College of
Medicine, 116 Reynolds Medical Bldg., TAMUS HSC, 1114 TAMU, College Station, TX 77843-1114. Tel.: 979-862-3188; Fax: 979-862-3339; E-mail: aejohnson@tamu.edu.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M300173200
 |
ABBREVIATIONS |
The abbreviations used are:
ER, endoplasmic reticulum;
NBD, 7- nitrobenz-2-oxa-1,3-diazole;
NBD-Lys, N
-[6-(7-nitrobenz-2-oxa-1,3-diazo-4-yl)aminohexanoyl]-Lys;
NBD-RNC, NBD-labeled RNC;
pBiP, pre-BiP;
pPI, preproinsulin;
pPL, preprolactin;
pPLss, preprolactin signal sequence;
RNC, ribosome-nascent chain complex;
SRP, signal recognition particle;
BiP, heavy-chain immunoglobulin binding protein..
 |
REFERENCES |
1.
|
Walter, P.,
and Johnson, A. E.
(1994)
Annu. Rev. Cell Biol.
10,
87-119[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Keenan, R. J.,
Freymann, D. M.,
Stroud, R. M.,
and Walter, P.
(2001)
Annu. Rev. Biochem.
70,
755-775[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Wild, K.,
Weichenrieder, O.,
Strub, K.,
Sinning, I.,
and Cusack, S.
(2002)
Curr. Opin. Struct. Biol.
12,
72-81[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Johnson, A. E.,
and van Waes, M. A.
(1999)
Annu. Rev. Cell Dev. Biol.
15,
799-842[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
von Heijne, G.
(1985)
J. Mol. Biol.
184,
99-105[Medline]
[Order article via Infotrieve]
|
6.
|
Walter, P.,
Ibrahimi, I.,
and Blobel, G.
(1981)
J. Cell Biol.
91,
545-550[Abstract]
|
7.
|
Siegel, V.,
and Walter, P.
(1988)
EMBO J.
7,
1769-1775[Abstract]
|
8.
|
Gaut, J. R.,
and Hendershot, L. M.
(1993)
J. Biol. Chem.
268,
7248-7255[Abstract/Free Full Text]
|
9.
|
Wolin, S. L.,
and Walter, P.
(1993)
J. Cell Biol.
121,
1211-1219[Abstract]
|
10.
|
Falcone, D.,
and Andrews, D. W.
(1991)
Mol. Cell. Biol.
11,
2656-2664[Medline]
[Order article via Infotrieve]
|
11.
|
Krieg, U. C.,
Walter, P.,
and Johnson, A. E.
(1986)
Proc. Natl. Acad. Sci. U. S. A.
83,
8604-8608[Abstract]
|
12.
|
Thrift, R. N.,
Andrews, D. W.,
Walter, P.,
and Johnson, A. E.
(1991)
J. Cell Biol.
112,
809-821[Abstract]
|
13.
|
Krieg, U. C.,
Johnson, A. E.,
and Walter, P.
(1989)
J. Cell Biol.
109,
2033-2043[Abstract]
|
14.
|
Walter, P.,
and Blobel, G.
(1983)
Methods Enzymol.
96,
84-93[Medline]
[Order article via Infotrieve]
|
15.
|
Siegel, V.,
and Walter, P.
(1985)
J. Cell Biol.
100,
1913-1921[Abstract]
|
16.
|
Janiak, F.,
Walter, P.,
and Johnson, A. E.
(1992)
Biochemistry
31,
5830-5840[Medline]
[Order article via Infotrieve]
|
17.
|
Crowley, K. S.,
Reinhart, G. D.,
and Johnson, A. E.
(1993)
Cell
73,
1101-1115[Medline]
[Order article via Infotrieve]
|
18.
|
Johnson, A. E.,
Adkins, H. J.,
Matthews, E. A.,
and Cantor, C. R.
(1982)
J. Mol. Biol.
156,
113-140[Medline]
[Order article via Infotrieve]
|
19.
|
Crowley, K. S.,
Liao, S.,
Worrell, V. E.,
Reinhart, G. D.,
and Johnson, A. E.
(1994)
Cell
78,
461-471[Medline]
[Order article via Infotrieve]
|
20.
|
Liao, S.,
Lin, J.,
Do, H.,
and Johnson, A. E.
(1997)
Cell
90,
31-41[Medline]
[Order article via Infotrieve]
|
21.
|
Dell, V. A.,
Miller, D. L.,
and Johnson, A. E.
(1990)
Biochemistry
29,
1757-1763[Medline]
[Order article via Infotrieve]
|
22.
|
Husten, E. J.,
Esmon, C. T.,
and Johnson, A. E.
(1987)
J. Biol. Chem.
262,
12953-12961[Abstract/Free Full Text]
|
23.
|
Ye, J.,
Esmon, N. L.,
Esmon, C. T.,
and Johnson, A. E.
(1991)
J. Biol. Chem.
266,
23016-23021[Abstract/Free Full Text]
|
24.
|
Sperrazza, J. M.,
Russell, D. W.,
and Spremulli, L. L.
(1980)
Biochemistry
19,
1053-1058[Medline]
[Order article via Infotrieve]
|
25.
|
Bock, P. E.,
and Shore, J. D.
(1983)
J. Biol. Chem.
258,
15079-15086[Abstract/Free Full Text]
|
26.
|
Abrahamson, J. K.,
Laue, T. M.,
Miller, D. L.,
and Johnson, A. E.
(1985)
Biochemistry
24,
692-700[Medline]
[Order article via Infotrieve]
|
27.
|
Janiak, F.,
Dell, V. A.,
Abrahamson, J. K.,
Watson, B. S.,
Miller, D. L.,
and Johnson, A. E.
(1990)
Biochemistry
29,
4268-4277[Medline]
[Order article via Infotrieve]
|
28.
|
Bock, P. E.,
Olson, S. T.,
and Björk, I.
(1997)
J. Biol. Chem.
272,
19837-19845[Abstract/Free Full Text]
|
29.
|
Johnson, A. E.,
Woodward, W. R.,
Herbert, E.,
and Menninger, J. R.
(1976)
Biochemistry
15,
569-575[Medline]
[Order article via Infotrieve]
|
30.
|
Kurzchalia, T. V.,
Wiedmann, M.,
Girshovich, A. S.,
Bochkareva, E. S.,
Bielka, H.,
and Rapoport, T. A.
(1986)
Nature
320,
634-636[Medline]
[Order article via Infotrieve]
|
31.
|
Chapman, R.,
Sidrauski, C.,
and Walter, P.
(1998)
Annu. Rev. Cell Dev. Biol.
14,
459-485[CrossRef][Medline]
[Order article via Infotrieve]
|
32.
|
Walter, P.,
and Blobel, G.
(1981)
J. Cell Biol.
91,
557-561[Abstract]
|
33.
|
Ogg, S. C.,
and Walter, P.
(1995)
Cell
81,
1075-1084[Medline]
[Order article via Infotrieve]
|
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