Correspondence to: Reid Gilmore, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655-0103. Tel:(508) 856-5894 Fax:(508) 856-6231 E-mail:reid.gilmore{at}umassmed.edu.
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Abstract |
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The Sec61 complex performs a dual function in protein translocation across the RER, serving as both the high affinity ribosome receptor and the translocation channel. To define regions of the Sec61 complex that are involved in ribosome binding and translocation promotion, ribosome-stripped microsomes were subjected to limited digestions using proteases with different cleavage specificities. Protein immunoblot analysis using antibodies specific for the NH2 and COOH terminus of Sec61 was used to map the location of proteolysis cleavage sites. We observed a striking correlation between the loss of binding activity for nontranslating ribosomes and the digestion of the COOH- terminal tail or cytoplasmic loop 8 of Sec61
. The proteolyzed microsomes were assayed for SRP-independent translocation activity to determine whether high affinity binding of the ribosome to the Sec61 complex is a prerequisite for nascent chain transport. Microsomes that do not bind nontranslating ribosomes at physiological ionic strength remain active in SRP-independent translocation, indicating that the ribosome binding and translocation promotion activities of the Sec61 complex do not strictly correlate. Translocation-promoting activity was most severely inhibited by cleavage of cytosolic loop 6, indicating that this segment is a critical determinant for this function of the Sec61 complex.
Key Words: endoplasmic reticulum, protein targeting, protein translocation, translocon structure, protein topology
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Introduction |
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Proteins that are translocated across or integrated into the ER are cotranslationally recognized by the 54-kD subunit of the signal recognition particle (SRP)1 when the NH2-terminal signal sequence emerges from the exit site on the large ribosomal subunit (for review see initiates the release of the signal sequence from SRP, and results in the attachment of the ribosomenascent chain complex (RNC) to the translocation channel (
The nascent polypeptide is subsequently transported across the ER membrane through a protein-lined aqueous pore in the membrane (, Sec61ß, and Sec61
(
once the ribosomenascent chain complex engages the translocation channel (
, TM2 and TM7 are the targets for photoreactive cross-linking agents when a nascent prepro-
-factor chain is targeted to the yeast SEC complex (
In addition to serving as the conduit for nascent polypeptide transport across the RER, the Sec61 complex functions as a high affinity ribosome receptor ( are responsible for the high affinity binding of the ribosome to the translocation channel, nor is it certain that Sec61ß and Sec61
do not contribute to the affinity between the ribosome and the Sec61 complex.
Ribosomenascent chain complexes can bind to unoccupied Sec61 complexes in an SRP-independent reaction that is thought to be driven by the affinity between the ribosome and the Sec61 complex ( (
We have used limited proteolysis to sever cytoplasmically exposed segments of RER membrane proteins. The protease-digested microsomes were assayed for SRP-independent translocation activity and for the ability to bind nontranslating ribosomes or ribosomenascent chain complexes to determine which cytoplasmic segments of the Sec61 complex contribute to the various functions of the Sec61 complex. We have obtained evidence that SRP-independent translocation is not obligatorily dependent upon high affinity binding of the ribosome to the Sec61 complex. Cytoplasmic segments of the Sec61 complex that are important for high affinity ribosome binding map to COOH-terminal cytoplasmic segments of Sec61.
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Materials and Methods |
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Preparation of Rough Microsomes (RM), SRP, the SR Fragment, and Protease-digested PK-RM
Rough microsomes (RM) and SRP were isolated from canine pancreas as described by fragment was prepared as described previously (
Aliquots of the PK-RM (500 µl) were digested at a concentration of 2 eq/µl for 1 h. Trypsin (030 µg/ml) and chymotrypsin (0200 µg/ml) digestions were done on ice and terminated with 1 mM PMSF, followed by a 15-min incubation on ice and adjustment to 10 µg/ml of aprotinin. Digestion with endoproteinase Glu-C (200 µg/ml) was for 1 h at 37°C, and was terminated with 1 mM 3,4-dichloroisocoumarin. Thermolysin digestions (050 µg/ml), which were done in the presence of 1 mM CaCl2 at either 25°C or on ice, were terminated by the addition of 2 mM EDTA. The protease-digested PK-RM were adjusted to 550 mM potassium acetate and centrifuged for 30 min at 100,000 g in a Beckman type 50 rotor. The membranes were resuspended at a concentration of 0.1 eq/µl in membrane buffer, and centrifuged for 30 min at 100,000 g in a Beckman type 50 rotor. After repeating the preceding resuspension and centrifugation steps, the protease-digested PK-RM were adjusted to a concentration of 1 eq/µl in membrane buffer, and stored at -80°C.
Isolation of Ribosomes and 125I-Labeling of Ribosomes
Ribosomes were isolated from wheat germ cytosol (
The canine ribosomes were labeled with iodine-125 by incubating 26 pmol of ribosomes with 450 µCi of 125I-Bolton-Hunter reagent (Amersham Pharmacia Biotech) for 2 h on ice. Radiolabeling was terminated by adjusting the sample to 10 mM Tris-Cl, pH 7.5. The 125I-labeled ribosomes were separated from unincorporated radiolabel by centrifugation on a 520% sucrose gradient in 50 mM Tris-Cl, pH 7.5, 50 mM KCl, 2.5 mM MgCl2 in an SW40 rotor for 3 h at 200,000 gav.
Glycerol Gradient Centrifugation and Superose 12 Chromatography
20 eq of protease-digested PK-RM was mixed with 180 µl of a detergent high salt buffer to obtain the following final conditions: 20 mM Tris-Cl, pH 7.4, 500 mM NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM DTT, 1x PIC (protease inhibitor cocktail as defined in
A 150-µl sample of the clarified detergent extract was applied to a 5-ml 830% glycerol gradient in 20 mM Tris-Cl, pH 7.4, 500 mM NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM DTT, 1x PIC, 0.125% digitonin, and 25 µg/ml egg yolk phosphatidylcholine. The gradients were centrifuged for 18 h at 85,000 gav in an SW50.1 rotor and separated into 1315 fractions using a density gradient fractionator (ISCO). Clarified detergent extracts were applied to a 23.6-ml Superose 12 column equilibrated in 20 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM DTT, 1x PIC, 0.125% digitonin, and 25 µg/ml egg yolk phosphatidylcholine. Fractions of 0.5 ml were collected as the column was eluted with equilibration buffer.
Ribosome Binding Assays of Protease-digested PK-RM
Ribosome binding assays were performed by mixing a constant amount of 125I-labeled ribosomes (typically 0.18 pmol) with 06.7 pmol of unlabeled canine ribosomes. The ribosomes were incubated with TX-PK-RM, CX-PK-RM, VX-PK-RM, or THX-PK-RM for 20 min on ice in TKMD. The 30-µl sample was applied to a 1.3-ml Sepharose CL-2B column equilibrated in TKMD. The column was eluted with 270 µl of TKMD, followed by 1.5 ml of 50 mM TEA, 500 mM potassium acetate, 5 mM magnesium acetate, and 1 mM DTT. The ionic strength of the elution buffer was raised to reduce nonspecific absorption of unbound ribosomes to the Sepharose beads. 100-µl fractions of the eluate were collected, and the amount of 125I-labeled ribosomes in each fraction was determined using a Beckman -counter. The elution positions of membrane-bound ribosomes (0.30.6 ml) and unbound ribosomes (0.61.5 ml) were determined in control experiments wherein 125I-labeled ribosomes were fractionated after incubation in the presence or absence of PK-RM. The binding data was fit to the following equation using a nonlinear least squares program: RB = (BM · RF)/(Kd + RF), where RB and RF are the concentrations of bound and free ribosomes, respectively; Bm is the number of binding sites; and Kd is the dissociation constant.
SRP-dependent and SRP-independent Translocation Assays
A full-length mRNA encoding preprolactin (pPL) and truncated mRNAs encoding the NH2-terminal 86 residues of preprolactin (pPL86) and the NH2-terminal 77 residues of firefly luciferase (ffLuc77) were isolated from preparative scale transcriptions as described previously ( 52-kD fragment. Translocated prolactin was resolved from preprolactin by PAGE in SDS.
To assay SRP-independent translocation, the truncated pPL86 mRNA transcript was translated at 25°C for 15 min in a wheat germ system that contained [35S]methionine as described previously (
The factor of 1.33 corrects for the loss of the NH2-terminal methionine residue from pPL86 upon signal sequence cleavage.
Binding of RNCs bearing 35S-labeled pPL86 to the protease-digested PK-RM was assayed by centrifugal flotation on discontinuous sucrose gradients as previously described (
Protein Immunoblots
The procedure for protein immunoblots using enhanced chemiluminescence has been described ( and Sec61ß was provided by Dr. Christopher Nicchitta (Duke University Medical Center, Durham, NC) and Dr. Tom Rapoport (Harvard University, Cambridge, MA), respectively. An antibody was raised in rabbits against the COOH terminus of Sec61
(KEQSEVGSMGALLF) using standard procedures.
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Results |
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Sensitivity of Sec61 to Proteolysis
Sec61 is integrated in the ER with a topology that places four loops (L2, L4, L6, and L8) plus the NH2- and COOH-terminal tails on the cytoplasmic face of the membrane (Fig 1 A). The ß and
subunits of the Sec61 complex are integrated by single TM spans located near the COOH terminus. Membrane-bound ribosomes effectively block access of proteases to Sec61
and, to a lesser extent, to Sec61ß (
, was used to determine which cytoplasmic segments of Sec61
are accessible to proteases. Protease cleavage sites in Sec61
were mapped to cytoplasmic loops 6 and 8 by comparison to COOH-terminal Sec61
truncation products (
contains predicted cleavage sites for at least two of the proteases tested, we observed a remarkable difference in the sensitivity of these regions to protease digestion (Fig 1 B). Loop 8 and the COOH terminus were the most protease-sensitive regions of Sec61
, followed by loop 6. The NH2-terminal tail, which is proposed to be an amphipathic
-helix aligned with the membrane surface (
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Oligomeric Stability of the Protease-digested Sec61 Complex
As visualized by electron microscopy, the purified Sec61 complex forms oligomers that are composed of three to four Sec61 heterotrimers ( (a) was resolved from SRß (e), a subunit of the 100-kD SRP receptor, and from ribophorin I (f), a subunit of the oligosaccharyltransferase (OST). As expected, Sec61ß (b) and Sec61
(not shown) cosedimented with Sec61
. The OST serves as a useful internal sedimentation marker (peak in 911,
11S) corresponding to a protein molecular mass of
300 kD (
(fractions 69) relative to the OST would be consistent with a Sec61 oligomer composed of three to four 70-kD Sec61 heterotrimers. Sec61
subunits severed in loop 8 (L8NTF) and loop 6 (L6NTF) cosedimented precisely with intact Sec61
when the T1-PK-RM were analyzed (Fig 2 A, c). The sedimentation rate of Sec61
was also not altered by quantitative cleavage of Sec61
in loops 6, 8, and the COOH terminus by trypsin (d). When the intact Sec61 complex was solubilized with Triton X-100, and then resolved on the digitonin high salt glycerol gradient, Sec61ß (h) sedimented far less rapidly and was well resolved from Sec61
(g). Resolution of Sec61
and Sec61ß is consistent with a Triton X-100induced dissociation of the Sec61 oligomer into individual subunits. Because Sec61
accounts for 75% of the protein molecular mass of the Sec61 complex, the Triton X-100treated Sec61
subunit provides an approximate sedimentation marker for a Sec61 heterotrimer. Aliquots of the intact and protease-digested Sec61 complexes were mixed before glycerol gradient centrifugation (Fig 2 B). The intact Sec61
subunits, which were derived from the undigested PK-RM, cosedimented with the NH2-terminal 22-kD fragment, which was derived from the C50-PK-RM (a) or from the Th25 PK-RM (b). As observed in A, the protease-digested Sec61 complexes were well resolved from the Triton X-100treated Sec61
(c).
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A dissociation of the translocation channel into Sec61 heterotrimers would result in a simultaneous decrease in mass and an alteration in shape. To insure that a compensatory shape change did not mask the conversion of an oligomeric ring into heterotrimers, we analyzed the intact and protease-digested Sec61 complexes by gel filtration chromatography in a digitonin high salt buffer (Fig 2 C). Detergent-solubilized Sec61 complexes, from undigested membranes (sample a), eluted in the same fractions as the NH2-terminal fragments of Sec61 that were derived by trypsin digestion in loops 6 or 8 (samples b-d), indicating that the Stokes radius of the particle was not altered. Taken together with the glycerol gradient centrifugation data, the gel filtration chromatography experiments demonstrate that the Sec61 oligomer does not dissociate into heterotrimers or isolated subunits when Sec61
is severed in cytoplasmic loops 6 and 8, and the COOH terminus.
Ribosome Binding Is Abrogated by Proteolysis of Sec61
Now that we have defined which segments of the Sec61 complex are susceptible to protease digestion and have shown that Sec61 oligomers remain intact, we assayed the protease-digested PK-RM for ribosome binding activity to investigate the role of the cytoplasmic domains of the Sec61 complex. Ribosome binding to the protease-digested PK-RM was assayed by incubating the membranes with a fixed amount of 125I-labeled ribosomes and increasing amounts of unlabeled ribosomes (Fig 3 A). A physiological ionic strength buffer (150 mM potassium acetates) was used in these assays to minimize nonspecific binding of ribosomes to other RER membrane proteins ( 18 nM), which is a value comparable to previous reports (
in the chymotrypsin-digested PK-RM (Fig 3 B). The inhibition of ribosome binding activity by chymotrypsin digestion correlated quite well with digestion of Sec61
. The reduction in ribosome binding activity observed for the C1-PK-RM was of particular interest because low concentrations of chymotrypsin sever Sec61
uniquely within cytoplasmic loop 8, yielding an NH2-terminal fragment (L8NTF) and a COOH-terminal fragment (L8CTF; Fig 3 C). More extensive digestion of the PK-RM (e.g., C30-PK-RM) cleaves Sec61
within loops 6 and 8 and causes a loss of COOH-terminal immunoreactivity.
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The number of ribosome binding sites was reduced fourfold when 55% of the Sec61 was digested in the T1-PK-RM (Fig 3 D). Conceivably, the more dramatic effect of trypsin digestion on the ribosome binding activity of the Sec61 complex could be explained by the liberation of cytoplasmic Sec61
segments, which are crucial for ribosome binding. One limitation of mapping protease cleavage sites by protein immunoblot analysis is that we cannot determine whether the Sec61
subunits have single or multiple cleavage sites within loops 6 and 8. Two cleavages within a single loop would release a soluble tryptic peptide. The V200-PK-RM did not bind ribosomes (not shown).
Previous studies that predated the identification of the Sec61 complex as the ribosome receptor had shown that trypsin digestion of ribosome-stripped microsomes inhibits the subsequent rebinding of 80S ribosomes ( do not bind ribosomes in a hypotonic buffer (not shown). The latter result confirms the previous reports concerning the trypsin sensitivity of ribosome binding sites in mammalian RER (
RNC Interactions with Protease-digested Sec61 Complexes
Two distinct interactions are thought to be responsible for attachment of an RNC to the RER (
To determine whether RNC binding to the protease-digested PK-RM was reduced, we took advantage of the observation that RNCs will bind to vacant translocation channels in an SRP-independent reaction ( subunits are severed.
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Chromatography on a Sepharose CL-2B gel filtration column was used as a second method to separate membrane bound and unbound RNCs. More than 60% of the pPL86 RNCs coeluted with the undigested T0-PK-RM in the void volume of the gel filtration column that was equilibrated in 150 mM potassium acetate (Fig 4 B). The C5-PK-RM, which had shown a threefold reduction in the binding of nontranslating ribosomes, displayed a twofold decrease in RNC binding. The C30-PK-RM and the T10-PK-RM, which had a 10-fold or greater defect in ribosome binding (Fig 3), bound 2.7-fold and 5-fold less RNCs than the undigested PK-RM. RNCs that bind to intact Sec61 complexes resist extraction with 0.5 M potassium acetate, which is consistent with nascent chain insertion into the translocation channel (Fig 4 C). The majority (80%) of the pPL86 RNCs that bind to the protease-digested PK-RM (C30-PK-RM) were also insensitive to high salt extraction (Fig 4 C). To determine whether binding of RNCs to the Sec61 complex was signal sequenceindependent, we incubated intact and protease-digested microsomes (T10-PK-RM and C30-PK-RM) with RNCs that were assembled by translation of a truncated firefly luciferase mRNA (ffLuc77). As reported previously (
When the nascent polypeptide is inserted into the translocation channel, it resides in an environment that is inaccessible to proteases ( are severed.
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SRP-independent Translocation across Protease-digested PK-RM
Based upon the results described above, we anticipated that the extensively digested PK-RM would be inactive in an SRP-independent translocation assay, whereas the microsomes that retained significant ribosome binding activity (e.g., T1-PK-RM or C5-PK-RM) would show SRP-independent translocation defects that were proportional to the fold reduction in ribosome binding or RNC binding activities. To simplify the interpretation of SRP-independent translocation assays, we used the TRAM proteinindependent substrate pPL86 for these experiments (3 pmol of Sec61 oligomers) and 10 µl of wheat germ translation products (4.5 pmol of ribosomes,
250 fmol of pPL86 RNCs) are shown in Fig 5 (A and B). Surprisingly, trypsin or endoproteinase Glu-C digestion of Sec61
reduced SRP-independent translocation of pPL86 by, at most, twofold (Fig 5 A, solid bars). Even more striking, chymotrypsin digestion of Sec61
caused no more than a 20% reduction in SRP-independent translocation activity (Fig 5 B, solid bars). Binding of nontranslating ribosomes to the protease-digested membranes was more severely inhibited than the SRP-independent translocation activity (Fig 5A and Fig B, shaded bars). Control experiments demonstrated that the processed PL56 was sequestered within microsomal vesicles.
The trypsin-digested membranes (T1-PK-RM, T10-PK-RM, and T30-PK-RM) were assayed for SRP-independent translocation activity under conditions where 80S ribosomes were present in excess relative to RNC binding sites in the undigested PK-RM (Fig 5 C). Translocation of pPL86 across the protease-severed Sec61 channels was proportional to the quantity of added microsomes. When limiting amounts of protease-digested PK-RM were assayed (1 eq), the extensively digested PK-RM (T10-PK-RM and T30-PK-RM) were threefold less active than undigested PK-RM in SRP-independent translocation of pPL86. Thus, even when the RNCs are in excess relative to the TX-PK-RM, the fold reduction in translocation activity is considerably less than the observed reduction in the ribosome binding or RNC binding activities.
We next used a sensitive competition assay to determine whether the protease-digested Sec61 complexes retain residual binding determinants for 80S ribosomes that were not detected using the classical ribosome binding assay shown in Fig 3. Nontranslating 80S ribosomes compete with RNCs for binding to the Sec61 complex, hence, they act as competitive inhibitors of the SRP-independent targeting pathway (Lauring et al., 1995; 5% of the high affinity ribosome binding sites detectable in intact PK-RM (Fig 3 D), might be less sensitive to inhibition by 80S ribosomes when assayed for SRP-independent translocation activity. To allow a direct comparison of the effect of competing ribosomes, the competition assays were adjusted to obtain comparable translocation activity in the absence of the competitor. As observed previously, the addition of 80S ribosomes caused a concentration-dependent decrease in SRP-independent translocation across the undigested PK-RM (Fig 5 D, triangles). When the protease-digested T3PK-RM were assayed, we observed that the 80S ribosomes were twofold less effective as inhibitors of SRP-independent translocation across the T3-PK-RM (Fig 5 D, squares). Nonetheless, the 80S ribosomes did interfere with RNC binding to the Sec61 complex, suggesting that the protease-severed Sec61 complexes do retain residual affinity for the nontranslating ribosomes.
Thermolysin Dissection of Sec61
Given the dramatic reduction in ribosome binding activity caused by cleavage of Sec61 at multiple sites, we incubated the PK-RM with thermolysin on ice or at 25°C to achieve more selective digestion of Sec61
(Fig 6 A). The protein immunoblots using the NH2-terminalspecific antibody to Sec61
revealed proteolytic fragments for the 25°C digestions that were similar to those obtained with chymotrypsin (compare Fig 6 A with Fig 3 C). Proteolysis within loops 8 and 6 yielded 30- and 22-kD immunoreactive fragments of Sec61
, respectively. A more rapid loss of Sec61
immunoreactivity was observed when the blots were probed with the COOH-terminalspecific antibody (Fig 6 A). Smaller immunoreactive fragments of Sec61
were not detected with either antibody. Proteolysis within the 14-residue COOH-terminal tail should abolish immunoreactivity without substantially altering the gel mobility of Sec61
. When thermolysin digestions were performed on ice, the NH2-terminal antibody revealed limited digestion of Sec61
within loop 8. Selective cleavage of the COOH-terminal tail was readily apparent, as shown by the loss of COOH-terminal immunoreactivity for the Th20-PK-RM and Th50-PK-RM (Fig 6 A). Control immunoblots using the antibody to ribophorin I (Fig 6 A) showed that differences in Sec61
COOH-terminal immunoreactivity could not be explained by differential recovery of the protease-digested membranes during preparative procedures. Quantification of the protein immunoblots disclosed the percentage of intact Sec61
that was recognized by the COOH-terminal antibody, and the intact-sized Sec61
that was recognized by the NH2-terminal antibody (Fig 6 B). The difference between the NH2- and COOH-terminal values indicates the percentage of Sec61
that was selectively cleaved in the COOH-terminal tail.
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The thermolysin-digested PK-RM were assayed for ribosome binding activity (Fig 6 B). The quantity of ribosome binding sites (solid bars) was compared with the amount of intact Sec61 recognized by the NH2-terminal (shaded bars) and COOH-terminal (diagonal bars) antibodies. A comparison of the ribosome binding activities of the Th2-PK-RM, Th20-PK-RM, and Th50-PK-RM revealed the critical importance of loop 8 and the COOH-terminal tail of Sec61
. The loss of ribosome binding activity by these three membrane preparations cannot be ascribed to digestion of loop 6, which remained intact, but instead must be dependent upon digestion of either loop 8, the COOH terminus or both segments. The importance of the COOH-terminal tail of Sec61
is evident upon comparison of Th2-PK-RM and Th50-PK-RM. Although the extent of digestion within loop 8 was comparable, Th50-PK-RM lack the COOH-terminal tail of Sec61
, and display threefold fewer ribosome binding sites. The latter result demonstrates that an intact Sec61
COOH terminus is important for the interaction between an 80S ribosome and the translocation channel.
The thermolysin-digested membranes were assayed for SRP-dependent translocation activity using the TRAM-independent substrate preprolactin ( fragment to reconstitute SRP receptor activity, as protein immunoblots had shown that the ThX-PK-RM lack intact SR
(not shown). Translocation of preprolactin across the membrane is accompanied by signal sequence cleavage to yield processed prolactin. Quantification of the results (bottom) shows that the SRP-dependent translocation activity correlated quite well with the ribosome binding activity. Translocation of preprolactin was abolished when Sec61
was severed in cytoplasmic loop 6 (e.g., Th25-PK-RM) and was strongly inhibited when Sec61
was cleaved in loop 8 (Fig 7 A). Similar results were obtained when we assayed SRP-dependent integration of op156, a ribosome-tethered nascent chain derived from bovine opsin (data not shown).
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The thermolysin-digested PK-RM were also assayed for SRP-independent translocation activity (Fig 7 B). Quantification of the data revealed that the ribosome binding and SRP-independent translocation-promotion activities of the Sec61 complex are not strictly linked. The COOH-terminal tail of Sec61 is dispensable for SRP-independent translocation activity. The most severe reduction in the SRP-independent translocation of pPL86 was observed when Sec61
was cleaved in both loops 6 and 8.
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Discussion |
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Proteolysis of Sec61
The topology of Sec61 in the mammalian ER membrane was initially deduced using hydropathy algorithms (
and the experimentally verified topology model for S. cerevisiae Sec61p (
. Based upon the presence of potential cleavage sites for trypsin, chymotrypsin, and endoproteinase Glu-C in each of the four loops and two termini, it was conceivable that each cytoplasmic segment of Sec61
would be susceptible to cleavage by all three proteases, with the exception of loop 6 which lacks a predicted cleavage site for endoproteinase Glu-C. Instead, we observed that the COOH-terminal half of Sec61
was much more sensitive to proteolysis than the NH2-terminal tail, whereas loops 2 and 4 were not digested by any protease we tested. Although a lack of digestion within loop 4 can be explained by the short length of this segment, loop 2 is comparable in length to loop 6, so size alone cannot be the explanation. The relative insensitivity of the NH2 terminus is consistent with the hypothesis that this segment of canine Sec61
is embedded on the membrane surface as an amphipathic
-helix as has been proposed for S. cerevisiae Sec61p (
is protected by another mechanism. The selective digestion of the COOH-terminal tail by thermolysin on ice indicates that this segment of Sec61
is highly exposed on the surface of the Sec61 oligomer.
Proteolysis of Sec61 did not cause the translocation channel to dissociate into Sec61 heterotrimers or fragments thereof. The maintenance of the oligomeric structure was not unexpected as hydrophobic interactions between the TM spans of Sec61
, Sec61ß, and Sec61
are presumably responsible for the stability of the oligomer. Once we established that the oligomeric structure of the Sec61 complex was not compromised by proteolytic digestion of cytoplasmic loops, we assayed the protease-digested PK-RM for ribosome binding activity, SRP-independent translocation of pPL86, and SRP-dependent translocation of preprolactin.
Binding of Nontranslating Ribosomes to the Sec61 Complex
Several RER membrane proteins (Sec61 complex, p180 and p34) bind ribosomes in hypotonic solution when reconstituted into proteoliposomes (
Our results support the hypothesis that the ribosome binding activity of the Sec61 complex can be ascribed to Sec61. More than 50% of Sec61
remained intact in protease-digested membranes that lacked detectable ribosome binding (e.g., T5-PK-RM and V200-PK-RM), demonstrating that Sec61
is not sufficient for ribosome binding activity. The extreme sensitivity of Sec61ß to thermolysin digestion (not shown) yielded the Th2-PK-RM that lack intact Sec61ß yet retain considerable ribosome binding activity.
help stabilize the association between the ribosome and Sec61
.
As the ultrastructural evidence indicates that the ribosome is tethered to the Sec61 complex via a single visible junction ( is the ribosome-binding site. In support of the hypothesis that the most protease-accessible regions of Sec61
might correspond to the ribosome binding site, we observed that Sec61 complexes that lack intact Sec61
subunits do not bind nontranslating ribosomes. A comparison of 12 eukaryotic Sec61
sequences reveals that loops 6 and 8 are highly conserved, particularly with respect to the location and number of charged amino acids. Because the COOH terminus of Sec61
is one of the least conserved cytoplasmic segments of the Sec61 complex, an important role for the COOH terminus of Sec61
in ribosome binding was unexpected. The results obtained with the C1-PK-RM and the Th2-PK-RM strongly support the hypothesis that loop 8 is required for ribosome binding to the Sec61 complex. Selective cleavage of the COOH-terminal tail of Sec61
, by thermolysin on ice (Th50-PK-RM), showed that this segment of Sec61
is crucial for the binding of a nontranslating ribosome to the translocation channel.
Assuming that proteolysis of the translocation channel results in a random digestion of Sec61 subunits in a tetramer of Sec61 heterotrimers, the partially digested membranes should contain a mixture of translocation channels that have between zero and four intact Sec61
subunits. One unexpected result was the observation that ribosome binding to the protease-digested PK-RM requires more than one intact Sec61
subunit per translocation channel. Consider an example of membranes that retain
50% intact Sec61
(e.g., C1-PK-RM or Th2-PK-RM). A random 50% digestion of tetrameric translocation channels would yield a binomial distribution of channels that contain zero to four intact Sec61
subunits (6.25% with zero intact, 25% with one intact, 37.5% with two intact, 25% with three intact, and 6.25% with four intact). The number of ribosome binding sites we detect in C1-PK-RM or Th2-PK-RM (4050% of that present in PK-RM) is much greater than the 6% of complexes that retain four intact Sec61
subunits, and is much less than the 94% of complexes that retain at least one intact Sec61
subunit. Instead, our results are best explained by a model that requires multivalent contact between the ribosome and two or three Sec61
subunits per Sec61 oligomer. In this regard, our demonstration that the Sec61 oligomer remains intact following proteolysis was a critical observation. Based upon the minimal protein bridge that tethers a ribosome to the yeast Sec61 oligomer (
subunits in a tetrameric translocation channel could be proteolyzed without reducing ribosome binding activity. Our interpretation of this apparent paradox is that physiological salt-insensitive binding of the ribosome to the canine Sec61 complex requires one or more secondary contact points that were not observed in the three-dimensional reconstructions of the S. cerevisiae ribosome-Sec61 complex.
The Signal Sequence Contributes to the Specificity and Affinity of RNC Attachment
The three independent methods we used to analyze RNC binding provided evidence that protease-severed Sec61 complexes bind RNCs with a reduced capacity and a reduced affinity relative to intact Sec61 complexes. Nonetheless, RNC binding to the Sec61 complex cannot be directly equated with high affinity ribosome binding activity. Attachment of the pPL86 RNCs to Sec61 complexes that lack high affinity ribosome binding activity is most readily explained by the hypothesis that the signal sequence of the nascent polypeptide is a second ligand that contributes significantly to the specificity and affinity of the interaction between an RNC and the Sec61 complex. RNCs that lack a signal sequence (e.g., ffLuc77) bind poorly to the protease-digested Sec61 complexes. How can we rationalize this conclusion with the previous data showing that 80S ribosomes compete with RNCs for SRP-independent binding to the translocation channel? As noted previously ( that is inaccessible to proteases, hence, it is distinct from the cytoplasmic loops that contact the ribosome.
SRP-independent Translocation of Polypeptides through Protease-digested Sec61 Complexes
SRP-independent translocation through the Sec61 complex is thought to accurately mimic the RNC binding, nascent chain insertion and transport phases of the translocation reaction. When RNCs are targeted by the SRP-independent pathway, binding of the RNC to the Sec61 complex is signal sequenceindependent (
When we assayed SRP-independent translocation across the protease-digested PK-RM, we made several unexpected observations. Protease-digested PK-RM that lack binding sites for nontranslating ribosomes remain competent for SRP-independent translocation of pPL86. These results indicate that a functional interaction between a nascent polypeptide and the translocation channel is not strictly dependent upon an initial high affinity binding of the ribosome to Sec61 complex. The most definitive resolution of the ribosome binding and the translocation promotion activities of the Sec61 complex was obtained by limited digestion of Sec61 with thermolysin. Cleavage of the COOH terminus of Sec61
drastically reduced ribosome-binding activity while having a relatively modest effect upon SRP-independent translocation of pPL86.
The interaction between an RNC and the Sec61 complex progresses through several distinct stages as the nascent polypeptide increases in length (. Salt-resistant RNC attachment occurs upon further elongation when the signal sequence is inserted into a protease-inaccessible environment in the translocation channel (
. RNCs bearing pPL86 did not remain attached to the protease-digested PK-RM on sucrose flotation gradients in a physiological ionic strength buffer (Fig 4 A). Gel filtration chromatography, which avoids exposure of the sample to 2 M sucrose and high centrifugal fields, provided evidence that the RNCs were bound to the protease-digested PK-RM (Fig 4B and Fig C). Further evidence that the RNCSec61 interaction was altered was provided by the finding that the junction between the ribosome and the membrane was not sufficiently tight to prevent access of a macromolecular probe (proteinase K) to the nascent polypeptide.
Regions of Sec61 Implicated in RNC Binding and Protein Translocation
All of the protease-digested PK-RM described here were also assayed for SRP-dependent translocation activity using the procedure shown in Fig 7 A ( in either cytoplasmic loop 6 or loop 8 leads to a complete block in the SRP-dependent translocation pathway. The restrictive block of the SRP-dependent targeting pathway is most readily explained by the accumulation of an upstream translocation intermediate that precedes transfer of the RNC from SRP54 to Sec61
(
; cleavage within loops 6 and 8 reduced RNC binding and nascent chain translocation. Although the moderate reduction in SRP-independent translocation activity probably reflects the reduced affinity of the translocation channel for the ribosome, our results strongly suggest that the translocation-promoting function of the Sec61 complex resides in a protease-inaccessible region of Sec61
. A molecular genetic dissection of Sec61p has suggested that an intact cytoplasmic loop 6 is crucial for the in vivo function of the Sec61 complex (
appears to be crucial for translocation of proteins across the ER. Our results indicate that loop 8 and the COOH terminus are required for high affinity binding of ribosome to the Sec61 complex. We propose that these two segments cooperate to form a ribosome-binding platform that is responsible for both the primary and secondary contacts between the translocation channel and the ribosome. Whereas a detailed description of the ribosome-binding site in Sec61
will require further ultrastructural and molecular genetic analysis, the results described here show that the COOH-terminal half of Sec61
should be the focus for further scrutiny.
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Footnotes |
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1 Abbreviations used in this paper: CX-PK-RM, chymotrypsin-digested PK-RM; ECL, enhanced chemiluminescence; NAC, nascent chainassociated complex; OST, oligosaccharyltransferase; PIC, protease inhibitor cocktail; PK-RM, puromycin high saltwashed RM; RM, rough microsomes; RNC, ribosome nascent chain complex; SR, signal recognition particle receptor; SRP, signal recognition particle; TEA, triethanolamine acetate, pH 7.5; TM, transmembrane; TX-PK-RM, trypsin-digested PK-RM; ThX-PK-RM, thermolysin-digested PK-RM; VX-PK-RM, endoproteinase Glu-Cdigested PK-RM.
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Acknowledgements |
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We thank Gert Kreibich, Chistopher Nicchitta, Tom Rapoport, and Peter Walter for providing antibodies to ribophorin I, Sec61, and Sec61ß, and SRß, respectively. We thank Kennan Kellaris (Georgetown University, Washington, DC) for raising antisera to the COOH terminus of Sec61
while she was a postdoctoral fellow in this lab.
This work was supported by National Institutes of Health grant PHS GM 35687.
Submitted: 12 November 1999
Revised: 31 May 2000
Accepted: 31 May 2000
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References |
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