(Received for publication, October 11, 1994; and in revised form, December 23, 1994)
From the
Apolipoprotein (apo) B is either co-translationally assembled into lipoproteins, or becomes associated with the membrane of the endoplasmic reticulum (ER) and is subsequently degraded. It has been proposed that apoB undergoes a novel process of translocation which generates cytoplasmically exposed apoB in the ER of hepatic and non-hepatic cells.
Transmembrane forms of apoB can also be generated by in vitro translation (Chuck, S. L., and Lingappa, V. R.(1992) Cell 68, 9-21), which might explain the origin of untranslocated apoB in vivo. Here we have investigated a protocol which generates transmembrane forms of apoB during in vitro translation of truncated RNA transcripts. We observe that apoB can become transmembrane at sites of ribosome pausing and be held in this configuration by persistence of tRNA on the peptide chains. Ribosome pausing also occurs at these same sites in the absence of acceptor microsomes. Transmembrane topology can be generated at sites of ribosome pausing in a cytosolic protein, sea urchin cyclin when fused to a signal sequence. Mapping of the ribosome pause sites in apoB and in cyclin revealed no amino acid sequence homology. Chimeric constructs with engineered downstream glycosylation sites showed no evidence that ribosome pause sequences affect translocation of transcripts with termination codons. Transmembrane forms of apoB and cyclin were not generated during translocation into the ER in transfected COS cells.
Apolipoprotein B100 is co-translationally assembled into
lipoprotein particles(1) . It is unresolved whether the nascent
particle in the rough endoplasmic reticulum (ER) ()is fully
mature (2) or whether it acquires further lipid during
maturation in the smooth ER and in the Golgi
apparatus(3, 4, 5) . Subsequently very low
density lipoprotein is secreted to distribute triglyceride from the
liver to peripheral tissues(3) . A proportion of newly
synthesized apoB100 does not exit from the ER, but remains
membrane-associated until subjected to presecretory
degradation(6, 7, 8) .
The topology of the membrane-associated apoB, and the mechanisms whereby apoB is destined either for lipoprotein secretion or presecretory degradation are controversial. Several studies suggest that a proportion of newly synthesized apoB chains is exposed on the cytoplasmic face of microsomes from primary rat (6) or chick (7) hepatocytes, liver tissue(9) , or HepG2 cells(10, 11) . It has been further reported that following stable expression in Chinese hamster ovary cells, apoB53 (the N-terminal 53%) is rapidly degraded, without undergoing complete translocation into the ER lumen while apoB15 can be translocated and secreted(12) . In contrast, incomplete translocation of apoB53 was not confirmed in transiently transfected COS cells(13) .
Studies in intact cells have suggested that incompletely translocated apoB may be present on the cytoplasmic leaflet of the ER. Parallel studies using in vitro translation and translocation have also detected transmembrane forms of apoB, and it has been suggested that these arise by translocational pausing at topogenic sequences(14, 15) . Although the proposed transmembrane sites in intact cells and the cell-free systems do not coincide, it has been suggested that the phenomena are related and derive from an unusual mechanism of apoB translocation which operates in vivo(11, 16) .
Previously we could not confirm that transmembrane intermediates are produced during in vitro translation of apoB15, therefore we proposed that this region of apoB is co-translationally translocated into the ER(17, 18) . The results presented in this study provide additional support for this conclusion. Recently, however, a protocol has been described for ``trapping'' apoB transmembrane intermediates by in vitro translation of transcripts lacking termination codons(15) . Here we confirm that transmembrane structures can be generated by in vitro translation of transcripts lacking termination codons. However, we propose a model for their generation based on ribosome pausing rather than signal-mediated translocational pausing. We therefore suggest that the events which occur during cell-free translation of transcripts lacking termination codons do not reflect the true apoB translocation pathway which operates in vivo.
cDNA encoding: 1) cyclin(21) ; or 2) and 3) apoB (20) was expressed after fusion to the apoB signal sequence by ligation of polymerase chain reaction amplified cDNA into SstII and XbaI digested vector pUBSS (Fig. 1). Vector pUBSS was constructed as follows. The apoB signal sequence was amplified with mismatched oligonucleotides: C AAA CAG AGC TCC ATG GAC CCG and GAC ATT TTC CCG CGG TTC CTC TT. This yielded a fragment with: (i) a 5` SstI site (underlined), (ii) an NcoI site spanning Met(-27) (underlined), and (iii) a 3` SstII site (underlined) created in-frame after the third codon of the mature protein. After subcloning into SstI and SstII digested plasmid pKS, the fragment was excised with NcoI and SalI to include the pKS multiple cloning region. This was ligated into a pGEM plasmid containing the T7 RNA polymerase promoter and nucleotides 259 to 837 of the encephalomyocarditis 5`-untranslated region(22) . The resulting plasmid pUBSS encodes Met-Ala-apoB (-27 to 3), and allows expression of cDNA ligated in-frame into the SstII site.
Figure 1: Constructs for protein expression. A, vector pUBSS (constructed in plasmid pKS+) has a T7 RNA polymerase promoter (T7), the encephalomyocarditis 5`-untranslated region (EMCV.UTR), the apoB signal sequence (BSS), an SstII site (Ss), and a polylinker (PL). This was used to express cDNAs fused in-frame at the SstII site to generate the apoB, and cyclin (CYC) constructs as described in the text. Residue numbers refer to the mature natural proteins. Key restriction sites for linearization for transcription or for cDNA manipulation are shown (B, BamHI; Bg, BglII; H, HindIII; Hp, HpaI; Nc, NcoI; St, StuI). Asparagine residues forming potential N-linked glycosylated sites are marked N, and when shown in bold have been introduced by mutagenesis. Termination codons are also shown (ter). In clone SKA.B`B`cyc two copies of the proposed 11-residue core B` sequence (B`) are present (see (23) ). cDNAs encoding apoB15.cyc fusion proteins were expressed in the eukaryotic expression vector pSV7d. BUTR, apoB 5`-untranslated region.
1) A cDNA in pGEM1 encoding Met-Ala and
then residues 13 409 of Arbacia punctulata cyclin was a
gift from Drs. Mike Howell and Tim Hunt (Imperial Cancer Research Fund,
Clare Hall). This was amplified with a mismatched 5`-oligonucleotide to
create an SstII site (underlined), and with a
3`-oligonucleotide spanning the vector SalI site (underlined):
TT GGT CCG CGG ATG GCC ATG GCT CAT GGT and CTG CAG GTC GAC TCT AGA GGA
TCC. The fragment was subcloned into vector pUBSS to yield plasmid
ss.cyc (Fig. 1). Full-length transcripts with termination codons
were made after SalI digestion. Truncated transcripts were
made by linearization at internal sites as described under
``Results.''
2) ApoB cDNA encoding residues 187 424
was amplified with the mismatched 5`- and 3`-oligonucleotides: TGT CCG
CGGTTC ATG CCC ATG CGC ACA GGC ATC and TGT CTA GAG TTA GTT GTT GAC CGC
GTG GCT, respectively, to introduce a 5` SstII site and two
additional methionine codons (underlined), and a 3` termination codon
and XbaI site (also underlined). This was subcloned into
vector pUBSS to generate plasmid SX.B5 which encodes no glycosylation
sites. A further derivative of plasmid SX.B5, SX.B5glc (Fig. 1),
was made by digesting with StuI and SalI and fusing a
distant segment of apoB cDNA encoding glycosylation sites, in-frame at
the StuI(1196) site (residue 330). To achieve this, apoB cDNA
encoding residues Leu-3037 to Ala-3208 was amplified with mismatched
5`- and 3`-oligonucleotides G CCT ACG TTA ACA AAC TTA ACA GGG AAG ATA
GAC TTC and T GGG GAG GTC GAC GTG AGA TTT TTA AGC TT. The
5`-oligonucleotide introduces an HpaI site (underlined), and
the 3`-oligonucleotide introduces a termination codon and an SalI site (also underlined).
3) ApoB cDNA was polymerase chain reaction amplified between residues Val (228) and Gly (330). The 5`-oligonucleotide was mismatched to introduce an SstII site and two additional methionine residues (underlined): TG GCA CCG CGG ATG GGC ATG GGC ATG GAG CAA CAC CTC TTC. This fragment was extended from the StuI(1196) site with apoB cDNA encoding residues Leu-475 and Leu-781, and subcloned into vector pUBSS to create plasmid SXA.Bg which lacks glycosylation sites. Plasmid SXA.Bg was cleaved at the BglII(1956) site and at the polylinker SalI site to insert either: (a) cDNA encoding the B` sequence and downstream reporter glycosylation sites derived from apoB cDNA (SXA.B`apoB) (Fig. 1) or (b) two tandem copies of the B` sequence followed by reporter glycosylation sites in the context of cyclin cDNA (SXA.B`B`cyc) (Fig. 1) as described below.
Subsequently plasmid B15.cyc (Fig. 1) was created from B17.HS. The full-length cyclin polymerase chain reaction product described in section 1 of ``Transcripts for in Vitro Translation'' was digested with SstII and SalI and subcloned into SstII and SalI digested B17.HS.
A further derivative, plasmid B15.B`B`cyc (Fig. 1), was made by inserting two tandem copies of the putative core B` sequence, LKKTKNSEEFA (23) between the HindIII and SstII sites of B15.cyc. A double stranded oligonucleotide with two tandem copies of the B` sequence was prepared as described under ``Transcripts for In Vitro Translation'' except that a new 5`-oligonucleotide TCA ACA AAA GCT TTG CTG AAG AAG ACC AAG AAC TCC (replacing oligonucleotide i) was used to introduce a HindIII site (underlined).
For labeling, cells from 4
75 cm
flasks were detached with 2 mM EDTA
in phosphate-buffered saline, collected by low speed centrifugation,
and then suspended in 3 ml of methionine-free Dulbecco's modified
Eagle's medium (Life Technologies, Inc.). After preincubation for
30 min at 37 °C, 125 µCi/ml
[
S]methionine (Tran
S-label, ICN)
was added and the incubation was continued for times specified in the
text. The cells were collected and washed by low speed centrifugation
at 4 °C and then were either (i) directly solubilized for
immunoprecipitation or (ii) dispersed in isotonic sucrose and
homogenized.
Figure 2: ApoB9 becomes transmembrane at a ribosome pause site. A, apoB9 RNA with (lanes 1 and 2) or without (lanes 3-6) a termination codon (ter) was translated for 75 min in the presence (lanes 1-4) or absence (lanes 5 and 6) of canine microsomes (mic). Aliquots were trypsinized (trp) for 0 or 60 min and then resolved by SDS-PAGE on 8-15% gels. ApoB9 (B9) with a termination codon is fully protected, but without a termination codon is cleaved to a product comigrating with pause product A. A peptidyl-tRNA product (B9-tRNA) is seen when apoB9 is translated without a termination codon. Unglycosylated and faster migrating forms of apoB9 and of pause products A and B are produced in the absence of membranes (lane 5), and are digested with exogenous protease (lane 6). B, apoB9 RNA without a termination codon was translated in the presence of microsomes. After trypsinization for 0 or 60 min, products were resolved by SDS-PAGE on 8-15% gels. ApoB9 is cleaved to products comigrating with both pause products A and B. C, apoB9 RNA without a termination codon was translated in the presence (lanes 2 and 3) or absence (lanes 1 and 4) of microsomes. Where indicated, N-acetyl-Asn-Tyr-Thr-carboxamide (NYT, (31) ) was added to inhibit glycosylation. Microsome processed pause products A and B migrate faster with inhibitor (3) than without(2) , indicating glycosylation. The unglycosylated processed products (3) migrate slightly faster than unprocessed apoB9 (1 and 4), owing to signal cleavage.
Figure 6:
ApoB9, transmembrane at a ribosome pause
site, completes translocation after treatment with EDTA, or RNase A but
not puromycin. A, apoB9 RNA lacking a termination codon (lanes 1-4), or RNA encoding a transmembrane control
protein from MMTV (lanes 5-8) was translated in the
presence of microsomes. Aliquots were trypsinized (trp) either
without(-) further treatment (lanes 1, 2, 5, and 6) or after (+) successive treatment with emetine,
puromycin, and EDTA at 30 °C and then with Mg on
ice (EmPuEdMg) (lanes 3, 4, 7, and 8).
Aliquots were dissolved in SDS-PAGE buffer + 2.0 µg of RNase A
and resolved by SDS-PAGE on 8-15% gels. Without treatment apoB9
was transmembrane at pause A, but after treatment was translocated to a
fully protected form. Inclusion of RNase A verifies that the digestion
assays really are determining protein topology, rather than the
presence of esterified tRNA. Translation of MMTV revealed a major
product, and smaller amounts of glycosylation variants. These products
were cleaved by 2 kDa (the cytoplasmic domain), and treatment with
EmPuEdMg was without effect. B, apoB9 RNA lacking a
termination codon was translated in the presence of microsomes, then
treated with RNase A for 0 min at 0 °C (lanes 1 and 2) or for 30 min at 37 °C (lanes 3 and 4). Aliquots were then trypsinized for 0 or 60 min. Without
RNase treatment, apoB9 was transmembrane at pause sites A and B, but
became fully translocated after RNase A treatment. C,
transcripts encoding apoB9 or apoB11, in both cases lacking termination
codons, were translated in the presence (+) or absence(-) of
microsomes (mic). Aliquots were treated with EDTA (+, lanes 2, 4, 6, and 8) under conditions which promote
post-translational translocation of apoB9, or were untreated (-, lanes 1, 3, 5, and 7). Samples were boiled
immediately in SDS loading buffer to minimize spontaneous hydrolysis of
peptidyl-tRNA and loaded onto 8-15% SDS-PAGE gels. ApoB9 samples
contain peptidyl-tRNA except after promotion of post-translational
translocation with EDTA (lane 2). Under identical conditions,
a much smaller proportion of peptidyl-tRNA is present in apoB11
samples. D, apoB9 RNA lacking a termination codon was
translated for 30 min in the presence (+, lanes
1-5) or absence (-, lanes 6 and 7)
of microsomes (mic). In lane 3, puromycin (pur) was present co-translationally (c). Incubations
were continued for a further 30 min either with puromycin added
post-translationally (+, lanes 1, 2, 4, and 6)
or without puromycin addition (-, lanes 5 and 7). One translation reaction was digested with trypsin (trp) for 0 or 60 min (lanes 1 and 2). The
other translation reactions were undigested (-, lanes
3-7). Aliquots of the reaction mixtures were boiled
immediately in SDS loading buffer to minimize the spontaneous
hydrolysis of peptidyl-tRNA and loaded directly onto 8-15%
SDS-polyacrylamide gels. ApoB9 is cleaved to a product comigrating with
pause A (lane 2). Post-translational addition of puromycin
does not affect the quantity of peptidyl-tRNA detected either in the
presence or absence of membranes (lanes 5 and 7). In
the absence of membranes, apoB9 and the pause products are
unglycosylated and hence migrate faster (lanes 6 and 7).
Oligonucleotide-directed RNase H cleavage of transcripts mapped ribosome pause A between residues 252 and 280 (Fig. 3A) and indicated that B is the carboxyl-terminal of residue 305 (Fig. 3A). These sequences are not only present in apoB9 but also in apoB6, B11, B13, and B15. However, translation of the latter proteins from transcripts lacking termination codons did not generate transmembrane proteins, suggesting that pause sites A and B do not influence membrane topology in all contexts (Fig. 3, B and C).
Figure 3: The transmembrane topologies of expressed apoB proteins are not determined by simple topogenic sequences. A, apoB9 RNA lacking a termination codon or apoB9 RNA cleaved with RNase H and oligonucleotides ALW, ASU, or T432 were translated for 75 min in the presence of microsomes. Aliquots of the translated products from the cleaved templates (apoB 1-305 (lane 1), 1-280 (lane 2), and 1-252 (lane 4)) were electrophoresed alongside translocated and trypsinized apoB9 (B9) (3) on 12% gels. ApoB9 pause transmembrane site A is localized between residues 252 and 280. Pause product B was not precisely mapped but migrates more slowly than apoB (1-305). B, apoB11 RNA lacking a termination codon was translated (trn) in the presence of microsomes for 10-30 min, then trypsinized (trp) for 0 (lanes 1-4) or 60 (lanes 5-8) min. The proportion of pause products A and B was low. There was no evidence that apoB11 becomes transmembrane at pause A or B at any time point examined since losses of full-length apoB11 were accompanied by parallel losses in the pause products. C, apoB15 (lanes 1 and 2), apoB13 (3 and 4), or apoB6 (lanes 5 and 6) RNA lacking a termination codon was translated in the presence of microsomes for 60 min then trypsinized for 0 (lanes 1, 3, and 5) or 60 (lanes 2, 4, and 6) min. There was no evidence that apoB6, 13, or 15 were transmembrane. Pause products were clearly visible in the apoB15 translation, but did not increase on proteolysis.
Interpretation of proteolysis experiments can
be complicated by loss of total material as observed in Fig. 3, B and C. We interpret this as resulting from vesicle
instability since it is also observed with soluble secretory proteins
such as yeast mating factor (not shown). To control for protein
loss and hence to confirm that complete co-translational translocation
of this region of apoB occurs, construct SXB.B5glc was produced (Fig. 1). This construct encodes apoB residues 187-330
(including pauses A and B) and then has two downstream reporter
glycosylation sites. In vitro translation of SXB.B5glc with
microsomes yielded two products glycosylated at one or both sites (Fig. 4A), and both products were equally protected
against protease (Fig. 4B). No evidence was found for
unglycosylated protease-sensitive translocation intermediates generated
by sequences A and B. No protected glycosylated fragments were detected
upon proteolysis which if present would indicate a polytopic
conformation. We therefore deduce that this region of apoB is
co-translationally translocated under normal conditions of translation
when a termination codon is present.
Figure 4:
Ribosome pausing between residues 252 and
280 does not result in detectable intermediates of translocation from
transcripts with termination codons. A, clone SXB.B5glc
encodes apoB residues 187-330 (i.e. including the mapped
pause A and B sites) followed by downstream reporter glycosylation
sites. Protein SXB.B5glc was translated in the presence or absence of
microsomes for the times indicated and the products were resolved by
SDS-PAGE on 15% gels. In the absence of membranes a single band
(prepeptide) is observed. In the presence of membranes at all time
points examined the predominant products (glc1 and glc2) migrate more slowly than the prepeptide owing to
glycosylation, confirming that the protein has been translocated beyond
the pause A and B sites. Traces of prepeptide (1%) are present in
the presence of microsomes since in vitro processing is not
completely efficient, however, there is no evidence of abundant
translocation intermediates. (In additional experiments it was verified
that a clone starting at apoB residue 187 does pause at sequences A and
B when truncated at an internal restriction site (not shown), i.e. residues 1-187 are not required for the expression of
ribosome pause sites A and B.) B, protein SXB.B5glc was
translated in the presence of membranes for 30 min with (lanes 1 and 2) or without (lanes 3 and 4)
peptide inhibitor of glycosylation (NYT), then subjected to
proteolysis for 0 or 60 min. Products were resolved on a 15% SDS-PAGE
gel. The unglycosylated (lanes 1 and 2) and singly
and doubly glycosylated (lanes 3 and 4) forms of the
protein were equally protected (confirmed by scintillation counting of
the radioactive bands). No evidence was seen for an unglycosylated,
protease-sensitive form of the protein in lanes 3 or 4, which would be indicative of intermediates of
translocation.
Figure 5: Cyclin can adopt a transmembrane configuration at two distinct sites when translocated into microsomes. A, transcripts of signal peptidyl cyclin with a termination codon (ter) (lanes 1 and 2) or transcripts from the cyclin cDNA cleaved at the ScaI(1202) (lanes 3 and 4) or BamHI(1074) (lanes 5 and 6) sites were translated for 75 min with microsomes to generate full-length cyclin, cyclin (amino acid 373), and cyclin (amino acid 330), respectively. While full-length cyclin and cyclin 373 were protected from trypsin (trp), cyclin 330 showed tRNA persistence (330-tRNA) and was transmembrane at pause site II. B, transcripts of the cyclin cDNA cleaved at the Csp45 (238) site were translated in the presence of microsomes and trypsinized for 0 (lane 1) or 60 min (lane 2). Membranes were collected by ultracentrifugation and the products were resolved on a 16.5% Tricine gel. The truncated cyclin (amino acid 51) was cleaved to products comigrating with pause products III and IV. (The presence of peptidyl-tRNA was confirmed on separate gels.)
We noted that truncations of apoB (Fig. 6C) or cyclin (Fig. 5A) which adopt a transmembrane configuration show tRNA persistence while truncations which are fully translocated have negligible or absent tRNA. We confirmed that there are differences in tRNA persistence between apoB9 and B11 when translated either in the presence or absence of microsomes (Fig. 6C), i.e. these differences are not dependent on a membrane receptor. Furthermore, when post-translational translocation of apoB9 is induced with EDTA, the tRNA moiety is lost (Fig. 6C).
Puromycin alone did not promote post-translational translocation of transmembrane apoB9 (Fig. 6D). However, we observed that puromycin added post-translationally could not displace tRNA from apoB9, even though it inhibited protein synthesis co-translationally (Fig. 6D). This suggests that the ribosomes have dissociated from the apoB9 chains after translation, and are not available to displace esterified tRNA with puromycin. Loss of ribosomes was confirmed by ultracentrifugation (32, 33) of the lysates (not shown).
If differences in tRNA persistence determine whether truncated proteins are stabilized in transmembrane configurations at ribosome pause sites, this would explain the co-translational translocation of apoB9 with a termination codon (Fig. 2A) and of SXB.B5glc with a termination codon (Fig. 4, A and B) since tRNA would be released immediately on completion of these proteins.
We were unable to trap transmembrane proteins in reticulocyte translation after RNase H-mediated truncation of messages to generate proteins ending at residues 150 or 250 (not shown). To examine the possible function of the B` sequence under normal conditions of translation in reticulocyte lysates, we generated a construct (SXA.B`apoB, Fig. 1) with reporter glycosylation sites downstream from the B` sequence and located in the adjacent apoB sequence (residues 100-137). Translation of protein SXA.B`apoB without membranes generated a single product. With microsomes and glycosylation inhibitor together, a faster migrating product resulted owing to signal cleavage. Translation with membranes alone yielded three products: processed unglycosylated protein and protein glycosylated at one or both sites. Proteolysis revealed that each processed form of the peptide was protected to a similar extent (Fig. 7A). There was no evidence for selective loss or cleavage of the unglycosylated form of the protein. Thus in reticulocyte lysates, this region is co-translationally translocated, in contrast to previous results in wheat germ lysates(14) .
Figure 7:
Effect of the B` sequence on protein
translocation. A, construct SXA.B`apoB has a carrier domain,
downstream of which is the B` sequence in its natural context followed
by two reporter glycosylation sites. SXA.B`apoB transcripts with a
termination codon were translated for 60 min in the presence (lanes
2-4) or absence (lane 1) of microsomes. In lane
2, a peptide inhibitor of glycosylation (NYT) was also
present. Products were resolved by SDS-PAGE on 15% gels. Processed,
unglycosylated SXA.B`apoB (lane 2) migrated more quickly than
preSXA.B`apoB (lane 1) owing to signal cleavage. In the
presence of membranes three forms of the protein were detected
representing glycosylation at 0, 1, or 2 sites (lane 3). Each
form was equally protected against trypsin digestion (lane4), and there was no evidence of cleavage of the
unglycosylated protein to yield a protected fragment of 23 kDa
consistent with it becoming transmembrane at the B` site. B,
construct SXA.B`B`cyc consists of a carrier domain, two tandem copies
of the B` sequence and then cyclin as a downstream reporter domain with
two glycosylation sites. Transcripts from construct SXA.B`B`cyc with a
termination codon were translated for 60 min in the presence (lanes
1, 3, and 4) or absence (lane2) of
microsomes. Products were resolved by SDS-PAGE on 15% gels. In the
presence of membranes, three forms of the protein were detected
representing glycosylation at 0, 1, or 2 sites (lane1). PreSXA.B`B`cyc(2) was intermediate in mobility
between the signal cleaved unglycosylated, and singly glycosylated
forms. Aliquots of the protein were trypsinized for 0 or 60 min (lanes 3 and 4). All three forms of the protein were
equally protected and there was no evidence that the unglycosylated
protein was cleaved to a 23-kDa product transmembrane at the B` site.
Transcripts from SXA.B`B`cyc cDNA linearized at the BglII site
lack a termination codon. Following translation with microsomes, then
digestion for 0 or 60 min (lanes 5 and 6), the
resulting protein showed persistence of tRNA (lane5)
and was transmembrane at a ribosome pause site (pause V) (lane6). C, COS cells were co-transfected with apoB
expression plasmid B17 and either with B15.cyc (lanes
1-3) or with B15.B`B`cyc (lanes 4-6). Cells
were labeled for 15 min with [S]methionine.
Membranes (0.5-1.8 M sucrose) were prepared and
trypsinized for 0 min (lanes1 and 4) or 60
min (lanes2 and 5) or for 60 min plus 0.2%
saponin (lanes3 and 6). Digests were
stopped with soybean trypsin inhibitor followed by ultracentrifugation
to collect the membranes. Pellets were redissolved prior to
immunoprecipitation with anti-apoB antiserum. In parallel experiments
membranes from untransfected COS cells were digested for 0 (lane7) or 45 min (lane8) with antiserum
against the EGF receptor or for 45 min in the presence of 0.2% saponin (lane9). Products were resolved by SDS-PAGE on
5-15% gels. The B15.cyc and B15.B`B`cyc proteins were equally
protected against proteolysis when compared with B17 as a measure of
vesicle integrity. Under these conditions of proteolysis the EGF
receptor (EGF-R) is cleaved from the 170-kDa mature protein to
the 100-kDa luminal domain (LD).
To exclude the possibility that residues 100-137 of apoB supply specific restart information (15) which counteracts the B` sequence in SXA.B`apoB, plasmid SXA.B`B`cyc (Fig. 1) was also constructed. The encoded protein has two tandem copies of the core B` sequence (23) followed by cyclin in which two potential glycosylation sites are located. Translation of SXA.B`B`cyc in the presence of membranes yielded three processed products: unglycosylated protein and protein glycosylated at one or both sites. All three species were once again similarly protected against proteolysis when translated with a termination codon (Fig. 7B). We conclude that the B` sequence can be translocated in a heterologous context where specific restart information would not be predicted. Truncation at the BglII site in the cyclin moiety of SXA.B`B`cyc (i.e. upstream of the termination codon) yielded a protein which showed tRNA persistence and which became transmembrane at a ribosome pause site (pause V in Fig. 7B). Trapping at or very close to this site was previously observed with cyclin cDNA alone truncated with BglII (Fig. 5B) and does not depend on the B` sequence. These results establish definitively that demonstrating trapping in a construct without a termination codon does not imply that the transmembrane region identified will exert any measurable effect on the topology of the full-length protein translated with a stop codon.
Figure 8:
Membrane topology of apoB36 in COS cells. A, COS cells were co-transfected with apoB36 and apoB15.cyc
encoding plasmids, and then 48 h later were labeled for 15 min with
[S]methionine. Isolated microsomes were digested
with trypsin for 0 (lane 1) or 45 (lane2)
min or for 45 min plus 0.2% saponin (lane3).
Membranes were collected and then immunoprecipitated with anti-apoB
antiserum. Products were resolved by SDS-PAGE on 5-15% gels.
ApoB36 and B15.cyclin proteins are similarly protected in intact
microsomes (2) but become susceptible to proteolysis when the
membranes are disrupted with saponin(3) . An additional band (X) is present which is also precipitated with anti-apoB
antiserum but is not apoB (see (8) ) and which is also
expressed in untransfected COS cells (not shown). Protein X also shows
similar protection to apoB36. B, transcripts encoding apoB30
were translated in the presence (lanes 3 and 4) or
absence (lanes1 and 2) of microsomes and
trypsinized for 0 (lanes1 and 3) or 60 min (lanes2 and 4). Products were resolved by
SDS-PAGE on 5% gels. ApoB30 was fully digested by trypsin when
translated in the absence of membranes (lane2) but
showed protection in the presence of membranes (lane4), confirming there is no block to its translocation
into heterologous microsomes. No new bands were generated on
trypsinization, suggesting that topogenic information is not present in
this protein.
Transmembrane domains of integral membrane proteins typically consist of 20 consecutive hydrophobic amino acids(37) . In some instances the hydrophobic sequence may be interrupted with charged or polar amino acids as in glycophorin (38, 39) or the T cell receptor (40) . However, hydrophobic amino acids alone may not be sufficient to direct membrane insertion. Thus trypsinogen with 22 consecutive hydrophobic amino acids is wholly secretory(37) . Adjacent stretches of charged amino acids may be required to direct proper membrane insertion of the hydrophobic region. Thus altering the charge distribution around the transmembrane domain of certain prokaryotic membrane proteins can reverse their orientation in the membrane(37, 41, 42) . For certain eukaryotic proteins it has been suggested that specific charged sequences, stop transfer effectors, direct their proper integration into membranes(43) .
Structure prediction based on the
primary sequence of apoB has revealed extensive regions of amphipathic
helix and
sheet but no domains that would be predicted to
span a phospholipid bilayer(20, 44, 45) .
These amphipathic structures bind to and stabilize the surface of
lipoprotein particles, where a phospholipid monolayer surrounds a
hydrophobic core of neutral lipid. While classical transmembrane
sequences may be absent from apoB, it has been suggested that specific
charged sequences are present which have similarities with eukaryotic
stop transfer effector sequences(15) . In the absence of
adjacent hydrophobic sequences, it is assumed that they cannot direct
stable bilayer insertion of apoB but that they stall the protein in the
translocation pore(15) . To facilitate the detection of pause
transfer signals in apoB, a ribosome trapping protocol was proposed
based on translation of transcripts lacking termination
codons(15) . It was assumed that ribosomes would remain in
situ at the ends of these truncated messages so that a frozen
image of particular stages of the translocation process might be
obtained. The locations of pause transfer sequences in apoB could be
inferred from the sites at which it becomes transmembrane. Two distinct
sequences in apoB were postulated to have this function by interacting
with a membrane receptor: residues 86-99 and
305-318(15) . A consensus sequence of LKK-SE might
be proposed for a pause transfer signal by aligning these two sequences (Table 2).
Initially we truncated apoB RNA to yield apoB9
without a termination codon. Two sites in apoB9 (at 35 and 37 kDa from
the NH terminus) could adopt a transmembrane configuration.
When mapped, the 35-kDa transmembrane site, which is invariably
expressed in truncated apoB9, was 4 kDa upstream of the previously
proposed topogenic sequence (305-318), and so is presumably not
determined by it. Distinct transmembrane structures could also be
established in signal peptidyl cyclin by truncation at two separate
sites in the cyclin cDNA. Comparison of the transmembrane sequences in
apoB and cyclin failed to reveal primary sequence homology which if
present might suggest interaction with a membrane receptor (Table 2). Moreover, we observed that both the apoB and the
cyclin transmembrane sites exactly corresponded to ribosome pause
sites. Since these ribosome pause sites are expressed in the absence of
acceptor membranes, their production may not depend on the action of a
postulated membrane receptor.
We therefore propose that under the modified (15) conditions of in vitro translation, the nascent peptide is continuously translocated into microsomes until at certain points the ribosomes stall. If translation reinitiates, it can become uncoupled from translocation generating cytoplasmically exposed protein (Fig. 9). There is a precedent for the uncoupling of translation from translocation in wheat germ lysates, where the artifactual production of transmembrane structures has been ascribed to inefficient coupling between animal microsomes and plant ribosomes (46) . Coupling between rabbit ribosomes and canine microsomes is stronger and so reticulocyte translations are reported to be a better predictor of protein topology in intact cells than wheat germ translations(46) . However, we suggest that uncoupling can also occur during the reticulocyte translation of ``difficult'' regions of RNA where ribosome pausing occurs.
Figure 9:
Model for the generation of transmembrane
structures during in vitro translation. Ribosomes continue
along the mRNA and the protein product is co-translationally inserted
into microsomes (hatched). A site of ribosome pausing
() is reached. There is a variable efficiency of reinitiation
from the pause site, and subsequent translation may become uncoupled
from translocation so that protein synthesized after reinitiation is
cytoplasmic (unfilled). When aliquots of the translation
reactions are digested under conditions where reinitiation is efficient (left), the decrease in the full-length protein is accompanied
by a clear increase in the pause product. When reinitiation is
inefficient (right), then the decrease in the full-length
product is associated with only a modest proportional increase in the
pause product.
A striking feature of the data that formulated the pause transfer model of apoB translocation is that truncation at some, but not all, sites leads to transmembrane proteins. This was previously interpreted as suggesting restart sequences(15) . Alternatively, we suggest that it is differences in tRNA persistence on the peptide chains which determines whether a specific truncation yields a transmembrane protein. Esterified tRNA may stabilize transmembrane topology by steric interference with translocation. We do not understand why tRNA remains esterified to a greater proportion of certain peptide chains than to others.
As described previously (15) transmembrane apoB9
undergoes post-translational translocation after EDTA treatment,
showing that the protein is transmembrane in the translocation pore
rather than integrated into the bilayer. If the presence of tRNA
determines protein topology it might be predicted that puromycin alone
would induce translocation. Indeed the lack of effect of puromycin has
previously been used to support the presence of topogenic signals in
apoB and to discount steric effects of esterified tRNA(23) .
However, in our hands, puromycin did not displace tRNA from the apoB9
chains, presumably because the ribosomes had dissociated. It appears
that the extent of ribosome dissociation varies between different
reticulocyte lysates and/or transcripts. Thus persistence of tRNA but
not ribosomes on truncated globin peptides was observed by Minshull and
Hunt (47) , ()while continuing ribosome attachment
has been observed by other
workers(31, 48, 49, 50) . However,
ribosome persistence was not demonstrated in the previous study, and on
the contrary, it was reported that protein samples were treated to
hydrolyze esterified tRNA before electrophoresis despite prior
treatment with puromycin(15) .
While transmembrane structures were observed in apoB and cyclin when translated without termination codons, with termination codons the proteins appeared to be co-translationally translocated. To confirm that the ribosome pause transmembrane sequences we had mapped to the apoB5 region have no measurable effect on translocation under standard conditions of translation (i.e. from transcripts with stop codons), we engineered downstream glycosylation sites. Translocation intermediates were not detectable. Using standard criteria for translation experiments, we suggest that this region of apoB undergoes a completely normal process of translocation.
It was recently suggested that transmembrane topology near residues 86-99 (the B` region) is independent of ribosome pausing, because presynthesized chimeric apoB peptides became transmembrane when presented post-translationally to microsomes(23) . However, for post-translational translocation to occur in these experiments, a ribosome must be present on the peptide chain to allow targeting to the microsomal membrane(48) . The attached ribosome will occlude the final 30-40 amino acids at the COOH terminus of the polypeptide. Since the end of the chimeric apoB proteins was about this distance from the apoB moiety it might be argued that in the previous study (23) the chimeric protein was forced to become transmembrane in the apoB moiety by virtue of the attached ribosome. When we analyzed the B` region in reticulocyte lysate by engineering downstream reporter glycosylation sites we failed to see evidence of translocation intermediates. We also expressed the B` sequence in a chimeric apoB15.cyc construct in COS cells and found no evidence of altered translocation relative to a construct lacking the B` sequence. Shelness et al.(13) also noted that the B` region does not result in transmembrane topology in COS cells. However, these workers did not exclude the possibility of adjacent restart sequences. In agreement with Shelness et al.(13) we also did not find evidence that a failure of translocation accounts for the lack of apoB lipoprotein secretion from transiently transfected COS cells. Rather, in agreement with Gordon et al.(51) we find that absence of luminal apoB lipidation by microsomal triglyceride transfer protein account for the lack of apoB secretion from heterologous cells(52) .
Finally, it should be noted that the current studies relate to short-term expression of the amino terminus of apoB by translation or transfection. This seems a legitimate approach to determine whether, as previously proposed, discrete topogenic sequences interact with a putative membrane receptor to gate translocation. These results do not, however, exclude other translocation events which may give rise to incompletely translocated apoB in hepatocytes (6, 7, 9) or hepatoma cells (10, 11) and which may be selected for during stable expression in heterologous cells(12) .