©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Studies on the Translocation of the Amino Terminus of Apolipoprotein B into the Endoplasmic Reticulum (*)

(Received for publication, October 11, 1994; and in revised form, December 23, 1994)

Richard J. Pease (§) James M. Leiper (¶) Georgina B. Harrison James Scott

From the Medical Research Council Molecular Medicine Group, Royal Postgraduate Medical School, Du Cane Road, London W12 ONN, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Apolipoprotein B100 is co-translationally assembled into lipoprotein particles(1) . It is unresolved whether the nascent particle in the rough endoplasmic reticulum (ER) (^1)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.


EXPERIMENTAL PROCEDURES

Transcripts for in Vitro Translation

Transcripts encoding yeast alpha mating factor were obtained from Promega. cDNA encoding a type II membrane protein of MMTV type 7 (19) was a gift from Drs. A. Knight and J. Dyson, Medical Research Council Clinical Sciences Centre, London, United Kingdom. Transcription of RNA encoding apoB9 and apoB17 from plasmids EB9 and EB17 was described previously(17) . Plasmid EB17 was elongated with apoB cDNA (20) to encode apoB30 with a termination codon at XbaI(4194) and transcripts were prepared after linearization of the construct at a vector SalI site. ApoB9 transcripts lacking termination codons were generated by linearizing plasmid EB17 at the BglII(1412) site of the cDNA. In some cases apoB17 transcripts were truncated by hybridization with antisense oligonucleotides and enzymatic scission of the heteroduplex. Typically, 50 ng of oligonucleotide and 1 unit of RNase H (Promega) were incubated with 6 µl of transcription reaction for 20 min at 37 °C in 20 mM Tris, pH 7.4, 1 mM MgCl(2). Cleaved template was used without further treatment in translation reactions. The oligonucleotides used were: ALW, 5`-ATT TTG CTC AGA GAT GGT TAG-3`; ASU, 5`-GGT GCT CTC AAA TGC GAG GCC-3`; T432, 5`-A CAA GTG ACA CAG ACT T-3`.

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.

SXA.B`apoB

ApoB cDNA encoding residues Thr (71) to Ser (177) (i.e. the B` sequence (15) in its natural context) was amplified with mismatched oligonucleotides ACC CTG AAG GAT CCG TAT GGC TTC and CTG CCC GTC GAC TCA TTT AAA GGA TAT TTC TGT TGT CAC ATT to introduce a 5` BamHI site (underlined), and a 3` glycosylation site, termination codon, and SalI site (all underlined). This was subcloned into plasmid SXA.Bg digested BglII and SalI to yield plasmid SXA.B`apoB.

SXA.B`B`cyc

A double stranded oligonucleotide with a 5` BamHI site (underlined), with two tandem copies of the putative minimum core pause transfer sequence LKKTKNSEEFA(23) , and with a 3` SstII site (underlined) was created by annealing and filling in the following 3 single stranded oligonucleotides: (i) TCA ACA AAG GAT CCG CTG AAG AAG ACC AAG; (ii) CTG AAG AAG ACC AAG AAC TCC GAG GAG TTC GCA CTC AAA AAG ACG AAA AAT TCG GAA GAA TTC GCG GCG; (iii) CGC CCG CGG CGC GAA TTC TTC CGA ATT TTT C. The double stranded product was digested with SstII and was ligated to the 5` SstII site of an SstII digested cyclin cDNA fragment. The cyclin fragment was obtained by amplifying cyclin cDNA between residues 13 and 206 with the following oligonucleotides: TT GGT CCG CGG ATG GCC ATG GCT CAT GGT and CAC CGA ATG GTC GAC AAG TTA TCT GTC GAT CA. The 5`-oligonucleotide introduces an SstII site (underlined) and the 3`-oligonucleotide introduces a termination codon at residue Phe (207) and a SalI site (underlined). The core B` sequence-cyclin ligation product was digested with BamHI and SalI and subcloned into BglII and SalI digested plasmid SXA.Bg to encode SXA.B`B`cyc.

In Vitro Translation Reactions

Aliquots of transcription reactions (3 µl volume, 0.5 µg of RNA) were added to 30 µl of rabbit reticulocyte lysate (Promega), with 3 µl of canine pancreatic microsomes (Promega) and 20 µCi of [S]methionine (ICN). For protease protection assays, translation mixtures were adjusted to 25 mM Tris-HCl, pH 7.5, 2 mM tetracaine HCl, and incubated for 60 min at 0 °C with 400 µg/ml tosylphenyl chloromethyl ketone-treated trypsin (Worthington). Digests were inactivated with 100 µg of soybean trypsin inhibitor. ApoB post-translational translocation assays were as described(15) , except that MgCl(2) was substituted for CaCl(2). In some instances translation reactions were preincubated with 150 milliunits of potato apyrase (Sigma) for 30 min at 37 °C before post-translational translocation was induced. Translated products were resolved on SDS/Tris glycine (24) or SDS/Tris-Tricine (25) polyacrylamide gels.

Constructs for Cellular Expression by Transfection

The preparation of SV40 based apoB17 and apoB36 expression vectors has been described previously(8, 26) . Additionally, an apoB15.cyclin (B15.cyc) fusion construct in vector pSV7d (27) was made as follows. First an SstII site was created downstream of the natural HindIII(2279) site in the apoB17 cell expression plasmid (26) (yielding plasmid B17.HS). To achieve this, plasmid apoB17 was amplified with a mismatched 5`-oligonucleotide flanking the HindIII(2279) site to introduce an SstII site (underlined), and with a 3`-oligonucleotide complementary to the pSV7d polylinker sequence: GT CAA CAA AGC TTT CCG CGG GTT AAT GGT G and GAT CAT TAC TTA TCT AGG T. This product was digested HindIII-SalI and used to replace the existing HindIII(2279) to SalI (polylinker) fragment in apoB17, allowing cDNA to be fused at the introduced SstII site at the end of apoB15.

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).

Metabolic Labeling of Cultured Cells

COS I cells were maintained in Dulbecco's modified Eagle's medium (Flow), supplemented with 10% heat-inactivated fetal calf serum (Sigma), and were used at 70% confluency. Cells were transfected with apoB17, apoB36, or apoB15.cyc expression vectors (8, 26) as follows. Cells were trypsinized, then resuspended at between 3 times 10^6 and 10^7 cells/ml in 0.6 ml of phosphate-buffered saline with 25 µg of each plasmid. Cells were electroporated on a Bio-Rad gene pulser apparatus (960 microfarads at 250 V with 0.4-cm path length), then plated for 48-60 h prior to use.

For labeling, cells from 4 times 75 cm^2 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 (TranS-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.

Preparation of Membranes from Cultured Cells

Dispersed cells (in 6-8 ml of 0.25 M sucrose with 20 mM imidazole, pH 7.4) were homogenized with 15-20 passes through a ball bearing homogenizer(28) . The total homogenate (5 ml) was layered directly onto a discontinuous gradient of 1.0 ml of 0.5 M sucrose and 1.0 ml of 1.8 M sucrose (each with 20 mM imidazole) in Beckman Quickseal tubes, and centrifuged at 100,000 times g at 6 °C in an SW41 Ti rotor with adaptors. The 0.5-1.8 M sucrose membrane interface (containing rough ER, smooth ER, and Golgi membranes) was gently dispersed in 0.25 M sucrose with 2 passes in a 1-ml Dounce homogenizer. Membranes (typically 2.0 mg of protein in 0.8 ml final volume) were adjusted to 25 mM Tris-HCl, pH 7.5, and 2.0 mM tetracaine HCl (29) and then 200-µl aliquots were digested for 45 min at 25 °C with 50 µg of trypsin. Where indicated, saponin (Sigma) was added at the concentration shown in the text. Reactions were terminated with 200 µg of soybean trypsin inhibitor (Sigma). Membranes were collected by centrifugation in the TLA-100 rotor of a Beckman Optima TL ultracentrifuge and then dissolved in 1.5 ml of immunoprecipitation buffer with protease inhibitors (30) plus 100 µg of soybean trypsin inhibitor. Samples were immunoprecipitated with 20 µl of sheep antihuman apoB (Boehringer Mannheim) followed with 7 µg of protein A-Sepharose (Sigma) and the products resolved by SDS-polyacrylamide gel electrophoresis(24) .


RESULTS

Ribosome Pausing Generates Transmembrane Structures in ApoB and Cyclin

Transmembrane ApoB

ApoB9 (47 kDa) translated from RNA with a termination codon was fully translocated, as we previously observed (17) , suggesting co-translational translocation. In addition to full-length apoB9, minor bands of 35 and 37 kDa were observed (pause A and B, in Fig. 2A). ApoB9 translated without a termination codon showed increased pause products and an RNase-sensitive band of apoB9-tRNA. Proteolysis of these translation products reduced apoB9, with a concomitant increase in pause A. The increase in pause product A (Fig. 6, A and D) or both products A and B (Fig. 2B and Fig. 6B) upon cleavage of apoB9 is also apparent in subsequent experiments. Pause products A and B are observed when apoB9 is translated without microsomes, but their mobility is increased since they are unglycosylated (confirmed with a glycosylation inhibitor(31) , Fig. 2C). Taken together these results suggest that ribosome pausing may generate transmembrane structures independently of membrane function.


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 alpha 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.



Transmembrane Topology in a Cytosolic Protein

To investigate the consequences of ribosome pausing on membrane insertion in a cytosolic protein, we engineered a signal sequence onto cyclin. Full-length cyclin generated a 55-kDa glycosylated and signal cleaved protein which was resistant to protease digestion (Fig. 5A). Transcripts lacking termination codons were generated by cleavage of the cDNA at internal sites. The ScaI terminated protein was translocated, while the BamHI terminated protein became transmembrane at a ribosome pause site and showed marked persistence of peptidyl-tRNA. This site (pause II in Fig. 5A) was mapped by RNase H cleavage between residues 202 and 231 (not shown). In other experiments cyclin became transmembrane both at sites I and II, and additionally ribosome pausing also occurred at these same sites without microsomes (not shown). Cyclin became transmembrane at a second distinct pair of pause sites (III and IV) after truncation at the Csp45I (238) or the BglII (354) sites (Fig. 5B). Thus, truncations of cyclin mimic truncations in apoB. The resulting proteins can become transmembrane when the cDNAs are truncated at some but not all sites.


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.)



tRNA Persistence, Ribosome Pausing, and Transmembrane ApoB9

As described previously(15) , transmembrane apoB9 becomes post-translationally translocated after successive treatment with emetine, puromycin, EDTA, and metal ions, while an integral membrane protein from MMTV remained transmembrane (Fig. 6A). Subsequent experiments revealed that EDTA alone is necessary and sufficient to promote translocation and that there is no exogenous energy requirement to complete translocation (Table 1). RNase A also induces post-translational translocation, suggesting that either tRNA or ribosome persistence maintains the transmembrane state (Fig. 6B). As described below, tRNA persistence appears to be the determining factor.



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.

Co-translational Translocation of ApoB Residues 86-99 in Reticulocyte Lysates and Intact Cells

Analysis of a Second Putative Topogenic Region

Evidence has been presented that a distinct transmembrane sequence, B` residues 86-99, is present near the amino terminus of apoB(15, 23) . This sequence can be trapped in wheat germ lysates by translation of transcripts lacking termination codons(15) . More significantly, it also generates transmembrane structures in wheat germ lysates even from apoB15 transcripts having termination codons(14) .

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.

Analysis of ApoB Translocation in COS Cells

Since the B` sequence generates transmembrane structures in wheat germ lysates (14) but is co-translationally translocated in reticulocyte lysates, we examined its function by transfection to determine which lysate better reflects its behavior in intact cells. Construct B15.cyclin encodes a fusion between apoB15 and cyclin. In B15.B`B`cyc two tandem copies of the core B` sequence (23) are inserted at the fusion site (Fig. 1). Membranes were prepared and digested with trypsin. In three separate experiments there was no apparent difference in the protection of B15.cyclin and B15.B`B`cyc after proteolysis when corrected for the recoveries of apoB17 (co-transfected and used here as an internal control for vesicle integrity) (Fig. 7C). To verify the proteolysis conditions we cleaved an endogenous transmembrane protein, epidermal growth factor receptor (34, 35) (Fig. 7C). The above results support our translation data and suggest that the B` sequence has no measurable topogenic information in intact cells.

Topology of ApoB36 Transiently Expressed in COS Cells

It was previously reported that there is a specific block to apoB translocation in heterologous cells. Since apoB23 and smaller proteins are secreted from COS cells, while apoB29 and larger proteins cannot be secreted ((36) , and confirmed during these studies, not shown) this might imply topogenic sequences downstream from apoB23 and outside of the B15 region of the protein so far analyzed. We therefore examined the topology of transiently expressed apoB36 in COS cell microsomes, using co-transfection with apoB15.cyc as an internal control for vesicle stability. ApoB36 was fully protected against proteolysis relative to B15.cyc (Fig. 8A) and this was confirmed in three separate experiments. We also confirmed that this region of apoB can be fully translocated into the ER of heterologous cells by in vitro translation into canine pancreatic microsomes (Fig. 8B). ApoB30 was translocated to a protected form and there was no evidence of cleavage to transmembrane products which, if they were present, might suggest that topogenic sequences were acting.


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.




DISCUSSION

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 alpha helix and beta 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(2) 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) , (^2)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) .


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

Recipient of a Medical Research Council Research Studentship.

(^1)
The abbreviations used are: ER, endoplasmic reticulum; MMTV, mouse mammary tumour virus; apo, apolipoprotein; Tricine, N-2-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis.

(^2)
T. J. Hunt, personal communication.


ACKNOWLEDGEMENTS

We are very grateful for continuing advice and encouragement from Dr. Tim Hunt. We acknowledge the excellent craftsmanship of Graham Faulkner (Bioengineering, MRC Clinical Research Centre) who constructed the ball bearing homogenizer. We thank Drs. Shoumo Bhattacharya, Shailendra Patel, and Anne Soutar for advice. We thank Lesley Sargeant for excellent secretarial help.


REFERENCES

  1. Boren, J., Graham, L., Wettesten, M., Scott, J., White, A., and Olofsson, S.-O. (1992) J. Biol. Chem. 267, 9858-9867 [Abstract/Free Full Text]
  2. Rusinol, A. E., Verkade, H. J., and Vance, J. E. (1993) J. Biol. Chem. 268, 3555-3562 [Abstract/Free Full Text]
  3. Gibbons, G. F. (1990) Biochem. J. 268, 1-13 [Medline] [Order article via Infotrieve]
  4. Cartwright, I. J., and Higgins, J. A. (1992) Biochem. J. 285, 153-159 [Medline] [Order article via Infotrieve]
  5. Higgins, J. A. (1988) FEBS Lett. 232, 405-408 [CrossRef][Medline] [Order article via Infotrieve]
  6. Davis, R. A., Thrift, R. N., Wu, C. C., and Howell, K. E. (1990) J. Biol. Chem. 265, 10005-10011 [Abstract/Free Full Text]
  7. Dixon, J. L., Chattapadhyay, R., Huima, T., Redman, C. M., and Banerjee, D. (1992) J. Cell Biol. 117, 1161-1169 [Abstract]
  8. White, A. L., Graham, D. L., LeGros, J., Pease, R. J., and Scott, J. (1992) J. Biol. Chem. 267, 15657-15664 [Abstract/Free Full Text]
  9. Wilkinson, J., Higgins, J. A., Groot, P. H. E., Gherardi, E., and Bowyer, D. E. (1992) FEBS Lett. 304, 24-26 [CrossRef][Medline] [Order article via Infotrieve]
  10. Furukawa, S., Sakata, N., Ginsberg, H. N., and Dixon, J. L. (1992) J. Biol. Chem. 267, 22630-22638 [Abstract/Free Full Text]
  11. Du, E. Z., Kurth, J., Wang, S. L., Humiston P., and Davis, R. A. (1994) J. Biol. Chem. 269, 24169-24176 [Abstract/Free Full Text]
  12. Thrift, R. N., Drisko, J., Dueland, S., Trawick, J. D., and Davis, R. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9161-9165 [Abstract]
  13. Shelness, G. S., Morris-Rogers, K. C., and Ingram, M. F. (1994) J. Biol. Chem. 269, 9310-9318 [Abstract/Free Full Text]
  14. Chuck, S. L., Yao, Z., Blackhart, B. D., McCarthy, B., and Lingappa, V. R (1990) Nature 346, 382-385 [CrossRef][Medline] [Order article via Infotrieve]
  15. Chuck, S. L., and Lingappa, V. R. (1992) Cell 68, 9-21 [Medline] [Order article via Infotrieve]
  16. Dixon, J. L., and Ginsberg, H. N. (1993) J. Lipid Res. 34, 167-179 [Abstract]
  17. Pease, R. J., Harrison, G. B., and Scott, J. (1991) Nature 353, 448-450 [CrossRef][Medline] [Order article via Infotrieve]
  18. Pease, R. J., Harrison, G. B., Leiper, J. M., and Scott, J. (1992) Nature 356, 116-117
  19. Knight, A. M., Harrison, G. B., Pease, R. J., Robinson, P. J., and Dyson, P. J. (1992) Eur. J. Immunol. 22, 879-882 [Medline] [Order article via Infotrieve]
  20. Knott, T. J., Pease, R. J., Powell, L. M., Wallis, S. C., Rall, S. C., Jr., Innerarity, T. L., Blackhart, B., Taylor, W. H., Marcel, Y., Milne, R., Johnson, D., Fuller, M., Lusis, A. J., McCarthy, B. J., Mahley, R. W., Levy-Wilson, B., and Scott, J. (1986) Nature 323, 734-738 [Medline] [Order article via Infotrieve]
  21. Pines, J., and Hunt, T. (1987) EMBO J. 6, 2987-2995 [Abstract]
  22. Kaminski, A., Howell, M. T., and Jackson, R. J. (1990) EMBO J. 9, 3753-3759 [Abstract]
  23. Chuck, S. L., and Lingappa, V. R. (1993) J. Biol. Chem. 268, 22794-22801 [Abstract/Free Full Text]
  24. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  25. Schagger, H., and von Jagow, G. (1987) Anal. Biochem. 166, 368-379 [Medline] [Order article via Infotrieve]
  26. Graham, D. L., Knott, T. J., Jones, T. C., Pease, R. J., Pullinger, C. R., and Scott, J. (1991) Biochemistry 30, 5616-5621 [Medline] [Order article via Infotrieve]
  27. Truett, M. A., Blacher, R., Burke, R. L., Caput, D., Chu, C., Dina, D., Hartog, K., Kuo, C. H., Masiarz, F. R., Merryweather, J. P., Najarian, R., Pachl, C., Potter, S. J., Puma, J., Quiroga, M., Rall, L. B., Randolph, A., Urdea, M. S., Valenzuela, P., Dahl, H. H., Favalaro, J., Hansen, J., Nordfang, O., and Ezban, M. (1985) DNA (N. Y.) 4, 333-349 [Medline] [Order article via Infotrieve]
  28. Balch, W. E., and Rothman, J. E. (1985) Arch. Biochem. Biophys. 240, 413-425 [Medline] [Order article via Infotrieve]
  29. Scheele, G. (1983) Methods Enzymol. 96, 94-111 [Medline] [Order article via Infotrieve]
  30. Cardin, A. D., Witt, K. R., Chao, J., Margolis, H. S., Donaldson, V. H., and Jackson, R. L. (1984) J. Biol. Chem. 259, 8522-8528 [Abstract/Free Full Text]
  31. Kassenbrock, C. K., Garcia, P. D., Walter, P., and Kelly, R. B. (1988) Nature 333, 90-93 [CrossRef][Medline] [Order article via Infotrieve]
  32. Waters, M. G., Chirico, W. J., and Blobel, G. (1986) J. Cell Biol. 103, 2629-2636 [Abstract]
  33. Mueckler, M., and Lodish, H. F. (1986) Nature 322, 549-552 [Medline] [Order article via Infotrieve]
  34. Renfrew, C. A., and Hubbard, A. L. (1991) J. Biol. Chem. 266, 21265-21273 [Abstract/Free Full Text]
  35. Waterfield, M. D., Mayes, L. V., Stroobant, P., Bennet, P. L. P., Young, S., Goodfellow, P. N., Banting, G. S., and Ozanne, B. (1982) J. Cell. Biochem. 20, 149-161 [Medline] [Order article via Infotrieve]
  36. Yao, Z., Blackhart, B. D., Linton, M. F., Taylor, S. M., Young, S. G., and McCarthy, B. J. (1991) J. Biol. Chem. 266, 3300-3308 [Abstract/Free Full Text]
  37. Jennings, M. L. (1989) Annu. Rev. Biochem. 58, 999-1027 [CrossRef][Medline] [Order article via Infotrieve]
  38. Marchesi, V. T., Furthmayr, H., and Tomita, M. (1976) Annu. Rev. Biochem. 45, 667-698 [CrossRef][Medline] [Order article via Infotrieve]
  39. Ross, A. H., Radhakrishnan, R., Robson, R. J., and Khorana, H. G. (1982) J. Biol. Chem. 257, 4152-4161 [Abstract/Free Full Text]
  40. Bonifacino, J. S., Cosson, P., and Klausner, R. D. (1990) Cell 63, 503-513 [Medline] [Order article via Infotrieve]
  41. Boyd, D., and Beckwith, J. (1990) Cell 62, 1031-1033 [Medline] [Order article via Infotrieve]
  42. von Heijne, G. (1989) Nature 341, 456-458 [CrossRef][Medline] [Order article via Infotrieve]
  43. Yost, C. S., Lopez, C. D., Prusiner, S. B., Myers, R. M., and Lingappa, V. R. (1990) Nature 343, 669-672 [CrossRef][Medline] [Order article via Infotrieve]
  44. Yang, C.-Y., Gu, Z.-W., Weng, S.-A., Kim, T. W., Chen, S.-H., Pownall, H. J., Sharp, P. M., Liu, S.-W., Li, W.-H., Gotto, A. M., Jr., and Chan, L. (1989) Arteriosclerosis 9, 96-108 [Abstract]
  45. Yang, C.-Y., Chen, S.-H., Gianturco, S. H., Bradley, W. A., Sparrow, J. T., Tanimura, M., Li, W.-H., Sparrow, D. A., DeLoof, H., Rosseneu, M., Lee, F.-S., Gu, Z.-W., Gotto, A. M., Jr., and Chan, L. (1986) Nature 323, 738-742 [Medline] [Order article via Infotrieve]
  46. Spiess, M., Handschin, C., and Baker, K. P. (1989) J. Biol. Chem. 264, 19117-19124 [Abstract/Free Full Text]
  47. Minshull, J. (1989) Cyclins in Xenopus laevis , Ph.D. Thesis, University of Cambridge, United Kingdom
  48. Perara, E., Rothman, R. E., and Lingappa, V. R. (1986) Science 232, 348-352 [Medline] [Order article via Infotrieve]
  49. Roitsch, T., and Lehle, L. (1988) Eur. J. Biochem. 174, 699-705 [Abstract]
  50. Caulfield, M. P., Duong, L. T., and Rosenblatt, M. (1986) J. Biol. Chem. 261, 10953-10956 [Abstract/Free Full Text]
  51. Gordon, D. A., Jamil, H., Sharp, D., Mullaney, D., Yao, Z., Gregg, R. E., and Wetterau, J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7628-7632 [Abstract]
  52. Leiper, J. M., Bayliss, J., Pease, R. J., Brett, D. J., Scott, J., and Shoulders, C. C. (1994) J. Biol. Chem. 269, 21951-21954 [Abstract/Free Full Text]

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