Cell, Vol. 66, 9-21,
10, 1992, Copyright
0 1992 by Cell Press
Pause Transfer: A Topogenic Sequence in Apolipoprotein B Mediates Stopping and Restarting of Translocation Steven L. Chuck’t and Vishwanath R. Lingappats *Department of Microbiology and Immunology tDepartment of Medicine *Department of Physiology University of California, San Francisco San Francisco, California 94143-0444
Summary Previously, we described the stepwise translocation of a large amino-terminal fragment of apolipoprotein B (apo 815) in which the nascent secretory chain translocates through a series of distinct, nonintegrated transmembrane intermediates with large domains exposed to the cytoplasm. Thus, apo 815 appears to stop and restart translocation at several points. We have identified a sequence of amino acids in apo B15 that confers this behavior on a heterologous chimeric protein. In addition, we dissect pausing into two distinct steps, stopping and restarting, thereby trapping otherwise transient intermediates. Finally, we demonstrate the function of a second “pause transfer” sequence over 200 amino acids downstream in apo B15 that restarts translocation posttranslationally, suggesting that multiple pause transfer sequences are involved in the biogenesis of apolipoprotein B. Introduction Since nascent secretory proteins are generally translocated into the lumen of the endoplasmic reticulum (ER) cotranslationally, large domains are not usually exposed to the cytoplasm once translocation has begun (Blobel and Dobberstein, 1975). However, apolipoprotein B (apo B) is one exception. This large protein (molecular mass over 500,000 daltons) contains an amino-terminal signal sequence and is secreted from human cells in single copy on lipoprotein particles including very low density lipoproteins and low density lipoproteins (Young, 1990). apo B forms a backbone to which other apoproteins bind and is a ligand for the low density lipoprotein receptor (Brown and Goldstein, 1986). It is an extremely hydrophobic molecule; when chemically separated from lipid, it precipitates in aqueous solutions (Fisher and Schumaker, 1966). Despite this overall [email protected]
, apo B contains only short uninterrupted stretches of hydrophobic amino acids, none of which are predicted to be membrane-spanning domains (Knott et al., 1986; Yang et al., 1966). Previously, we observed that translocation of the amino-terminal 717 residues of apo B (a 70 kd protein, termed apo B15), in sharp contrast to the translocation of typical secretory proteins, generates a series of distinct transmembrane forms with large carboxy-terminal domains exposed to the cytosol. Upon digestion of nascent apo 815 synthesized in vitro with proteinase K, several smaller amino-terminal lengths
are protected (Chuck et al., 1990). Over time, progressively longer amino-terminal domains become protected from protease until the chain becomes fully protected, indicating that these transmembrane forms are sequential translocational intermediates. This stepwise translocation can occur at least in part posttranslationally. Therefore, at several points during the biogenesis of apo B15, translocation of the nascent secretory chain appears to stop and then restart. Despite spanning the membrane, however, the nascent chains of apo 815 are readily extracted by sodium carbonate at pH 11.5. Hence, unlike conventional membrane proteins, these transmembrane forms are not integrated in the bilayer, consistent with the ideathat these transmembrane forms are transient translocational intermediates in an aqueous protein- conducting channel (Gilmore and Blobel, 1965; Simon and Blobel, 1991; Lingappa, 1991). One hypothesis to explain these unusual findings is that multiple specific sequences of amino acids throughout apo 815 may direct the chain to stop and then restart translocation across the ER membrane. In a manner similar to signal and stop transfer sequences (Lingappa et al., 1984; Yost et al., 1983) such a sequence might confer its functional characteristics when engineered into the coding region of a heterologous chimeric protein that lacks this translocational behavior. The function of such a topogenic sequence should be marked by three cardinal features: it should stop translocation and leave an intermediate that spans the membrane of the ER; the transmembrane intermediate should be extractable from the membrane with sodium carbonate at pH 11.5; and translocation should subsequently restart. Here we identify such a “pause transfer” sequence of 33 amino acids within apo B15 and demonstrate that this sequence confers this translocational behavioron a heterologouschimeric protein. In addition, we dissociate the stopping and restarting functions directed by this sequence. Finally, we provide evidence that the biogenesis of apo B involves the sequential action of multiple pause transfer sequences by demonstrating the function of a second pause transfer sequence, over 200 amino acids downstream of the first sequence, that restarts translocation posttranslationally in its native context. These results have implications for the function of the ER in the assembly of complex secretory proteins. Results Localization of 33 Amino Acids in apo 815 That Mediate Transmembrane Topology To localize a topogenic sequence mediating a pause in translocation, we truncated plasmid DNA encoding apo B15 downstream of the SP6 promoter (Figure 1A) and studied the topology of progressively shorter proteins expressed by transcription with SP6 RNA polymerase followed by translation using a cell-free wheat germ lysate supplemented with canine pancreas microsomes. When the plasmid is truncated at the first BsmAl site (corre-
Bsu $6 Bsm
1 Nco Bgl 2
Apo B15 truncations
Lane lvlbs PK Det
1 2 +--
+ + +
+ + _
11 + _
12 + + +
Figure 1. Truncations of apo 815 (A) Map of truncations of apo 815. The black bar depicts apo 815, a mature protein of 697 amino acids (its signal sequence of 30 amino acids is shown as a white box). The diagram shows the number of amino acids (top) corresponding to the restriction sites (bottom) used to create the proteins in this paper. (B) Truncations of apo 815 at Bsu98 and BsmAl. The piasmid DNA encoding apo 815 was digested with Bsu99 (lanes l-5) or BsmAi (lanes&9,12) and expressed in vitro. At 90 min, aiiquotsof the translation reactions were proteoiyzed and then visualized by SDS-PAGE and autoradiography as described in Experimental Procedures. An aiiquot of apo 815 linearized after the termination codon (lane 11) was proteoiyxed to generate a predominanceof transmembranefragments (lane 10) for comparison with the two truncations. Molecular mass markers are shown to the left of lane 1 (see note in Experimental Procedures). The three precursors are identified by downward pointing arrowheads in lanes 2,7, and 11. In lane 4, the fully protected signaicleaved species of apo B truncated at Bsu99 is identified wfth an upward pointing arrowhead. The smallest protected fragments generated upon proteoiysis of apo B truncated at BsmAl and apo B15 are identified in lanes 9 and 10 with upward pointing arrowheads (lanes 10 and 11 are from a lighter exposure than lanes l-9 and 12 but are shown in their exact position on the original gel). The translations of apo B truncated at Bsu39 were treated with emetine at 90 min and puromycin at 70 min to allow complete signal cleavage of the translocated proteins. Mbs, membranes; PK, proteinase K; Det, detergent.
sponding to the amino-terminal 137 amino acids of mature apo B), a short protein is synthesized (Figure 1, lanes 6-9, 12). Some of these chains are transmembrane with a fragment protected from proteolysis (upward pointing arrowhead, lane 9) that comigrates with the smallest amino-terminal fragment corresponding to a transmembrane intermediate of apo B15 (upward pointing arrowhead, lane 10). These small transmembrane fragments
(lanes 9 and 10) are recognized by antipeptide antibody directed against residues 12-27 of mature apo B (data not shown). On the other hand, the mature protein synthesized when the plasmid is truncated at the first Bsu36 site (corresponding to amino acid 77 of mature apo 6) is slightly smaller than the smallest amino-terminal fragment corresponding to a transmembrane intermediate of apo 815 (compare lane 4 with lanes 9 and 10). This protein appears completely protected from proteinase K in the absence of detergent (lane 4), as expected for a secretory protein. These data suggest that a sequence necessary for the transient transmembrane topology of a translocational intermediate of apo B15 exists in the 60 amino acid residues between the BsmAl and Bsu36 sites. To localize this region more precisely, we constructed chimeric proteins in which a passenger domain from chimpanzee alpha globin with an engineered glycosylation site (termed glycoglobin; see Experimental Procedures) was fused downstream of apo B at glutamatem (at the first Bsu36 site) and after valineIII. The topology of the first construct appeared wholly secretory, whereas some chains of the second fusion protein were transmembrane (data not shown). These results suggest that the intervening 33 amino acid residues that differentiate the two chimerit proteins (termed the B’ region) may mediate transient transmembrane topology. A Truncated Chimeric Protein Confirms the Identity of a Pause Transfer Sequence and Dissociates Stopping from Restarting The demonstration of a pause transfer sequence presents two problems. First, how can one detect the transmembrane intermediates, given theirtransient nature? Second, how can one distinguish such a sequence from an inefficient stop transfer sequence or from a region that is simply translocated slowly? One solution to both problems is to find conditions under which stopping is dissociated from restarting. When the chain is stopped under these conditions, otherwise transient intermediates would be detected, and a pause would be differentiated from inefficient stop transfer or slow translocation. We discovered that truncation of the coding region of a heterologous chimeric protein containing an insert of the codons for the B’sequence fulfilled these experimental conditions. We engineered an SPG-based plasmid encoding the signal sequence of bovine prolactin, beta lactamase, the B’ region of 33 amino acids, and glycoglobin (SLB’gG). The plasmid DNA was cleaved at the BamHl site 24 amino acids into glycoglobin (immediately after the onlyglycosylation site) and expressed in vitro with truncated secretory (SLgGIBam) and integral membrane chimeric proteins (SLSTgGIBam, which contains a stop transfer sequence) encoding identical amino- and carboxy-terminal passenger domains. Transcripts synthesized in vitro from these truncated plasmids lack termination codons and generate proteins in which the ribosome remains attached to the carboxyl terminus with the last approximately 40 amino acids yet to emerge from the ribosome (Blobel and Sabatini, 1970; Perara et al., 1966). Treatment with puromycin releases truncated proteins from their ribosomes (Traut
and Monro, 1964; Pestka, 1971). In the case of secretory proteins, the puromycin-released chains are then translocated into the ER lumen (Redman and Sabatini, 1966; Siuta-Mangano and Lane, 1961). Puromycin treatment alone does not remove the ribosome from the ER membrane, and it leaves the translocational channel open (Simon and Blobel, 1991). In contrast, treatment with EDTA substantially disassembles the ribosome (Sabatini et al., 1966) and treatment with high concentrations of potassium after puromycin strips ribosomes from the ER membrane (Adelman et al., 1973). We hypothesized that by truncating SLB’gG at BamHI, the entire B’ region can be translated, but only an initial portion of it would be exposed from the attached ribosome and thereby able to interact with the ER membrane and its machinery; the remainder of the B’ sequence and the 24 amino acids of glycoglobin would be covered by the ribosome. By adding puromycin to detach the truncated chain from the ribosome without removing the ribosome from the membrane, the truncated B’ chimera would be free to slip into the ER lumen unless translocation was stopped. In a second step, by stripping off the ribosome with either EDTAor potassium, the full B’sequencewould be exposed to the membrane, and translocation may restart. In contrast, the chimera lacking the B’sequence should not stop translocation, whereas the chimera containing a stop transfer sequence should not restart. This logic formed the basis for our experimental approach. Using the three truncated chimeric proteins, we first showed that the chimera encoding the B’sequence stops translocation, in contrast to the chimera lacking the B’ insert. The three truncated constructs were expressed in vitro in the presence of microsomal vesicles and treated with puromycin to release the nascent chains from their ribosomes. Transcription and translation to the truncation point was verified by glycosylation of SLgG and recognition of all three proteins by antipeptide sera directed against amino acids 7-24 of glycoglobin (data not shown). Subsequently, a tripeptide competitor of glycosylation was included cotranslationally to simplify the pattern of bands. Proteolysis revealed that the truncated secretory chimera (SLgGIBam) is fully protected (Figure 2A, lane 4; Figure 3) whereas the truncated integral membrane chimera (SLSTgGIBam) is cleaved to yield a single fragment recognized by antibody to lactamase but not antipeptide sera directed against glycoglobin (Figure 2C, lane 4; Figure 3). The majority of the truncated B’chimera (SLB’gGIBam) is also transmembrane (Figure 2B, lane 4), and immunoprecipitation confirmed that the carboxy-terminal domain was digested (Figure 3). These results suggest that the B’sequence has stopped translocation of the truncated B’ chimera efficiently, since the majority of chains stop. Moreover, the truncated B’chimera has truly stopped and is not simply translocated slowly, since over 90% of the transmembrane chains remain stopped 40 min after the addition of puromycin (data not shown). In a second step, translocation of the transmembrane chains of the truncated 8’ chimera restarts. Following the logic described earlier, we then treated the puromycinreleased transmembrane chains with either EDTA or 540
mM potassium acetate. The topology of the secretory and integral membrane controls did not change after treatment with EDTA (Figures 2A and 2C, lane 7). However, the truncated B’ chimera became fully protected from proteinase K after either of these two treatments (Figure 2B, lanes 7 and 10). In a second step, therefore, translocation of the truncated B’ chimera is restarted efficiently. Thus, stopping and restarting are distinct stages of a pause in translocation as evidenced by their dissociation; the transmembrane chains of the truncated B’ chimera remain stopped until treated with either EDTA or potassium. Similar experiments were performed with the codons for a region of apo B at which a pause is not detected (just upstream of the Bsu36 site) inserted into the identical chimeric context. The behavior of this chimeric protein under these experimental conditions was indistinguishable from the chimera lacking an insert (data not shown). This result further confirms the specificity of the B’ pause transfer sequence. Given the transient nature of the transmembrane intermediates directed by the B’sequence in its native context, we reasoned that truncation of these chimeric proteins at the BstEll site (92 codons downstream of the BamHl site) might allow the B’ sequence to restart translocation. Indeed, the chains of SLB’gGIBstEII were fully protected from digestion with proteinase K even in the absence of treatment with EDTA or high concentrations of potassium to restart translocation (Figure 4, lanes 6-l 0), suggesting that these proteinsstopped and then restarted. The control proteins truncated at BstEll had the expected topologies: thesecretorycontrol (SLgGIBstEII) isfullyprotected (lanes l-5), and the integral membrane construct (SLSTgGI BstEll) is transmembrane (lanes 11-15). The Transmembrane Intermediate Is Not Integrated in the Bilayer We next demonstrated that the transmembrane intermediate of SLB’gGIBam is not integrated in the bilayer. SLB’gGI Barn, the secretory control (SLgGIBam), and the integral membrane control (SLSTgGIBam) were synthesized separately in vitro in the presence of microsomal membranes. Aliquots were mixed, and the pooled sample was divided in half and treated with either isotonic sucrose (pH 7.5) or 0.1 M sodium carbonate (pH 11.5) (Fujiki et al., 1962). After ultracentrifugation, the supernates were separated from the pelleted membranes. As expected, the two control proteins are found in the intact vesicles in the sample treated with Tris-buffered sucrose (Figure 5, arrowheads A and C, lane 1). One third of SLB’gGIBam, however, is found in the supernatant (arrowhead B, lane 2). After treatment with sodium carbonate, the truncated secretory chimera is found in the supernatant (position of arrowhead A, compare lanes 3 and 4) whereas the integral membrane protein is found with the pelleted membranes (position of arrowhead C, compare lanes 3 and 4). Despite spanning the membrane, however, SLB’gGIBam is not integrated in the bilayer. The protein is extracted in amounts comparable to the truncated secretory control (position of arrowhead B, compare lanes 3 and 4). Thus, the B’sequence confers onto an otherwise secretory heterologous chimera the
1 2 3 - - +
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C 20092 684530-
Lane Mbs Et
Figure 2. Chimeric Proteins Codons into Glycoglobin)
Plasmids encoding SLgG, SLSTgG, and SLB’gG were digested with BamHl and expressed in vitro in the presence of a tripeptide competitor of N-linked glycosyfation. Emetine was added to each reaction at 40
min, followed by puromycin at 50 min. At 70 min, aliquots were proteolyzed, and the remainder was treated with either EDTA or potassium acetate. At 90 min, the samples treated with EDTA or potassium acetate were proteolyzed. Aliquots were analyzed by SDS-PAGE and autoradiography. Molecular mass markers are shown to the left of lane 1 in all panels. The downward pointing arrowheads (lane 2) identify the uncleaved precursors of the three chimeric proteins synthesized in the absence of vesicles. The smaller upward pointing arrowheads (lane 4) identify the major product of proteolysis in the presence of rough microsomes prior to treatment with EDTA or potassium acetate. The diagram below each gel depicts our interpretation of the topology after treatment with emetine and puromycin (left of arrows) and after treatment with EDTA or potassium acetate (right of arrows). The large and small subunits of the ribosome are shown as two shaded gray ovals. Letters denote the residues corresponding to lactamase (L), the B’(B’) or stop transfer (ST) sequences, and glycoglobin (G); dashed lines indicate residues within the ribosome. Although the Bland ST regions are labeled inside the vesicles (black ovals), portions of each sequence may span the membrane. (A) SLgGlBam, the secretory control. After treatment with puromycin, the protein is detached from the ribosome and translocated into the lumen of the vesicle (left of arrow). Upon treatment with EDTA, the ribosome is removed, and the translocational channel closes without altering the topology of the fully translocated protein (right of arrow). (B) SLB’gGIBam. Despite treatment with puromycin. the protein remains stopped within the translocational channel, and the altered ribssome-membrane junction (see Discussion) permits digestion of the carboxyl terminus (left of arrow). After treatment with EDTA or potassium, translocation restarts, and the protein is found entirely within the lumen of the vesicles (right of arrow). In addition, upon treatment with EDTA, some of the restarted chains are glycosytated, confirming expression and translocation of the entire protein (data not shown). (C) SLSTgGilBam. the integral membrane control. Treatment with puromycin (left of arrow) followed by EDTA (right of arrow) does not change the transmembrane topology of this protein that is integrated into the lipid bilayer.
30 - .B.
1 2 34 L TP G N SLgGlBam
5 6 70 L TP G N SLB’gG/Bam
of the Three
Lane Mbs PK Det
9 10 1112 L TP G N SLSTgG/Bam
1 2 3 4 + + +--+++--+++--++ -
11 12 13 - +
cated at BamHl
Figure 4. Topology of the Three Chimeric Proteins coglobin at BstEll (116 Codons into Glycoglobin)
Proteolyzed translation products of the three truncated chimeric proteins in Figure 2 (samples from lane 4 in each panel) were immunoprecipitated with antibody against lactamase (L), glycoglobin (G) or nonimmune sera (N) and visualized by autoradiography. A smaller aliquot of proteolyzed translation products is shown adjacent to the immunoprecipitated samples.
Plasmids encoding SLgG, SLB’gG, BstEll and expressed in vitro. Aliquots treated with emetine and puromycin mass markers are shown to the left (SLgGIBstEll) is shown in lanes l-5, the integral transmembrane protein
three cardinal features that distinguish the translocation of apo 615 from that of either conventional secretory or integral transmembrane proteins. In addition, the samples treated with Tris-buffered sucrose provide evidence that SLB’gGIBam stops and restarts translocation independent of the proteolytic assay. By densitometry, one third of the signal-cleaved SLB’gGI Barn is found in the supernatant of the sample diluted in neutral isotonic buffer (arrowhead B, compare lanes 1 and 2). In contrast, the identical dilution and centrifugation of SLB’gGIBam treated with EDTA after puromycin resulted in nearly all of the nascent chains localizing in the membrane pellet (position of arrowhead 8, compare lanes 5 and 6). These data suggest that chains of SLB’gGIBam are initially associated with the membrane in a way in which they can be released into the cytosolic fraction during ultracentrifugation, but upon treatment with EDTA are translocated entirely into the lumen of the vesicle. This change in topology upon treatment with EDTA contrasts with that of the secretorychimeric protein (SLgGIBam) and the integral membrane chimera (SLSTgGIBam). Taken together, these results indicate that the chains of SLB’gGIBam span the ER membrane in an environment that is subject to complete disruption by high pH and in part by centrifugation alone. We speculate that the paused proteins reside in the aqueous, proteinaceous channel across the bilayer through which they are being translocated (Gilmore and Blobel, 1965; Simon and Blobel, 1991). Pause Transfer Sequences Appear to Exist at More Than One Point in apo 615 The translocation of apo B15 is marked by a series of distinct
terminal lengths protected from proteinase K(Chuck et al., 1990). Such a stepwise process of translocation could be explained if pause transfer sequences occurred through-
and SLSTgG were digested with of the translation reactions were exactly as in Figure 2. Molecular of lane 1. The secretory chimera SLB’gGlBstEll in lanes 6-l 0, and (SLSTgGIBstEII) in lanes 11-15.
200 664530 c,
1234 P s Tris
(EDTA) Figure 5. Carbonate cated at BamHl
of the Three
SLgGIBam, SLB’gGIBam, and SLSTgG/Bam were translated independently in the presence of microsomal vesicles and treated with emetine then puromycin as in Figure 2. At 70 min, aliquots of the three truncated proteins were mixed. The pooled sample was divided in half and treated with Tris-buffered isotonic sucrose (pH 7.5) (Tris) or 0.1 M sodium carbonate (pH 11.5) (Carb). At 70 min, other aliquots were incubated with EDTA, and at 90 min these samples were treated with Tris-buffered isotonic sucrose (pH 7.5) (Tris (EDTA)]. Following ultracentrifugation, the supernates (S) and pellets(P) were separated, and the proteins were visualized by SDS-PAGE and autoradiography. Letters mark the bands corresponding to signalcleaved SLgGlBam (A), SLB’gGIBam (B), and SLSTgGlBam (C).
out the protein. We identified a second region in apo 815 where stopping and restarting of translocation occurs as demonstrated in its native context rather than in achimeric protein. The coding region for apo 815 was truncated at
Lane Mbs-PK Det
3 4 + + +--4-+--k+--++ - - _
_ + (high K+)
Figure 6. apo 815 Truncated at Stul Plaamid DNA encoding apo B15 was digested with Stul and expressed in vitro. Emetine was added to the translation reactions at 66 min, followed by puromycin at 70 min to allow the chains to translocate as completely as possible. At 60 mitt, aliquots were proteolyzed immediately (lanes l-5) or treated with EDTA (lanes 6-6) or potassium acetate (lanes 3-I 1) for an additional 20 min before being proteolyzed. Samples were visualized by SDS-PAGE and autoradiography. The downward pointing arrowhead in lane 2 marks the precursor, the downward pointing tailed arrow in lane 3 identifies the glycosylated mature protein, and the small upward pointing arrowhead in lane 4 indicates the proteolyzed transmembrane fragment that comigrates with a transmembrane fragment in apo 815 (data not shown).
the first Stul site (see Figure lA), expressed in vitro, and the mature protein of over 300 amino acids was released from the ribosome by puromycin. Over half of these chains are transmembrane by proteolysis (Figure 6, lane 4) generating a large amino-terminal fragment that comigrates with one of the transmembrane bands in apo 615 (data not shown). These chains also become fully translocated following treatment with either EDTA or potassium. These results imply the existence of a second pause transfer sequence upstream of the first Stul site. Note that the lack of predominance of the shorter transmembrane fragments (which are seen in apo 815 when translation is ongoing) is due to the experimental protocol; by incubating with emetine and puromycin for a total of 30 min (see legend to Figure 6), translation is stopped, and the synthesized chains of apo B truncated at Stul have a “translocational chase period” in which they become as fully translocated as possible in the presence of the attached ribosome, emphasizing that the remaining transmembrane intermediates appear trapped. In addition, although over half of chains truncated at Stul are transmembrane, many are fully protected from proteases. These chains may have paused but restarted translocation if their ribosomes detached from the membrane. Alternatively, these proteins may not have stopped at the upstream pause transfer sequence because other undefined factors might regulate stopping at these sequences.
Restarting Translocatlon Is Affected by Context and Can Occur Posttranslatlonally In the chimeric context of SLB’gG, the B’ pause transfer sequence confers the three essential functions predicted of such a topogenic sequence. However, our studies on apo B15 also suggested that some or all pause transfer sequences could stay stopped for sufficiently long periods of time for restarting to be detected posttranslationally (Chuck et al., 1990). apo B should therefore remain stopped at some pause transfer sequences even after their complete emergence from the ribosome, implying that factors other than just exposure of a pause transfer sequence, such as flanking sequences, influence restarting of translocation. Indeed, truncations of apo 815 downstream of the Stul site reveal that the pause transfer sequence near Stul can remain stopped well after exposure of the sequence and restarts posttranslationally. When the coding region for apo B is truncated at the first Bglll site (73 codons downstream from the Stul site), over half of the nascent proteins are still transmembrane (Figure 7A, lane 4). The transmembrane fragments comigrate with the transmembrane fragments generated by proteolysis of the protein encoded by truncation at Stul and are likewise amino-terminal fragments (data not shown). These transmembrane forms also become fully protected from proteinase K after treatment with either EDTA or potassium (Figure 7A, lanes 7 and 10). The transmembrane intermediates are not simply translocated slowly, since over 90% remain stopped for over 2 hr, and yet they become fully translocated after treatment with EDTA (data not shown). This stopping and restarting of translocation in apo B is observed to the same extent in rabbit reticulocyte lysates as in wheat germ lysates (Figure 78) and is equally well detected with trypsin after stabilization of microsomes with tetracaine (Figure 7C). These results indicate that pause transfer occurs in a mammalian system and is not an artifact of proteolysis. Again, the lack of apparent shorter transmembrane bands results from the translocational chase period while the ribosome is still attached (see above). Furthermore, translocation can restart posttranslationally, since the stopped chains of apo B truncated at Bglll restart long after they are synthesized (Figure 7). In apo B, this pause transfer sequence near the Stul site restarts translocation efficiently after synthesis of domains downstream of 89111. Truncation of the plasmid encoding apo 815 at the first Ncol site (66 codons beyond the first Bglll site) yields a protein that is now fully protected from proteinase K under the same conditions as Figure 6 and Figure 7 (unpublished data). This suggests that tram&cation of this longer protein restarts efficiently after stopping near the Stul site; the transmembrane intermediate seen when apo B is truncated at Stul and Bglll is no longer trapped. Yet, the information to trigger restarting is in the pause transfer sequence upstream of the Stul site and not between the Bglll and Ncol sites. First, the transmembrane chains truncated at either the Stul or Bglll sites become fully protected from proteolysis upon addition of EDTA or high concentrations of potassium (Figure 6; Figure 7). Second, when synthesized under the same protocol as
Pause Transfer 15
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the Stul and Bglll truncations to allow for translocation to proceed as completely as possible, a fusion protein encoding a domain of prolactin downstream of apo Bat the Stul site (apo B [Stull-Pt) is fully protected from proteolysis (unpublished data), suggesting that this fusion protein restarts translocation efficiently after stopping near the Stul site, in a manner analogous to SLB’gG truncated at BstEll. Therefore, no specific information in apo B beyond Stul must be necessary for restarting. So, it appears that the
2345678 + + --+,‘++ _
7. Restarting Translocation Posttranslationally
+ (EDTA) by Context
(A) apo 815 truncated at Bglll expressed in wheat germ lysate. Plasmid DNA encoding apo 815 was digested with Bglll, expressed in vitro, and treated with emetine and puromycin, followed by immediate proteolysis (lanes l-5). treatment with EDTA (lanes 5-9), or treatment with potassium acetate (lanes 9-11) exactly as in Figure 6. The downward pointing arrowhead in lane 2 marks the precursor, the downward pointing tailed arrow in lane 3 identifies the glycosylated mature protein, and the small sideways arrowhead indicates the major proteolyzed transmembrane fragment in lane 4. (B) apo 815 truncated at Bglll expressed in rabbit reticulocyte lysate and digested with proteinase K. Plasmid DNA encoding apo 815 was digested with Bglll, expressed in vitro, and treated with emetine and puromycin, with or without EDTA or potassium acetate exactly as in (A). Aliquots of the samples were treated with calcium chloride prior to digestion with proteinase K exactly as in (A). The downward pointing arrowhead in lane 2 marks the precursor, the downward pointing tailed arrow in lane 3 identifies the glycosylated mature protein, and the small sideways arrowhead indicates the major proteolyzed transmembrane fragment in lane 4. (C) apo 815 truncated at Bglll expressed in rabbit reticulocyte lysate and digested with trypsin. Aliquots of the samples from the experiment in (B) were treated with tetracaine rather than calcium chloride and digested with trypsin. All other procedures were identical. The downward pointing arrowhead in lane 2 marks the precursor, the downward pointing tailed arrow in lane 3 identifies the glycosylated mature protein, and the small sideways arrowhead indicates the major protaolyzed transmembrane fragment in lane 4. The size of the transmembrane fragments generated by proteolysis with proteinase K and trypsin vary slightly as expected given the difference in cleavage sites.
pause transfer sequence near the Stul site restarts translocation, but the flanking sequence between Stul and Bglll inhibits restarting in a manner that can be relieved once the ribosome reaches the Ncol site in apo B. Taken together, these results suggest that: another pause transfer sequence that is just upstream of the Stul site mediates stopping and restarting of translocation as demonstrated in its native context; that restarting translocation can occur posttranslationally; and that factors other
Figure 8. Comparison of the B’ Pause Transfer and TV Heavy Chain Stop Transfer Sequences
-4 1 ..RDTTMPM ~;;9KALLKKTKNSEEFAAAMSRYELKLAIPEGKd;l
p Stop Transfer
than just exposure of the complete pause transfer sequence, such as flanking sequences, affect restarting. Finally, by dissociating stopping and restarting, an otherwise transient, transmembrane intermediate in apo B can be kept stopped, rendering it readily detectable under different conditions of proteolysis. Discussion Previously, we have shown that apo 615 is translocated through a series of transmembrane forms with large carboxy-terminal domains accessible to proteases on the cytosolic face of the membrane. These transmembrane intermediates were shown to be readily extracted from the ER membrane and to chase into intermediates with progressively longer amino-terminal lengths protected from proteinase K. Here we have identified a pause transfer sequence of 33 amino acids in apo B (the B’sequence, Figure 8) that confers stopping without integration followed by restarting of translocation on a heterologous chimeric protein. Furthermore, we have dissociated stopping from restarting. In addition, we have provided evidence for a second pause transfer sequence over 200 residues downstream from the B’ sequence in apo 815 and shown that it functions in its native context. Moreover, we have demonstrated that restarting the translocation of apo B15, while mediated by the pause transfer sequence that stops translocation, is governed by factors other than just the sequence itself and can occur posttranslationally. Under some experimental conditions, the topology of proteins may be dependent on the translation system used (Spiess et al., 1999; Lopez et al., 1990). We have demonstrated, however, that pause transfer in apo 815 occurs in both wheat germ and rabbit reticulocyte lysates.
The 33 amino acids of the B’ sequence (top; residues 79-l 11 in mature apo B) and 49 amino acids of the u stop transfer sequence (bottom) are shown in bold face type. The hy dropathy plots (Kyte and Dootittle, 1982) correspond to the bold-faced residues. The flanking residues in the corresponding truncated chimerit proteins, SLB’gGIBam and SLSTgGl Barn, are shown in smaller type. On the left are the 6 amino acids of lactamase prior to the fusion point and 1 methionine introduced by a Ncol linker (at the fusion point); on the right of the sequences in boldface type are 2 residues (EF) encoded by an EcoRl linker, followed by the 24 amino acids of glycoglobin truncated at the BamHl site ending with the glycosylation site (only the first 5 residues of glycoglobin are shown for SLSTgGIBam). During construction of SLBpG, glutamaters waschanged toalanine (underlined) at the fusion between lactamase and the B’sequence, and the initial methionine of glycoglobin was changed to alanine (right of EF encoded by EcoRl linker).
The First Stage in Pause Transfer: Stopping Translocatlon A growing body of evidence suggests that translocation of proteins occurs through an aqueous channel in the membrane (Gilmore and Blobel, 1985; Simon and Blobel, 1991). What is the relationship of paused chains to these protein-conducting channels? We believe that the transmembrane intermediates lie within these aqueous channels. These paused forms are thereby transmembrane yet extractable at high pH and can restart translocation subsequently (see diagram, Figure 28). Since the truncated chains studied here stop despite the absence of a hydrophobic domain, it is unlikely that the charged sequence stops by direct interaction with the lipid bilayer. Rather, it would seem that a pause transfer sequence interacts with a protein receptor that may be a part of the machinery involved in translocation or stop transfer. The protected fragment of SLB’gGIBam that is detected by anti-lactamass antibody (Figure 3) is larger than SLgGIBam, suggesting that the B’ sequence is either within the channel or reaches the ER lumen, environments where the chain may contact other proteins. Alternatively, the sequence may be recognized on the cytoplasmic side of the.membrane in a manner that protects the sequence from proteolysis. Using truncated proteins, it may be possible to identify receptor proteins that engage the pause transfer sequence. The Second Stage in Pause Tmnsfer: Restarting Tmnslocation The mechanism by which translocation restarts is perhaps more complex. Pause transfer sequences appear to be the primary determinant in apo B necessary to restart translocation. Removal of the ribosome from the mem-
brane is not likely to be the principal event in restarting translocation, since apo B truncated at the first Ncol site restarts translocation in the absence of treatments that strip ribosomes. However, EDTA and potassium may restart translocation by altering ribosome-membrane interactions. On the other hand, exposure of an entire pause transfer sequence may not always restart translocation. Although the chimeric protein SLB’gGIBam restarts when the entire sequence emerges from the ribosome, apo B truncated at Bglll remains stopped near the Stul site despite the exposure of 10 kd more of apo B, indicating that factors other than just exposure of a pause transfer sequence affect restarting. Flanking domains, such as the one between Stul and Bglll, might influence restarting by a change in conformation or altering the alignment of the pause transfer sequence with a receptor. Effects of context have previously been shown for signal and stop transfer sequences (Andrews et al., 1988; Kuroiwa et al., 1991; Paterson and Lamb, 1987). In the chimeric proteins SLB’gG and apo B(Stul)-Pt, pausing mediated by the pause transfer sequences may simply be unaffected by the heterologous flanking domains, which may explain the apparent discrepancy between the behavior of SLB’gG truncated at BstEll and apo B(Stul)-Pt compared with apo B truncated at Bglll. How might a pause transfer sequence mediate the switch from stopping to restarting translocation? Stopping and restarting could be caused by sequential interactions with separate receptors or engagement followed by disengagement of a single receptor. Alternatively, restarting could be governed by a cellular regulatory molecule, such as a GTPase (Bourne et al., 1990) that is toggled by stopping; such proteins are already implicated in the regulation of targeting to the ER membrane (Connolly et al., 1991). Variations in pause transfer sequences or their context may alter the set point of such a molecular regulator, thereby accounting for differences in restarting (e.g., between SLB’gGIBam and apo B/Bglll). Regardless of the molecular switch from stopping, restarting may involve either the resumption of translocation through a completely intact translocational channel or reassembly of a partially or wholly disassembled translocational channel. Pause Transfer Sequences and Other Topogenic Sequences The key amino acids in the 33 residue B’ sequence have not been defined. No region of primary sequence identity was found between the B’ pause transfer sequence and the region near the Stul site. However, 8 of 11 residues at the 5’ end of the B’ coding region can be aligned with 8 of 14 residues in the region upstream of the Stul site. This alignment of LKK-T-N-A suggests that a consensus motif for pause transfer may exist. However, more studies on additional pause transfer sequences are necessary to evaluate this hypothesis fully. Regardless, it is notable that both the B’ sequence and the region upstream of the Stul site are predicted to form positively charged helices rich in lysine residues; furthermore, there are discrete regions predicted to form charged helices that roughly cor-
respond with the distribution of the major transmembrane fragments generated by proteolysis of apo 815. Signal sequences have been found to be extremely degenerate in primary structure (Kaiser et al., 1987) and yet are recognized with a high degree of specificity by receptor proteins (Siegal and Walter, 1988). Pause transfer sequences may likewise be quite degenerate in sequence yet specific in interaction, as has been proposed for stop transfer sequences (Yost et al., 1990). Similarly, it is unclear whether the B’ sequence is organized into one or more functional domains. For example, two discrete domains may exist within the pause transfer sequence with the upstream domain directing the stop and the downstream domain signaling the restart. The topology of the chimeric protein SLB’gG truncated at BamHl versus BstEll (Figure 2; Figure 4) could be interpreted in support of this hypothesis. In this view, the ribosome trapped by truncating at BamHl masks the restart domain until treatment with EDTA or potassium, whereas truncating at BstEll allows both the stop and restart domains to be exposed in the absence of treatments to disassemble the ribosome. However, the topology of apo B truncated at Bglll (Figure 7) suggests that complete exposure of all domains in a pause transfer sequence does not always restart translocation in the presence of other factors. Therefore, stopping and restarting translocation does not appear to result simply from the sequential exposure of two domains in a pause transfer sequence. It is equally plausible that the two actions of stopping and restarting of translocation aretoggled byonlyonesequence.Additional studies of the B’ sequence may elucidate the significant amino acids or domains. What is the relationship of pause transfer sequences to other topogenic sequences? Signal sequences start translocation, pause transfer sequences restart translocation. Restarting would not appear to require targeting or the completeassemblyof atranslocational channel. However, if reassembly of part of the translocational machinery is needed to restart translocation, a pause transfer sequence may recognize a subset of receptors for signal sequences in the ER membrane. Pause transfer sequences share some features of stop transfer sequences. Both types of topogenic sequences mediate stopping initially and alter the close relationship between the ribosome and membrane (Blobel, 1980; Simon and Blobel, 1991) since both types of newly synthesized proteins are accessible to exogenous proteases. This alteration in the ribosome-membrane junction is depicted as tilting of the ribosomes in Figures 28 and 2C and may occur to allow the growing chain to accumulate in the cytoplasm. However, unlike a stop-transfer sequence, a pause transfer sequence does not mediate integration into the bilayer, perhaps because the channel does not disassemble as completely (Blobel, 1980; Simon and Blobel, 1991). Lack of integration does not imply that pause transfer sequences are simply weak stop transfer sequences; in fact, the paused proteins stop efficiently and remain stopped until translocation is restarted. Instead, it suggests that integration may be mediated by the long hy-
drophobic domain in a stop transfer sequence that is missing from a pause transfer sequence (Figure 8). Thus, the charged regions in pause transfer and stop transfer sequences may stop translocation, and the hydrophobic domains in stop transfer sequences may permit a stopped protein to integrate into the bilayer. The pause transfer sequence of apo 815 may share features in common with the stop-transfer effector sequence (STE) (Yost et al., 1990) of the prion protein (Prusiner, 1991). Both pause transfer and STE sequences can mediate stopping, and both contain a charged region rich in lysines. For the prion protein, however, some or all of the stopped proteins are integrated at the hydrophobic domain that follows STE. Perhaps the difference in their phenotype lies in the presence of a hydrophobic domain downstream of STE that allows integration of prion protein into the bilayer in a regulated manner (Lopez et al., 1990). Such regulation may not be manifest for pause transfer sequences within apo B simply because they are not followed by sequences of sufficient hydrophobicity to integrate into the membrane.
Pause Transfer and the Translocatlon of apo 815 Our previous work demonstrated that apo 815 translocates through a series of transient, transmembrane intermediates (Chuck et al., 1990). This transient transmembrane topology appears to result principally from the action of specific pause transfer sequences that exist at more than one point along the length of apo 615. Thus, multiple pauses in translocation at pause transfer sequences throughout apo 815 could account for the stepwise translocation of this protein. We have also shown that a pause transfer sequence functioning within apo B can stop translocation despite translation of downstream residues and that restarting translocation can occur posttranslationally, which may explain the posttranslational conversion of at least some of the transmembrane intermediates we observed during the biogenesis of apo 815 (Chuck et al., 1990). These results confirm predictions of our original model of the translocation of apo 815. Furthermore, by using truncation to dissociate stopping from restarting, we have established conditions whereby a transient, transmembrane intermediate of apt 815 can be kept stopped, hence simplifying its detection. Thus, under conditions of translocational chase where all other transmembrane intermediates are converted, a particular transmembrane intermediate can be kept stopped for hours, yet restarted by treatment with EDTA. Indeed, with this technique, these stopped intermediates are equally well detected when using rabbit reticulocyte lysates, tetraCaine, and trypsin, the same reagents used by other workers who did not detect transient, transmembrane intermediates in apo B (Pease et al., 1991; for our response, see our Scientific Correspondence, Nature, submitted). Moreover, the conversion of these chains to fully protected forms after translocation is restarted with EDTA strongly suggests that the transmembrane fragments do not arise from overproteolysis.
The Role of Pause Transfer Sequences For what purpose might a nascent secretory protein pause? Pauses in translocation may be associated with modifications, assembly, or regulation of secretion of a protein. In the ER lumen, some modifications of nascent proteins, such as cleavage of the signal sequence can occur during translocation (Blobel and Dobberstein, 1975). The complete translocation of some proteins may even require the completion of certain modifications; some protein domains may have to wait until they have been modified before continuing translocation. Alternatively, translocation may be orchestrated to allow sufficient time to modify the chains as they pass through the translocational machinery. Certain modifications occur at particular sequences, such as cleavage of the signal sequence by signal peptidase, N-linked glycosylation, and the addition of glycosyl phosphatidylinositol anchors (Cross, 1987). Pause transfer sequences may act similarly, either by being sites for modifications that are necessary to restart translocation or by pausing translocation to allow specific alterations of the nascent chain. For example, apo B is an extremely hydrophobic protein that may bind an initial complement of lipid during pauses. Such an association may be necessary to render the nascent chain soluble in the aqueous environment of the ER. Pauses in translocation may be necessary for the folding and assembly of complex proteins into specific structures. Both folding and assembly of a nascent protein may occur during translocation. The heavy chain-binding protein, which is thought to help refold nascent lumenal domains, appears to be essential for translocation in yeast (Vogel et al., 1990). In addition, the reorganizing of disulfide bonds by protein disulfide isomerase can occur during translocation (Bulleid and Freedman, 1988). Thus, similar to cotranslocational modifications of proteins, pausing could result from or allow proper folding and assembly. apo B is a large, complex protein that may require folding or the formation of disulfide bonds cotranslocationally. Since many of the transient translocational intermediates of apo 815 appear to have large domains exposed to the cytoplasm, heat shock proteins (Pelham, 1986; Chirico et al., 1988; Beckmann et al., 1990) or other chaperones found in the cytoplasm may have a role in the assembly of apo B, in addition to membrane-associated or lumenal factors. Pauses in translocation might permit regulation of secretion. In response to stimuli to decrease intracellular transport or secretion, some nascent proteins may remain stopped in the ER membrane whereupon they may be targeted for degradation (Lippincott-Schwartz et al., 1988; Amara et al., 1989) or remain in a pool from which they could be rapidly secreted. Conversely, when the cell is stimulated to secrete these proteins, pausing may be quite brief; restarting may occur quickly, or stopping may be bypassed. Thus, regulation of secretion may be achieved by controlling either stopping or restarting of translocation and by degrading proteins spanning the ER membrane. Regulation of translocation may be intertwined with modifications or assembly of certain proteins. Indeed, the secretion of apo B might be regulated by this
translocational process. Control of the secretion of apo B in cultured hepatocytes appears to occur posttranslationally (Borchardt and Davis, 1987) since mRNA and intracellular apo B levels do not change in response to metabolic stimuli (Pullinger et al., 1989), and a large proportion of newly synthesized apo B can be degraded in a preQolgi compartment (Sat0 et al., 1990). Therefore, the secretion of apo B could be regulated by controlling its translocation and degrading the chains left stopped in the membrane of the ER (Davis et al., 1990). If apo B secretion is regulated in this manner, drugs might be found that alter the regulation of pausing, decrease the secretion of lipoprotein particles, and thereby lower plasma levels of cholesterol. Candidates for this class of drugs could be screened using chimeric proteins containing one or more of the pause transfer sequences found in apo B in a translocational assay. If they are involved in modifications, assembly, or regulation, pause transfer sequences could be exploited to alter the behavior or fate of engineered proteins in a manner similar to signal and stop transfer sequences (Yost et al., 1983; Lingappa et al., 1984). Experimental
Plasmids All plasmids are derived from pSP64 (Promega) into which the 5’ untranslated region of Xenopus globin is inserted at the Hindlll site. Chimerit proteins were verified by sequencing and immunoprecipitation with antibodies directed against lactamase and glycoglobin. SLSTgG: This plasmid encodes a chimeric protein comprised of the signal sequence of bovine prolactin (Sp”), 100 residues of beta lactamase, an Ncol linker, 49 residues of the p heavy chain stop transfer sequence (Rothman et al., lQ88), an EcoRl linker, and 111 residues of chimpanzee alpha globin containing a six codon acceptor site for N-linked glycosylation (glycoglobin, Perara et al., 1986). SLgG: The plasmid encoding SLSTgG was linearized with Ncol in the presence of ethidium bromide (one Ncol site is at the amino terminus of the signal sequence, the other is at the junction of L and ST), treated with mung bean nuclease, and digested with Sphl. The fragment was purified by agarose gel electrophoresis and ligated into a vector prepared by cleavage of SLSTgG with EcoRI. treatment with mung bean nuclease. digestion with Sphl. and purification byagarose gel electrophoresis. SLB’gG: First, apt B3.lt was created by digesting the plasmid encoding apt 815 (Chuck et al., 1990) with Sac1 and ligating with T4 DNA ligase. This step brought the EcoRl site in the polylinker region immediately downstream and in-frame with the first Sac1 site in apo B. Second, an oligonucleotide encoding amino acids 102-I 11 in mature apo B was ligated into the Sac1 and EcoRl sites in apo B3.lt. Third, this construct was cut with Bsu36 and treated with mung bean nuclease, and a Ncol linker (GCCATGGC) was ligated into this site. Fourth, the B’fragment bordered by Ncol and EcoRl sites was purified by agarose gel electrophoresis and ligated into a vector prepared by digestion of SLSTgG with Ncol and EcoRl and by subsequent treatment with calf intestinal phosphatase to create the plasmid B’gG. Fifth, the Ncol fragment encoding SL was ligated back into the Ncol site of B’gG to create SLB’gG. Cell-Free Trsnscrlptlon, Translstlon, and Translocatlon Transcription by SPS RNA polymerase was carried out at 40°C for 1 hr (Melton et al., 1984; Krieg and Melton, 1984). We prepared and used cell-free lysates of wheat germ (Erickson and Blobel, 1963) or rabbit reticulocytes (Perara and Lingappa, 1985) to translate the synthesized mRNAs at 24OC for the times indicated in the figure legends. Micrcsomal membranes derived from the ER of canine pancreas were prepared (Walter and Blobel, 1983) and used at a concentration of 4 OD
per 10 nl of translation reaction. Some aliquots of translation products were incubated at 24OC with 0.5 mM tripeptide competitor of N-linked glycosylation (acetylaterl Asn-Tyr-Thr), 0.1 nM emetine, 2 mM puromytin, 10 mM EDTA, or 540 mM potassium acetate, as indicated in the text and figure legends. (The experiments in Figures 78 and 7C were performed at 30°C as well as at 24OC. and identical results were obtained.) Calcium chloride was added to a free concentration of 10 mM to all samples including those treated with EDTA. Aliquots of translation products were then digested with 0.4 mg/ml proteinase K for 30 min on ice in the absence or presence of 0.5% Triton X-100. (The samples in Figure 7C were treated with 2 mM tetracaine instead of calcium chloride and digested with 0.4 mg/ml trypsin instead of proteinase K. All other procedures were otherwise identical.) All products were subsequently transferred via pipette tips dipped in 200 mM phenylmethylsulfonylfluoride in dimethyl sulfoxide into boiling 0.1 M Tris (pli 8.9) with 1% SDS and boiled for 5 min. An aliquot of each sample was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) on 12%-17% gradient gels and visualized after fluorography with 2.5diphenyloxazole by autoradiography. In Figure 1 B, the apparent molecular mass of the transmembrane bands may not appear correct because of the sloping (“smile”) of the lanes. In addition, it has come to our attention that in Figure 18 of Chucket al., 1990, the 18 kd marker (lactoglobulin A) is mislabeled as 30 kd. Translation of plasmids truncated at particular sites yields several minor bands of much greater apparent molecular mass. These bands appear to represent truncated proteins linked to aminoacyl-tRNA as evidenced by precipitation with hexadecyltrimethylammonium bromide and their shift down to the principal bands by alkaline hydrolysis. Samples of these truncated proteins (apo Bl5/BsmAl and apo 8151 Bglll) were brought to pH 11.5 at 37OC for 30 min, then adjusted to pli 6.9 prior to SDS-PAGE to eliminate these minor bands without affecting the distribution of transmembrane and fully protected pro teins. Extraction with sodium carbonate: SLgGIBam. SLSTgGIBam, and SLBpGlBam were synthesized separately in vitro in the presence of microsomal vesicles. Aliquots were mixed, and the pooled sample was divided in half and diluted 76fold with either Tris-buffered isotonic sucrose (pH 7.5) or sodium carbonate (pH 11.5) (Fujiki et al., 1982). After incubating on ice for 30 min, the samples were centrifuged at 40,000 rpm in a Beckman Ti 70.1 rotor at 4OC for 3 hr. The supernates were removed with a micropipette, and the carbonate-treated supernatant was neutralized with glacial acetic acid. The proteins in the supernates were then precipitated with 15% trichloroacetic acid on ice for IO min, spun in a microfuge at 4OC for 10 min, washed once with 1: 1 ice-cold ethanol-ether, and spun in a microfuge at 4OC for 5 min. The dried precipitates and the pelleted membranes were dissolved in 100 ul 0.1 M Tris acetate (pH 8.9) with 1% SDS and boiled for 2 min, and equal aliquots were analyzed by SDS-PAGE followed by autoradiography. Aliquots of a terminated secretory protein and a terminated integral membrane protein were included in other experiments to verify the disposition of the truncated control proteins. Antlbodles and Immunopreclpltatlon The proteolyzed cell-free translation products (boiled in SDS) were diluted 26fold with TX-SWB (1% Triton X-l 00, 100 mM Tris [pH 8.01, 100 mM sodiumchloride, IOmM EDTA, 1 mM phenylmethylsulfonylfluoride), and l-2 pl of serum was added. After 6 hr on ice, 12 nl of protein A-Sepharose was added, and the tubes were rotated at 4OC for at least 2 hr. The beads were washed three times with TX-SWB and twice with 0.1 M Tris (pH 8.0) with 0.1 M sodium chloride, and the proteins were eluted by boiling in SDS-PAGE sample buffer. Antipeptide antibody against residues 12-27 of mature apo B was a gift from Thomas Innerarity. Acknowledgments We wish to thank Don Ganem for advice, Eve Perara for reading the manuscript, Fred and M. Clara Calayag for excellent technical help, and Loretta Yin Chuck for carefully reviewing the manuscript and for support. This work was supported in part by grant HL 45480 from the National Institutes of Health to VRL. SLC is a recipient of a National
Institutes of Health Physician-Scientist Award from the National Institute of Allergy and Infectious Diseases. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received
5, 1991; revised
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The data referred to as our Scientific Correspondence are now in press: Chuck, S. L., and Lingappa, V. R., Scientific Correspondence, Nature, in press.