Cell, Vol. 69, 343-352,

April 17, 1992, Copyright

0 1992 by Cell Press

Yeast Set Proteins Interact with Polypeptides Traversing the Endoplasmic Reticulum Membrane Anne Miisch, Martin Wiedmann,’ and Tom A. Rapoport Max-DelbrDck-Center for Molecular Robert-Riissle-Str. 10 O-l 115 Berlin Buch Germany

Medicine

Summary We show by photocross-linking that nascent secretory proteins, during their passage through the endoplasmic reticulum membrane of S. cerevisiae, are in physical contact with Sec6lp and Sec62p, two genetically identified membrane proteins that are essential for in vlvo translocation. Sec6lp seems to be in continuous contact, whereas Sec62p is involved only transiently. Translocation comprises both ATP-dependent and -independent phases of interaction with the Set proteins. The results suggest a direct role of the Set proteins in translocation. Introduction Translocation of proteins across the endoplasmic reticulum (ER) membrane is a decisive step in the biosynthesis of many classes of proteins in all eukaryotes. The process of specific targeting of proteins to the ER has been clarified to some extent (reviewed in Walter and Lingappa, 1986; Bernstein et al., 1989) but the mechanism by which they subsequently cross the membrane is unknown. Indirect evidence suggests that the environment that nascent polypeptides meet during translocation is formed at least in part by proteins (Gilmore and Blobel, 1985; Connolly et al., 1989). Proteins located in proximity to polypeptides translocating across the ER membrane can be identified by photocross-linking (Wiedmann et al., 1987a, 1989; Krieg et al., 1989; High et al., 1991; Thrift et al., 1991). Photoreactive chemical groups can be introduced into nascent polypeptides that are synthesized in vitro in the presence of microsomal membranes and trapped at specific points of their membrane passage. Irradiation results in cross-links to neighboring proteins. This approach has led to the identification of a glycoprotein of relative mass 35,000 (35 kd), termed the signal sequence receptor (SSR), in canine microsomes (Wiedmann et al., 1987a). Circumstantial evidence suggests a role for SSR in translocation (Hartmann et al., 1989). Other ER membrane components (signal peptidase complex, ribophorins, mp30) have also been implicated in the formation of a translocation complex (reviewed in Rapoport, 1990). Genetic studies of Saccharomyces cerevisiae have led to the identification of three ‘Present address: Dr. M. Wiedmann, Memorial Sloan-Kettering cer Center, Progam in Cellular Biochemistry and Biophysics, York Avenue, New York, New York 10021

Can1275

membrane proteins (SecGlp, Sec62p, Sec63p), encoded by essential genes, certain mutations in which result in defects of translocation (Deshaies and Schekman, 1987, 1989, 1990; Rothblatt et al., 1989; Sadler et al., 1989). None of the studies has demonstrated the direct involvement of a component in the mechanism of membrane passage of a protein. We now provide a link between the biochemical and genetic approaches. Employing photocross-linking for the in vitro translocation of nascent secretory proteins into S. cerevisiae microsomes, we demonstrate that they are in physical contact with the Set proteins, providing evidence for their direct role in translocation. The results give insight into the molecular events occurring in the membrane during translocation and suggest that the membrane environment of a nascent chain is more dynamic than previously assumed. Results Cross-Linking of Nascent Chains at Late Stages of Their Translocation through the ER Initial experiments were designed to identify membrane proteins in proximity of nascent prepro-a-factor (ppaF) chains at late stages of the translocation process. These were represented by a translocation intermediate with both cytoplasmic and lumenal domains of the nascent chain. To this end, a truncated ppaF mRNA coding for ppaF, which comprises 180 amino acids and lacks only the C-terminal 5 amino acids (ppaF,) (Figure l), was translated in a cell-free translation system in the presence of yeast microsomal membranes and chemically modified lysyl-tRNA. The latter contained a carbene-producing reagent in the side chain of the amino acid (Wiedmann et al., 1987b). Owing to the absence of a stop codon, the translating ribosome comes to a halt when the 3’ end of the mRNA is reached (Wiedmann et al., 1989; Krieg et al., 1989). The nascent chain remains attached to the ribosome with the carboxyl terminus linked to the tRNA, while the amino terminus is transferred into the lumen of the microsomal vesicles and is N-glycosylated at the three carbohydrate attachment sites (as shown by precipitation of triply glycosylated a factor chains by cetyltrimethylammonium bromide; data not shown). If one assumes that about 30-40 amino acids are buried inside the ribosome (Malkin and Rich, 1967; Blobel and Sabatini, 1970) and about 20 are required to span a membrane, two lysines of the ppaFt chain should be located within the membrane (see Figure 1) and may give rise to cross-links with integral ER proteins. The translocation intermediate produced with S. cerevisiae microsomes and labeled ppaFt chains was irradiated, and the total products were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and subsequent fluorography (Figure 2a, lane 2). Several cross-linked products wereobserved, which were not seen if irradiation was omitted (lane 1). They could be immunoprecipitated

Cell 344

membrane

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Figure 1. Scheme of the Predicted Translocation Intermediates Produced with ppaF Chains

cleavage sle

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by antibodies directed against pro-a-factor (lane 4 vs. 3). A major band of about 68 kd was found to be resistant to extraction of the membranes with carbonate (lane 6 vs. 5 and lane 8 vs. 7; indicated by dots), indicating that it contained an integral membrane protein (Fujiki et al., 1982). The other products were lost and thus probably contain proteins that are soluble or bound to the periphery of the membranes; they were not further analyzed. The 68 kd cross-linked product contained about 2% of the total

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The ppaF chains employed for cross-linking lacked either only the C-terminal 5 amino acids (ppaF,, 160aminoacids)or, in addition, internal regions (A24-69, A32-69, A52-69). The ribosome is assumed to occupy the C-terminal 30 or 40 amino acid residues, and the ER membrane, the preceding 20 residues. The positions of the lysines (K) carrying the photoreactive probes, of the carbohydrate attachment sites (CH), and of the signal peptidase cleavage site (arrow) are indicated.

radioactivity in ppaF. As expected, it was not seen in the absence of membranes (Figure 2b, lane 2 vs. 1). If prior to irradiation the nascent chains were released from the ribosome by treatment with puromycin, so that they were able to move through the membrane into the lumen (Connolly and Gilmore, 1986) the cross-links to the membrane protein were not observed (Figure 2b, lane 3). The same result was obtained if the nontruncated ppaF chains, which are expected to be completely translocated

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2. Photocross-Linking

4

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of Translocating

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Chains

to ER Membrane

Proteins

of S. cerevisiae

(a-c) A translocation intermediate was produced with labeled ppaF, chains (see Figure 1) containing photoreactive probes and S. cerevisiae microsomes, and it was irradiated. The total products (T) were analyzed after precipitation with trichloroacetic acid or after immunoprecipitation with cr factor antibodies (immun. prec.) by SDS-PAGE and fluorography. Cross-linked products of integral membrane proteins contained in the carbonate-extracted membrane pellet (P) were analyzed in the same manner. The exposure time of the X-ray film for the total products was one-third of that for products in the carbonate-extracted pellet. (d) Same experiment as in(b), but the products in the supernatant (S) after sedimentation of the membranes are shown (exposure time of the X-ray film was about one-tenth of that in lb]), The position of o factor chains with different numbers of attached carbohydrate chains (O-3) is indicated. act. peptide, acceptor peptide inhibiting glycosylation; ppaF, completed ppuF chains; ppaF,-m, ppuF, chains with a point mutation in the signal sequence.

Set Proteins 345

Are Close

a

to Translocating

Chains

prepro- a-factor

comp.pepude: Irradlatlon: endon:

-

+ -

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-

c

-

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-

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Figure 3. Cross-Linking Se&l p

pre-lnvertase

SeC

Sec61

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(a) Photocross-linked products obtained with translocating ppaF, chains and ER membrane proteins of S. cerevisiae were subjected to immunoprecipitation with antibodies to a factor and to the Set proteins. In lane ‘endo l-f”, immunoprecipitated products were treated with endoglycosidase H; In lane “camp. peptide”. a peptide against which the Sec61 antibodies were raised was added as competitor during the immunoprecipitation. (b) Cross-linked products obtained with transloeating invertase chains (Invpa9) and microsomes of S. cerevisiae were immunoprecipitated with Sec6lp antibodies.

46 K-

123

across the membrane (lanes 4 and 5), were employed for cross-linking. The cross-linked nascent ppaF, chains are glycosylated, since inhibition of N-glycosylation in the in vitro system by a competing acceptor peptide reduced the size of the cross-linked products (Figure 2c, lane 3 vs. 2) (the inhibitory effect of the peptide on glycosylation is shown in Figure 2d, lane 3 vs. 2). Thus, the cross-linked chains must have a trans-membrane configuration with a lumenal, glycosylated portion and a cytoplasmic, tRNAlinked portion. Appearance of the cross-linked products depends on a functional signal sequence of the ppaF. If a mutant was employed (ppaF,-m) that contains a Glu residue instead of an Ala residue in the hydrophobic core of the signal sequence (Allison and Young, 1988) both the efficiency of translocation (Figure 2d, lanes 4, 5) and the yield of cross-linked product (Figure 2c, lanes 4, 5) were greatly reduced. These data demonstrate that the cross-linked membrane protein(s) are only in proximity to nascent chains that are trapped in the process of traversing the membrane. The cross-linked products could be immunoprecipitated with peptide-specific and affinity-purified antibodies directed against Sec6lp (Figure 3a, lane 5). The immunoprecipitation was not entirely complete, as another -30% of the products could be brought down in a second round (data not shown). The immunoprecipitated 66 kd band was not observed if irradiation was omitted (lane 3) or if an excess of the peptide against which the antibodies were raised was added as competitor for immunoprecipitation (lane 4). The cross-link to Sec6lp was not observed with nascent chains released from the ribosome by puromycin (lane 7). Treatment of the cross-linked product with endoglycosidase H reduced its size (lane 6). Since Sec6lp is not glycosylated (Deshaies et al., 1991), the cross-linked nascent chain must have carried a carbohydrate chain(s) and must therefore have a portion in the lumen of the ER. Thus, cross-linking to Sec61 p has occurred with a nascent chain in transit through the membrane. No cross-linked

45

products were immunoprecipitated by antibodies against Sec62p or Sec63p (lanes 8 and 9; even on longer exposures no bands were visible). From the prominence of the 68 kd band among the total cross-linked products (Figure 2a) and from the fact that about equal amounts of the 68 kd product could be immunoprecipitated by antibodies directed against a factor (Figure 3a, lane 2) and against Sec6lp (lane 5) one may conclude that SecGlp is the major cross-linking partner of translocating ppaF, chains. Assuming that the triply glycosylated form of ppaFt (M, = 28 kd) gives rise to the major cross-linked species of 68 kd, the size of Sec61 p contained in it can be estimated to be about 40 kd. This estimate, although significantly smaller than expected from the sequence (53 kd), corresponds to its abnormally fast mobility in SDS gels (Deshaies et al., 1991). Results similar to those of ppaF were obtained with a relatively long fragment of preinvertase containing 249 amino acids (Inv,,,) (Figure 3b). Again, Sec61 p gave major cross-links, which disappeared upon treatment with puromycin (lane 2 vs. 3). Also, the nascent chains in the crosslinked product were glycosylated, as demonstrated by the size shift after treatment with endoglycosidase H (lane 4 vs. 5). Early Stages of Translocation of ppa-F To study early stages of translocation, we exploited the property of ppaF to be translocated posttranslationally across yeast microsomal membranes in an ATP-dependent manner (Hansen et al., 1986; Rothblatt and Meyer, 1986b; Waters and Blobel, 1986; Wiedmann et al., 1988). Sanz and Meyer (1989) had demonstrated for proOmpA that two phases can be distinguished, an ATP-independent binding step and a subsequent ATP-dependent translocation phase. ppaF competed for proOmpA for the binding, suggesting that it behaves similarly. We sought direct evidence that prebound ppaF can be subsequently translocated. ppaFt chains were synthesized in the absence of mem-

Cell 346

membranes: ATPinIstlncub.: ATPIn2nd

--+++ -

incub.:

-

-

- SP

-

-

j-

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+

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-3 -2 -1 -0

12

3

4

5

Figure 4. Posttranslational Translocation of ppaF Involves Independent Binding Step and a Subsequent ATP-Dependent cation Step

an ATPTranslo-

Radiolabeled ppaF, chains were synthesized in the absence of membranes and isolated with the ribosomes by centrifugation. They were incubated (1st incub.) with or without microsomes from S. cerevisiae in the absence of ATP, except for sample 5, which received ATP. All samples were centrifuged to sediment the membranes together with bound nascent chains through a sucrose cushion containing a high salt concentration. Lanes 1 and 2 show the supernatant (S) and pellet (P) fractions of an incubation in the absence of membranes. The membrane pellets (P) were resuspended and incubated (2nd incub.) in the absence or presence of ATP. The position of c factor chains carrying different numbers of carbohydrate chains (O-3) is indicated.

branes and isolated with the ribosomes by sedimentation. They were then incubated with or without yeast microsomes in the absence of ATP (Figure 4, samples 2-4). A control sample was incubated in the presence of microsomes and ATP (lane 5). All samples were centrifuged to sediment the microsomes through a sucrose cushion containing a high salt concentration. ppaF, chains were only seen in the pellet if microsomes were present (lanes 3, 4 vs. 2; lane 1 shows the supernatant of sample 2). The microsomescontaining bound ppaFt chains were then resuspended and again incubated in the absence or presence of ATP (lane 3 vs. 4). It may be seen that glycosylated products, indicative of translocation, were only seen after addition of ATP (lane 4). These data confirm results using a different protein (Sanz and Meyer, 1989) and show that the posttranslational translocation process can be divided into an ATP-independent binding step and a subsequent ATP-requiring translocation phase. We next analyzed cross-linked products produced in a posttranslational assay in the absence or presence of ATP. Cross-links to Sec61 p were observed only in the presence of ATP (Figure 5a, lane 3 vs. 4; the specificity of the immunoprecipitation was shown by addition of a competitor peptide, lane 2). The cross-linked products were generally more heterogeneous in posttranslational assays, owing to incomplete glycosylation of the nascent chains. If the products were treated with endoglycosidase H, they were reduced in size and migrated as a single band (Figure 5b, lane 2 vs. 1). Treatment of the sample with puromycin prior to irradiation resulted in the disappearence of the

cross-links (lane 3). These data indicate that in the presence of ATP a translocation intermediate is produced which, like the one produced in a cotranslational assay, has a transmembrane configuration. Cross-links of lower intensity were seen with Sec62p only in the absence of ATP (Figure 5a, lane 8 vs. 7). The immunoprecipitation was prevented by the addition of a competitor peptide (lane 6). Despite the low yield (about 0.4% of total radioactivity in ppaF), cross-linking to Sec62p seems to be specific, since it was not observed with a ppaF carrying a signal sequence mutation (Figure 5c, lane 6 vs. 3). These data suggest that the interaction of the nascent chain with Sec62p precedes that with Sec61 p. The release of the nascent chain from Sec62p was triggered by ATP (Figure 5d). Nascent chains interacting with Sec62p in the absence of ATP (lanes 2 and 3) were not cross-linked after addition of ATP (lane 4). ATP could not be replaced by the analogs ATPyS, AMPPNP, or AMPPCP (Figure 5e). ATPyS caused weak cross-links to Sec61 p (lane 4; 3% compared with ATP) and a marginal decrease in cross-linking to Sec62p (lane lo), but these effects may have been due to contaminating ATP (or ADP if rephosphorylation occurred in the system). ATP was maximally effective at concentrations as low as 10 r.rM (data not shown); the analogs were tested at 2 mM. The invertase fragment lnvzbs gave weak ATP-dependent cross-links to Sec6lp in a posttranslational assay (data not shown). Cross-links to Sec62p were not discernable even in the absence of ATP, perhaps owing to inefficient translocation of the fragment. To investigate early stages of the translocation process in a different manner, shorter polypeptide chains with fewer translocated amino acids were employed, which carried different deletions of the pro region of the ppaF (A24-89, A32-89, A52-89 [Rothblatt et al., 19871) (Figure 1). These were tested in a cotranslational translocation assay. Cross-links were produced with both Sec61 p and Sec62p, but the relative yields (SecGlplSec82p) decreased with the chain length (the ratio decreased from about 8:l to 1:5) (Figure 6). With the two shortest chains (A24-89 and A32-89), the cross-linked products contained unglycosylated ppaF chains, since they were resistant to endoglycosidase H treatment (lanes 3 vs. 2, 6 vs. 5, 9 vs. 8, and 13 vs. 12) (A24-89 does not contain a glycosylation site, and that in A32-89 is only poorly utilized [Rothblatt et al., 1987; see Figure 61). Of particular interest were the results with the construct A52-89. Weak crosslinks of glycosylated nascent chains to Sec61 p (lanes 17 vs. 16) and stronger ones of unglycosylated chains to Sec62p (lanes 21 vs. 20) were observed, again indicating that Sec62p precedes Sec6lp in interacting with transloeating chains. It should be noted that the lack of glycosylation per se is not responsible for the cross-links of a nascent chain to Sec62p, as shown by their absence with unglycosylated ppaF, chains synthesized in the presence of an acceptor peptide (data not shown). Treatment with puromycin resulted in the disappearerice of the cross-links obtained with the longest deletion mutant Ag52-89 to both Sec6lp and Sec62p (Figure 6, lanes 18 and 22) indicating that both membrane proteins

Set Proteins 347

Are Close

to Translocating

Chains

a

b

antibodies: endo H: puromydn:

Figure 5. Cross-Linking to the Set Proteins of Posttranslationally Translocated ppaF Depends on ATP

C

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(a) ppaF,-chains containing photoreactive groups were synthesized in the absence of membranes and isolated with the ribosomes by centrifugation. They were incubated with microsomes in the absence or presence of ATP and irradiated, as indicated, to produce crosslinks to membrane proteins. These were analyzed by immunoprecipitation with Sec61 p or Sec62p antibodies and SDS-PAGE. Controls for the specificity of the immunoprecipitations were carried out by addition of competing amounts of the antigens against which the antibodies were raised. (b) Cross-linked products containing SecGlp contain glycosylated chains, as demonstrated by the size shift after treatment with endogly cosidase H (endo H) (lane 2) and do not occur after release of the chains from the ribosome by puromycin (lane 3). (c) Photocross-linking to Sec62p was tested in parallel with wild-type ppaF, chains and with chains carrying a signal sequence mutation (ppaFt-m). (d) ppaF, chains were incubated with microsomes first without ATP at 0% then with ATP at 26OC to demonstrate their ATP-dependent release from Sec62p (lane 4). Controls are shown with ATP either present (lane 1) or absent (lane 2) in both incubations or with the second incubation carried out at O°C (lane 3). (e) Photocross-linking of ppaF, chains to SecGlp and Sec62p was tested with ATP analogs (2 mM) as indicated.

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ppaF, chains that carry deletions in the pro region (see Figure 1) were synthesized in the presence of microsomes of S. cerevisiae and modified lysyl-tRNA and photocross-linked to integral membrane proteins. The products were analyzed by immunoprecipitation with antibodies against SecGlp and Sec62p. Where indicated, the nascent chains were released from the ribosomes by puromycin prior to irradiation. “endo l-t” indicates treatment of the immunoprecipitated products with endoglycosidase H.

Cell 346

Sac61

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Figure 7. The Effect of ATP on the CrossLinking of ppaF Chains of Different Length to the Set Proteins

A521

ppaF chains of different length (see Figure 1) were synthesized in the absence of microsomes and isolated with the ribosomes by centrifugation. After incubation with microsomes from S. cerevisiae in the absence or presence of ATP, the samples were irradiated, as indicated, and analyzed by immunoprecipitation with antibodies against Sec6lp (a) or Sec62p (b).

+ -

-++-+*-++-•+

ion:

1

2

3

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5

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10 11 12

123

are only in proximity to polypeptides in transit through the membrane. For the shorter chains, however, treatment with puromycin had little or no effect (lanes 10, 14; data for A24-89 not shown). They apparently remain in the translocation site even after their release from the ribosome, perhaps because their uncleaved signal sequence confers on them a membrane affinity and/or because the lumenal domain of the polypeptides is too short to pull them inside. ATP-Dependent and ATP-Independent Phases of ppaF Translocation Additional studies of the effect of ATP on the juxtaposition of the nascent ppaF chains to the Set proteins were performed on shorter chains. In contrast to the long chains (ppaF, and A52-89), which gave cross-links to Sec8lp only in the presence of ATP (Figure 7a, lanes 8 vs. 9, 11 vs. 12) the shorter chains A24-89 and A32-89 actually gave stronger cross-links in the absence of ATP (lanes 3 vs. 2 and 8 vs. 5). This was particularly pronounced with the construct A32-89. ATP also had a reproducible effect on the gel mobility of the Sec81 p cross-links of the shorter chains. In the presence of ATP, a doublet of bands was seen (lanes 2 and 5), and the major band had a higher apparent molecular weight. These results indicate that the juxtaposition of the nascent chains to Sec6lp changes by the addition of ATP. Most likely, different cross-links between the molecules are produced that differ slightly in their gel mobility. Cross-linking to Sec82p occurred in the absence of ATP with all the chains (Figure 7b, lanes 3,8,9; see also Figure 5 for results on ppaF,). In contrast to the behavior of the long ppaF, chains (Figure 5) the shorter chains gave cross-links to Sec82p also in the presence of ATP (lanes 2, 5, 8). This result is in agreement with the fact that they all gave cross-links to Sec82p in a cotranslational system (Figure 8). It should be noted that the short chains A24-89 and A32-89 were transported (and glycosylated in the case of A32-89) to some extent even in the absence of ATP (data not shown), in analogy to their behavior in Sec81 p cross-linking. We have also investigated the ATP requirement of the last step in translocation, i.e., the membrane transfer of

456

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9

the C-terminal tail of a polypeptide with a large lumenal domain. Microsomes containing the long ppaFt chains in a trans-membrane orientation were reisolated and incubated in the absence of ATP with or without puromycin (Figure 8a, lanes 3 vs. 2). The Sec8lp cross-links disappeared even in the absence of ATP. (The efficiency of ATP depletion was demonstrated in a parallel posttranslational translocation assay; Figure 8b). It is therefore likely that the C-terminal tail is pulled inside the lumen of the ER simply by folding of the nascent polypeptide chain. Discussion We have demonstrated that the genetically identified Sec81 p and Sec82p proteins are in physical contact with nascent secretory polypeptides as they pass through the ER membrane of S. cerevisiae. The previous genetic experiments indicated that the Set proteins are needed for translocation through the ER membrane of a wide variety of proteins, but they did not exclude the possibility of an indirect effect. Now, our results provide evidence for a direct involvement of Sec81 p and Sec82p in the translocation process. The Set proteins seem to be part of a proteinaceous environment that secretory proteins meet during their membrane transfer. The only other protein for which both physical proximity to a translocating polypeptide and essentiality have been demonstrated is MOM38 (also known as general insertion protein/site, import site protein 42) a component of the outer membrane of mitochondria involved in protein import into the organelle (Vestweber et al., 1989; Baker et al., 1990; Kiebler et al., 1990; Sdllner et al., 1991). The agreement between the genetic and biochemical approaches also demonstrates the validity of the photocross-linking method. Our data give insight into the molecular events occurring in the membrane during translocation. Sec81 p turned out to be the major cross-linking partner of nascent chains that had reached a late stage in the translocation process. These polypeptide chains had a sizable portion in the lumen of the ER membrane, were glycosylated, and moved through the membrane when released from the ribosome by puromycin. Cross-links to Sec81 p were only seen with polypeptides that had initiated but not yet completed their membrane transfer. It is likely that cross-linking occurred

Set Proteins 349

Are Close

to Translocating

Chains

b -

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ATP:

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Figure 8. The Puromycin-Induced Links Does Not Depend on ATP

Disappearance

of Sec6lp

Cross-

(a) ppaF, chains were cotranslationally inserted into microsomes from S. cerevisiae, and the membranes were isolated by centrifugation to remove ATP and incubated in the absence of ATP with or without puromycin. After irradiation, as indicated, cross-links to integral membrane proteins were analyzed by immunoprecipitation with antibodies to Sec61 p. (b) To test the efficiency of ATP depletion, mock-treated microsomal membranes were incubated with ppaF, chains posttranslationally in the absence or presence of ATP. The position of the nascent chains with different numbers (g-3) of carbohydrate chains are indicated.

near or within the membrane. Deletion of two of the three first lysines of the chain expected to be in the lumen of the ER in the translocation intermediate produced with ppaF (see Figure 1) did not prevent cross-linking to Sec61 p (see results obtained with the construct A52-69). The only lumenal lysine left is close to the membrane (maximum distance 5 amino acids). Also, the sequence of Sec6lp indicates that it spans the membrane several times and does not contain any sizable domain in either the cytoplasm or the lumen (Stirling et al., 1992). It therefore appears that translocating polypeptides are in immediate proximity to Sec6lp within the membrane and that they move alongside Sec61 p through it. Consistent with such a function, SecGlp is abundant in yeast ER membranes (Deshaies et al., 1991). S. Sanders, K. M. Whitfield, J. P. Vogel, M. D. Rose, and R. W. Schekman (submitted) have also recently demonstrated that ppaF, arrested in its translocation through the yeast ER membrane by coupling avidin to its C-terminus, can be cross-linked to Sec61 p with a bifunctional reagent. They have also determined that mutations in Sec62p and Sec63p perturb this interaction. Sec62p seems to interact with nascent chains only transiently. In a posttranslational translocation system, Sec62p became cross-linked to long nascent ppaFchainsonly in the absence of ATP, conditions shown to allow only the initial binding of the precursor molecules. Addition of ATP, which results in their translocation, caused the disappearance of cross-links to Sec62p and the concomitant appearance of cross-links to Sec61 p. In a cotranslational system,

cross-links to Sec62p were only observed with short and not yet glycosylated nascent chains of ppaF. They were not seen for long chains representing a late stage of translocation. For the construct 852-89, the nascent chains were shown to be heterogeneous with respect to their stage of translocation; the non-glycosylated chains, corresponding presumably to an earlier stage, gave crosslinks to Sec62p, whereas the glycosylated chains gave cross-links to SecGlp. Sec6lp seems to be involved at early stages of the translocation process as well, since it gave cross-links to even the shortest nascent chains. We therefore favor a model with a transient involvement of Sec62p and a constant engagement of Sec61 p in both coand posttranslational translocation. Strong support for a transient function of Sec62p is provided by its presence in ER membranes in about one-tenth the amount of Sec6lp (Deshaies et al., 1991). One might argue that nascent chains giving cross-links to Sec62p may not represent true translocation intermediates, since they were not glycosylated and cannot be shown to be transferred through the membrane after release from the ribosome by puromycin. Also, in the posttranslational assay in the absence of ATP, membraneinserted long nascent chains may hit proteins that are distant from those of the translocation site. Several observations argue against these possibilities. First, crosslinking depended on an intact signal sequence of ppaF, and ATP triggered the release of the nascent chain from Sec62p. Second, even short nascent chains gave crosslinks to Sec62p. Third, the genetic evidence for the importance of Sec62p, its spatial proximity to Sec61 p (Deshaies et al., 1991), and the fact that mutations in it can perturb the interaction of Sec61 p with the nascent chain (Sanders et al., submitted) would also argue that Sec62p is a genuine component of the translocation site and is located close to translocating chains. It is possible that nascent chains encounter Sec62p earlier than Sec61 p because of its larger cytoplasmic domains (Deshaiesand Schekman, 1990). Such adifference in spatial disposition between Sec62p and Sec61 p may explain the observed changes in the yields of cross-links with chain length of ppaF, since these may have different sizes of the polypeptide sticking out into the cytosol. Sec62p probably does not perform its function in isolation. It is part of a membrane-bound multisubunit complex (Sec62p-Sec63p complex) that includes Sec63p, a glycoprotein of M, 31.5 kd (gp31.5) and a nonglycoprotein of M, 23 kd (Deshaies et al., 1991). Sec6lp seems to be more loosely associated, since it is only found in the complex after cross-linking with a bifunctional reagent (Deshaies et al., 1991). These data are consistent with our model, in which a transient interaction of Sec62p with Sec6lp is postulated. We have provided evidence that translocation comprises both ATP-dependent and -independent phases. The initial insertion of short nascent chains into the translocation site close to Sec6lp and Sec62p occurs in the absence of ATP. Also, ATP is not needed for the puromycin-induced translocation of polypeptides once a large lumenal domain has been formed. The preceding phase, however, i.e.,

Cdl 350

the membrane transfer of a sizable polypeptide domain, seems to require ATP. ATP triggered the release of long nascent chains from Sec62p and had an effect on the juxtaposition to Sec61 p of even the shortest chains. The ATP requirement apparently does not involve a cytosolic component. This is also indicated by the fact that even after repeated washing of the microsomes or of the ribosome-bound nascent chains, ATP was still required for translocation (unpublished data) and that denaturation of precursors by urea did not relieve this requirement (Sanz and Meyer, 1989). Our finding that addition of ATP is not needed for the initial membrane insertion of a short nascent chain is in line with the fact that the SRP-dependent translocation of short nascent chains across canine microsomes can occur with a nonhydrolyzable GTP analog in the absence of ATP (Connolly and Gilmore, 1986). The spatial relation of the nascent polypeptide chain to the other components of the Sec62p-63p complex is not yet clear. Cross-links of short ppaF chains have been observed to Sec63p and to a 32-34 kd glycoprotein that may be gp31.5 (our unpublished data). On the other hand, the glycoprotein cross-links could be produced by a homolog of the mammalian SSR, since in parallel experiments with canine pancreas microsomes, SSR was among the crosslinking partners (data not shown). A strikingly similar cross-linking pattern with microsomes from yeast and mammals was also obtained with long ppaF (ppaF1) chains (data not shown), suggesting that Sec6lp is ubiquitous. From the fact that different membrane proteins are found in proximity to translocating polypeptides at different stages, one may conclude that the membrane protein environment is more dynamic than previously assumed. It seems that the nascent chain is mobile and migrates to different proteins that are stationary in the bilayer, or vice versa, that different membrane proteins migrate into its proximity. If a protein-conducting channel indeed exists in the ER membrane (Blobel and Dobberstein, 1975; Rapoport, 1985; Simon and Blobel, 1991), it cannot be absolutely rigid. Experimental

Procedures

Transcription Thepiasmids pSPICaF(for synthesisof ppaF,)(Rothbiattet al., 1987), A24-89, A32-89, A52-89 (Rothbiatt et al., 1987), and pSP8CaFm (for synthesis of ppaf-m chains carrying a signal sequence mutation) (Allison and Young, 1988) were cleaved with Ncii. For synthesis of full-length ppaF, the piasmid pSP85-aF was cut with Sail. TranscriptionwascarriedoutwithSP8RNApoiymeraseessentiaiiyasdescribed by Melton et al. (1984). The transcripts were added to the translation mixture without purification. The piasmid pGEM2-Suc2-91 (for synthesis of inv,8) (Rothblatf and Meyer, 1988a) was transcribed with SP8 poiymerase. The transcripts were incubated for 2 min at 70°C followed by 5 min at room temperature with a 0.02 OD,&ri oiigonucleotide corresponding to positions 749-788 of the coding region. The hybrid was added directly to the translation mixture. Cotransiational Trensiocation Translation was performed in a 20 pl final volume with an extract prepared from Candida maltosa (Wiedmann et al., 1988) in the presence of microsomal membranes (0.2 Azao units) from S. cerevisiae prepared as described by Waters and Blobei (1988), %S-methionine (25 &i), trifluoromethyl-diazarinobenzoic acid (lDBA)-lysyl-tRNA (2.4

pmol) (Wiedmann et al., 1987b), and SP8 transcripts (3 ~1). The samples were incubated for 15 min at 28OC. Where indicated, glycosylation of nascent chains was inhibited with 1 mM acceptor peptide (Ac-Asn-Tyr-Thr-NH*) added during translation. Treatment with puromycin (2 mM) was carried out for 10 min at 28OC after 10 min translation. Posttranslational Translocation Translation was carried out in a 30 pl volume for 10 min as described before, except that microsomes were omitted. Cycioheximide (2 mM) was added and the sample layered over a 80 PI cushion of buffer C (50 mM HEPES-KOH [pH 7.51, 0.5 M sucrose, 0.5 M potassium acetate, 1 mM dithiothreitol (DTT), 2 mM cycioheximide). After centrifugation in an Airfuge (Beckman, rotor A-100) for 1 hr at 28 psi, the ribosome pellet was taken up in 50 mM HEPES-KOH (pH 7.5), 0.18 M potassium acetate, 2.4 mM magnesium acetate, 1 mM DTT, 2 mM cycioheximide. Samples were incubated with microsomai membranes (0.2 As80 units) in the presence or absence of 2 mM ATP (or ATP analogs) for 10 min at 28OC and irradiated as described above. For posttranslational binding, nascent chains were isolated with the ribosomes by centrifugation and incubated in the absence of ATP with microsomes. The samples were layered over a 40 pi cushion of buffer C and centrifuged for IO min at 20 psi in an Airfuge. The microsomes were resuspended as above and incubated with or without 2 mM ATP. For puromycin treatment in the absence of ATP, nascent chains were isolated with the microsomes as described above. The membranes were resuspended in 50 mM HEPES-KOH (pH 7.5), 0.18 M potassium acetate, 1 mM DTT, 2.4 mM magnesium acetate, 2 mM cycioheximide and incubated with 2 mM puromycin for IO min at 28OC. ATP depletion of the pelleted microsomes was checked in a mocktreated sample using isolated ribosome-attached nascent chains in a posttranslational translocation assay. Cross-Linking and Product Analysis After incubation with microsomesfrom S. cerevisiae, the samples were irradiated for 5 min on ice with a mercury lamp and a 320 nm bandpass filter (Wiedmann et al., 1987b). The total products were analyzed after precipitation with trichioroacetic acid. For analysis of cross-links to integral membrane proteins, the membranes were extracted twice with 0.1 M N&O, (pH 12.5) for 15 min on ice with repeated centrifugation in an Airfuge (Beckman) for 10 min at 20 psi. The pellets were dissolved in SDS buffer (0.1 M Tris-HCI [pH 7.5],1% SDS, 30 mM DTT) at 90°C. After 1:lO dilution in I buffer (IO mM Tris-HCI ]pH 7.51, 0.15 M NaCi, 1 mM EDTA, 1% Triton X-l 00), the samples were subjected to immunoprecipitation with antibodies directed against a factor (Rothbiatt and Meyer, 1988b) (1 pl of serum), Sec81 p (Deshaies et al., 1991; provided by C. Stirling) (2.5 pg of affinity-purified immunoglobuiin preadsorbed to protein A-Sepharose), or Sec82p (Deshaies and Schekman, 1990; provided by D. Feldheim) (2 ~1 of serum). A 50% suspension of protein A-Sepharose (20 ~1) was used to collect the immunoglobuiins. For competition in the immunoprecipitation reactions, the C-terminal pep tide (for SecGlp) or the P-gaiactosidase fusion protein (for Sec82p) against which the antibodies had been raised was added (100 pM and 100 nM final concentrations, respectively). Treatment with endogiycosidase H was carried out overnight at 37OC after dissolving the immunoprecipitates in SDS buffer and I:10 dilution in 0.1 M sodium citrate (pH 8.5), 1 mM phenylmethylsuifonyl fluoride. The proteins were precipitated with trichioroacetic acid, washed with aceton, and dissolved in SDS sample buffer (0.1 M Tris base, 1 mM EDTA, 10% glycerol, 3% SDS, 30 mM DlT). Proteins in the supernatant after sedimentation of the membranes were precipitated with trichloroacetic acid, washed with aceton, and processed for immunoprecipitation as described before. Binding to concanavaiin A-Sepharose of the material in the redissolved carbonate-extracted membrane pellet was performed as described by GBriich et al. (1990).

We thank A. S. Girshovich and S. Brunner for gifts of TDBA, D. Allison and D. Meyer for plasmids, D. Gbriich and E. Hartmann for SSR antibodies and advice, and particularly D. Feidheim, C. Stirling, J. Roth-

Set Proteins 351

Are Close

to Translocating

Chains

blatt, and R. Schekman for the generous supply of antibodies to the a factor and Set proteins. We also thank many colleagues for helpful comments on various versions of the manuscript. The work was supported by the DFG (grant Ra 518/l-2). 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

September

10, 1991; revised

January

Kiebler, M., Pfaller, R., Sdllner,T., Griffith,G., Horstmann, H., Pfanner, N., and Neupert, W. (1990). Identification of a mitochondrial receptor complex required for recognition and membrane insertion of precursor proteins. Nature 348, 610-616. Krieg, U. C., Johnson, A. E., and Walter, P. (1989). Protein translocation across the endoplasmic reticulum membrane: identification by photocross-linking of a 39 kD integral membrane glycoprotein as part of a putative translocation tunnel. J. Cell Biol. 109, 2033-2043. Malkin, L. I., and Rich, A. (1967). Partial resistanceof tide chains to proteolytic digestion due to ribosomal Biol. 26, 329-346.

22, 1992.

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Added

In Proof

The data referred to in the text as Sanders et al., submitted, may now be updated: Sanders, S. L., Whitfield, K. M., Vogel, J. P., Rose, M. D., and Schekman, R. W. (1992). Sec6lp and BiP Directly Facilitate Poly peptide Translocation into the ER. Cell 69, in press.