(Received for publication, November 20, 1995; and in revised form, January 9, 1996)
From the
We have measured the minimum number of residues in a
translocating polypeptide required to bridge the distance between the
P-site in endoplasmic reticulum-bound ribosomes and the lumenally
disposed active site of the oligosaccharyl transferase. The results
suggest that a nascent chain may traverse the ribosome/translocase
complex in a largely extended conformation, and that hydrophobic
stop-transfer segments have a more compact, possibly -helical
conformation in the translocase.
Protein translocation through the membrane of the endoplasmic
reticulum (ER) ()is catalyzed by a complex multisubunit
translocation machinery comprising cytoplasmic, integral membrane, and
lumenal components(1, 2) . It has been proposed that
the integral membrane components form a water-accessible channel in the
membrane through which nascent chains can pass(3) .
Experimental evidence based on a wide range of methods such as
electrophysiology (4) , urea extraction(5) ,
photo-cross-linking(6) , and fluorescence quenching (7, 8) all support this proposal. Although the
environment of nascent translocating polypeptide chains has thus been
quite well characterized, their conformation during passage through the
ER translocase is not known. In mitochondria, it has been shown that
nascent chains traverse the two mitochondrial membranes in a largely
extended conformation(9) .
Previously, we have found that the lumenally oriented active site of the oligosaccharyl transferase (OST) enzyme (itself part of the translocation complex; (10) ), is positioned at a well defined distance above the surface of the ER membrane and can thus be used as a fixed point of reference against which the location of various parts of a nascent polypeptide in the translocase can be determined(11, 12) . We now report measurements of the minimum length of polypeptide chain required to bridge the distance between the ribosomal peptidyl transferase site (P-site) on the cytoplasmic side of the ER membrane and the OST active site on the lumenal side. The minimum distance has been measured both for nascent chains corresponding to a globular protein domain that normally becomes fully translocated to the lumen of the ER and for nascent chains containing a hydrophobic stop-transfer sequence which interrupts translocation and ultimately becomes integrated into the lipid bilayer. Our results suggest that non-hydrophobic nascent chains may adopt a fully extended conformation during passage through the ribosome/translocase complex, whereas the hydrophobic stop transfer sequence appears to form a more compact, possibly helical, structure when located in the translocase.
Templates for in vitro transcription of truncated mRNA were prepared using the polymerase chain reaction (PCR) to amplify fragments from pGEM1 plasmids containing the desired DNA constructs. The 5` primer was the same for all PCR reactions and had the sequence 5`-TTCGTCCAACCAAACCGACTC-3`. This primer is situated 210 bases upstream of the translational start, and all amplified fragments thus contained the SP6 transcriptional promoter from pGEM1. The 3` primers were chosen according to the positions of the truncations and were designed to have approximately the same annealing temperature as the 5` primer. None of the 3` primers contained translational stop codons. Amplification was performed with a total of 30 cycles using an annealing temperature of 59 °C. The amplified DNA products were separated on a low melting-point agarose gel, excised, and purified using Wizard PCR purification resin (Promega) as described in the manufacturers protocol.
Translation in reticulocyte lysate in the presence of dog pancreas microsomes was performed as described(19) . Translocation of polypeptides to the lumenal side of the microsomes was assayed by resistance to exogenously added proteinase K and by prevention of N-linked glycosylation through competitive inhibition by addition of a glycosylation acceptor tripeptide (N-benzoyl-Asn-Leu-Thr-N-methylamide) as described(11) .
Figure 1: Topology of Lep in the ER membrane.
To measure the minimum glycosylation
length, mRNAs truncated in the P2 domain of Lep at various defined
positions C-terminal to the potential glycosylation acceptor site
(Asn-Ser-Thr) were generated by PCR and in vitro transcription. When translated in a wheat germ lysate expression
system in the presence of SRP, the resulting truncated proteins (which
do not contain a translational stop codon) remain bound to the
ribosomal P-site at their C-terminal end (21) , while the more
N-terminal parts of the P2 domain extend through the
ribosome/translocase complex into the lumen of the ER.
As shown in Fig. 2(panel B, lanes 1-3), when the truncation was 39 amino acid residues C-terminal to the acceptor site, no glycosylation was seen unless the nascent chain was released from the ribosome by treatment with puromycin, indicating that the protein had been correctly targeted to the ER membrane but did not extended sufficiently far from the P-site for the potential glycosylation site to reach the OST active site. Similar results were obtained for nascent chains truncated 51, 56, 61, 63, and 64 residues away from the potential glycosylation acceptor site (lanes 4-18). Finally, for truncations 65 and 66 residues away from the potential glycosylation acceptor site, a sharp increase in the amount of glycosylation in the absence of puromycin was observed (lanes 19-24). We conclude that a minimum of 65 amino acid residues is required to span the distance between the ribosomal P-site and the OST active site.
Figure 2:
65
residues are required to bridge the distance between the P-site and the
OST active site. A, topology of the translocation
intermediates generated by translation of truncated Lep mRNAs. B, truncated transcripts were translated in wheat germ lysate
in the absence or presence of dog pancreas microsomes (RM) and
puromycin, and immunoprecipitated by a Lep antiserum. Lanes
1-3, truncation 39 residues away from the glycosylation
acceptor site at Asn; lanes 4-6,
truncation 51 residues away; lanes 7-9, truncation 56
residues away; lanes 10-12, truncation 61 residues away; lanes 13-15, truncation 63 residues away; lanes
16-18, truncation 64 residues away; lanes
19-21, truncation 65 residues away; lanes
22-24, truncation 66 residues away. Band a,
non-glycosylated Lep; band b: glycosylated
Lep.
We thus
constructed a protein, Lep-ST (Fig. 3A), that contains
an engineered glycosylation site Asn-Ser-Thr replacing
amino acids 200-202 and the stop-transfer sequence
QQQL
VKKKK inserted between amino acid residues 215 and
220. When expressed in the presence of microsomes, Lep-ST was
efficiently glycosylated on Asn
(Fig. 3B, lane 2) and glycosylation was inhibited by inclusion of a
glycosylation acceptor peptide (N-benzoyl-Asn-Leu-Thr-N-methylamide) in the reaction
mixture (lane 3). A short fragment representing the
glycosylated loop between the second transmembrane segment and the
hydrophobic stop-transfer sequence was protected from proteinase K
digestion (lanes 4 and 5), demonstrating that the
topology of Lep-ST was as depicted in Fig. 3A.
Figure 3: Lep-ST spans the microsomal membrane three times. A, topology of Lep-ST. The hydrophobic stop-transfer sequence is shown in black. B, Lep-ST was translated in vitro in reticulocyte lysate in the absence (lane 1) or presence (lanes 2-5) of dog pancreas microsomes. A glycosylation acceptor peptide (AP) was included in lanes 3 and 5 to block glycosylation of the protein. Parts of the protein exposed to the outside of the microsomes were digested by proteinase K (lanes 4 and 5). Band a: Non-glycosylated Lep; band b, glycosylated Lep; band c, glycosylated proteinase-protected fragment; band d, non-glycosylated proteinase-protected fragment.
Truncations were made at different sites downstream of the stop-transfer segment, and glycosylation in the presence of microsomes was assayed as above. As shown in Fig. 4, for truncations 52, 66, and 69 residues away from the potential glycosylation acceptor site no glycosylation was seen in the absence of puromycin (lanes 1-9). For truncations 70 and 71 residues away from the potential glycosylation site, there was a sharp increase in the amount of glycosylation observed in the absence of puromycin (lanes 10-15), and complete glycosylation was observed when the truncation was 74 and 91 residues away (lanes 16-21). It thus appears that the presence of a long hydrophobic stretch makes the nascent chain spanning the translocase complex more compact.
Figure 4:
71 residues are required to bridge the
distance between the P-site and the OST active site in the presence of
an 18-residue-long stop-transfer sequence. A, topology of the
translocation intermediates generated by translation of truncated
Lep-ST mRNAs. The stop-transfer sequence is shown in black. B, truncated transcripts were translated in wheat germ lysate
in the absence or presence of dog pancreas microsomes (RM) and
puromycin, and immunoprecipitated by a Lep antiserum. Lanes
1-3, truncation 52 residues away from the glycosylation
acceptor site at Asn; lanes 4-6,
truncation 66 residues away; lanes 7-9, truncation 69
residues away; lanes 10-12, truncation 70 residues away; lanes 13-15, truncation 71 residues away; lanes
16-18, truncation 74 residues away; lanes
19-21, truncation 91 residues
away.
By measuring the minimum number of residues required to bridge the distance between the ribosomal P-site and the OST active site, we have sought to indirectly determine the conformation of a nascent chain in transit through the ER membrane. Our data show that a translocating nascent chain corresponding to a globular domain of a protein needs to be extended by a minimum of 65 amino acid residues from the P-site in the ribosome to be able to interact with the OST active site (Fig. 5). This length is increased to 71 residues when a 18-residue-long hydrophobic stop-transfer sequence is present in the nascent chain.
Figure 5:
Glycosylation efficiency as a function of
the length of the nascent chain between the glycosylation acceptor site
and the end of the truncated nascent chain for Lep (glycosylated at
Asn, unfilled circles) and Lep-ST (glycosylated
at Asn
, filled circles). Values were normalized
by the efficiency of glycosylation measured in the presence of
puromycin (this normalization is important only for Lep-ST truncated 71
residues from the glycosylation acceptor site where overall targeting
to the microsomes was inefficient, possibly due to a new batch of wheat
germ lysate; cf. Fig. 4B, lanes
13-15).
These results are broadly consistent with
previously reported data, but have a significantly higher precision. An
early study provided a rough estimate of the P-site/OST distance of
45-95 residues(22) . More recently, it has been shown
that protease digestion of nascent secretory polypeptides in detergent
permeabilized microsomes results in protected fragments of around 70
amino acid residues(23) , and that photochemically induced
cross-linking between a nascent chain and Sec61 is possible at a
maximum distance of
70 residues away from the ribosomal
P-site(6) . Finally, it has been reported that disulfide bonds
can form in a translocating nascent chain when it reaches a length of
50-60 residues(24) .
We have shown previously that at
least 15 spacer amino acid residues are needed after the end of the
hydrophobic core of a N-C
-oriented
transmembrane segment in order for a potential acceptor site for
N-glycosylation to be glycosylated(11) . Furthermore, around 40
amino acid residues of a nascent chain are protected by free ribosomes
from protease digestion (23, 25) , and photochemical
cross-links to Sec61
can be seen at positions in a nascent chain
only 30 residues away from the P-site(6) . Taken at face value,
these results together imply the geometrical relationships depicted in Fig. 6. However, depending on the dimensions of the putative
ribosome/translocase channel and the conformational flexibility of the
nascent chain, a precise match between the results of the different
assays may not be expected.
Figure 6:
Geometry of the ER translocase complex.
The OST active site is located 15 residues away from the end of
the hydrophobic transmembrane segment ((11) , data not shown).
70 residues of the nascent chain are protease-protected in
detergent-solubilized microsomes (23) and also represent the
most distal position that can be cross-linked to
Sec61
(6) , 65 residues are required to bridge the
P-site/OST distance (this paper), 40 residues are protease-protected in
isolated ribosomes(23) , and cross-linking to Sec61
is
possible from position 30(6) . From the minimum length of a
nascent chain required for processing by signal peptidase (SPase), one can estimate that roughly 100 residues are needed
to span the distance between the signal peptidase active site and the
P-site in the ribosome(6, 30, 31) , implying
that the OST and signal peptidase active sites are not immediately
adjacent in the complex.
Electron microscopy studies of intact
ribosomes suggest that nascent chains exit the large ribosomal subunit
at a point 150 Å from the
P-site(26, 27, 28) . Assuming a typical
50-Å-thick membrane, this gives a first-order approximation to
the physical P-site/OST distance of
200 Å, suggesting that
the nascent chain must be able to bridge the distance between the
ribosomal P-site and the OST active site in a largely extended
conformation (
3.1 Å/residue, to be compared with 3.5 and 1.5
Å/residue in fully extended and
-helical conformations,
respectively). Since these physical dimensions are only rough numbers,
we cannot formally exclude that a short segment of the nascent chain is
in fact helical, although we consider this unlikely.
In agreement
with the suggestion of an extended conformation, we find that the
introduction of an 18-residue-long hydrophobic stop-transfer segment
between the glycosylation acceptor site and the end of the nascent
chain leads to an increase in the minimum glycosylation length of
6 residues, suggesting a more compact conformation of the nascent
chain. Since the hydrophobic segment is located only 19 residues
downstream of the glycosylation acceptor site and since the acceptor
site must be at least 17 residues away from the hydrophobic segment for
glycosylation to be possible in this construct, (
)the
stop-transfer segment is located within the translocase complex in the
critical constructs (truncations 69-74 residues away from the
acceptor site). In the translocase, stop-transfer segments are believed
to be in a partially lipid-exposed environment (12, 29) and thus most likely in a helical
conformation. Indeed, if 18 residues in a nascent chain were forced
from a fully extended to an
-helical conformation,
10 extra
residues would need to be added to the chain in order to bridge a
defined end-to-end distance. The 6-residue shift in the minimum
glycosylation length that we observe is thus in reasonable agreement
with an extended conformation for polar nascent chains and a helical
conformation for hydrophobic segments.
In summary, a model where the nascent polypeptide chains passes through a water-accessible translocase channel in a largely extended conformation is consistent with all the available data. Further, hydrophobic stop-transfer sequences appear to have a more compact, possibly helical conformation when located within the translocase, suggesting a more lipid-exposed environment.