(Received for publication, May 29, 1996, and in revised form, October 4, 1996)
From the School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT United Kingdom
We have investigated the molecular details of the
membrane insertion of the multiple-spanning membrane protein opsin.
Using heterobifunctional cross-linking reagents the endoplasmic
reticulum (ER) proteins adjacent to a series of defined translocation
intermediates were determined. Once the nascent opsin chain reaches a
critical minimum length Sec61 is the major ER component adjacent to
the polypeptide. Using a homobifunctional reagent, the cross-linking partners from a single cysteine residue in the nascent chain were analyzed. This approach identified chain length-dependent
cross-linking products between nascent opsin and a 21-kDa ribosomal
protein, followed by Sec61
and finally with Sec61
. Our data
support a model where the sequential transmembrane domains of a
multiple-spanning membrane protein are integrated at an ER insertion
site similar to that mediating the insertion of single-spanning
membrane proteins.
Membrane proteins are targeted to the endoplasmic reticulum (ER)1 membrane by a signal sequence which is usually a stretch of up to 20 apolar amino acid residues (1). The targeting process involves the recognition and binding of these signals by the signal recognition particle (SRP) (2). At the membrane, SRP interacts with the SRP receptor and, in a GTP-dependent manner, presents the nascent chain to the translocation/insertion machinery (2, 3).
Cross-linking studies have been used to identify ER proteins
responsible for the translocation of secretory proteins and the insertion of single-spanning membrane proteins (reviewed in Ref. 4).
Using a photocross-linking approach, Sec61 was identified as a major
cross-linking partner of the secretory protein preprolactin (5-7) and
type I and type II membrane proteins (8-11). Sec61
was also
identified as a major cross-linking partner of secretory and membrane
proteins using bifunctional cross-linking reagents (10, 12, 13).
Sec61
is part of a protein complex, together with Sec61
and
Sec61
(14, 15), and reconstitution studies showed that this Sec61
complex plus the SRP receptor are essential components for secretory
protein translocation and membrane protein insertion (14, 16).
Cross-linking also identified a second component of the translocation
machinery, the translocating chain-associating membrane protein (TRAM)
(8, 9, 11, 17-19). Reconstitution studies showed that TRAM is required
for the efficient insertion/translocation of a subset of membrane and
secretory proteins (14, 16, 19). While initial studies allowed
cross-linking from a number of positions within the nascent chain, more
recently site-specific cross-linking techniques have been developed,
allowing the environment of particular regions of the nascent chain to
be investigated (6-8, 20, 21).
Previous cross-linking studies have concentrated on secretory proteins,
and simple, single-spanning membrane proteins. In this study we have
analyzed the biosynthesis of opsin, a 39-kDa multiple-spanning membrane
protein which is targeted to the ER membrane by SRP (22, 23). Truncated
mRNAs were used to generate "translocation intermediates" which
remain associated with the ER translocation site due to the presence of
the ribosome at the C terminus of the truncated polypeptide (24).
Hetero- and homobifunctional cross-linking reagents were then used to
identify cross-linking partners of the inserting nascent chain. With
heterobifunctional reagents the only component of the ER translocation
site cross-linked to nascent opsin chains was Sec61. This adduct was
only observed when the nascent chain reached a critical minimum length.
In contrast, using the homobifunctional reagents we observed discrete
chain length-dependent cross-linking products between the
nascent chain and a 21-kDa ribosomal protein, followed by Sec61
, and
finally Sec61
.
The plasmid coding for opsin, pGEM3OP, was kindly
provided by Reid Gilmore, University of Massachusetts, Worcester, MA.
BsaHI, used to generate the 155-amino acid truncation of
opsin and the Endoglycosidase H (recombinant fusion protein) (Endo Hf)
were from New England Biolabs (Hitchin, Herts).
[35S]Methionine was supplied by Amersham (Amersham,
Bucks) and T7 RNA polymerase by Promega (Southampton, Hants). All
cross-linking reagents were purchased from Pierce & Warriner (Chester,
Cheshire) and Apollo Chemicals Ltd. (Stockport, Cheshire). The
concanavalin A-Sepharose was purchased from Pharmacia Biotech (St.
Albans, Herts). All other chemicals were purchased from BDH/Merck
(Poole, Dorset) and Sigma (Poole, Dorset). The
antibodies specific for SRP54, Sec61, TRAM, and Sec61
(under
denaturing conditions), were a gift from Bernhard Dobberstein, ZMBH,
Heidelberg, Germany. The Sec61
antisera used under "native"
conditions, and the Sec61
antisera, were both raised by Research
Genetics Inc. (Huntsville, AL) using peptides encoding the C-terminal
(
) and N-terminal (
) 12 amino acids of the published
sequences.
The coding region of bovine opsin was
subcloned from pGEM3OP into the plasmid pGEM3z using an
EcoRI/HindIII fragment. Most of the templates for
the transcription of different truncated opsin mRNAs were prepared
by PCR (25). The resulting opsin translocation intermediates were
all efficiently membrane integrated, glycosylated, and
immunoprecipitated by an anti-opsin monoclonal antibody. The upstream
primer was 160 bases 5 of the T7 RNA polymerase promoter and had the
sequence 5
-GGGCCTCTTCGCTATTACGC-3
, antisense primers were designed to
make 5 truncations of 106, 127, 132, 137, and 150 amino acids. For the
106-amino acid truncation (OP106) the primer 5
-GGCCCAAAGACGAAGTACCC-3
was used, 127-amino acid truncation (OP127) 5
-GGACCACAGTGCAATTTCAC-3
,
132-amino acid truncation (OP132) 5
-GGCCAGGACCACCAAGGACCA-3
,
137-amino acid truncation (OP137) 5
-ACCACGTACCGCTCGATGGC-3
, and for
the 150-amino acid truncation (OP150) the primer
5
-CTCCCCGAAGCGGAAGTTGCT-3
was used. The PCR products were purified
directly from the reaction mixture using the Wizard PCR purification
kit (Promega, Southampton, Hants.). The template for the 155-amino acid
truncation (OP155) was made by cleavage of the plasmid within the
coding region using the restriction endonuclease BsaHI.
The cysteine residue at position 140 was altered to a glycine using the Clontech Laboratories site-directed mutagenesis kit (Palo Alto, CA). Subsequent sequencing revealed two discrepancies from the published sequence (26), these were single base changes in the codons for amino acids 101 and 118 and did not alter the amino acid residue encoded. These changes were also present in the original plasmid.
In Vitro Transcription and TranslationTranscription of the
purified DNA was carried out as described by the manufacturer (Promega,
Southampton, Hants.). Translation of the resulting transcripts in a
rabbit reticulocyte lysate system (Promega, Southampton, Hants.) was
carried out at 30 °C in the presence of
[35S]methionine and canine pancreatic microsomes as
described by the manufacturer. Translation initiation was inhibited
after 15 min by the addition of 4 mM 7-methylguanosine
5-monophosphate, and chain elongation allowed to continue for a
further 10 min until translation was inhibited by the addition of 2 mM cycloheximide.
For cross-linking with bifunctional reagents,
the membrane associated components were isolated by centrifugation
through a high salt/sucrose cushion (250 mM sucrose, 500 mM KOAc, 5 mM Mg(OAc)2, and 50 mM HEPES-KOH, pH 7.9) for 10 min at 4 °C and 55,000 rpm (100,000 × g in a TLA100.2 rotor, Beckman
instruments). The resulting pellet was resuspended in a low
salt/sucrose buffer (250 mM sucrose, 100 mM
KOAc, 5 mM Mg(OAc)2, and 50 mM
HEPES-KOH, pH 7.9), a sample taken for trichloroacetic acid
precipitation, then N-hydroxysuccinimidyl iodoacetate,
succinimidyl 4-(p-maleimidophenyl)butyrate, MBS, BMP, or BMH
added to a final concentration of 1 mM, or S-MBS added at
0.12 mM (27). Following incubation for 10 min at 26 °C,
samples were quenched with 100 mM glycine, 5 mM
2-mercaptoethanol or 5 mM 2-mercaptoethanol alone (BMP and
BMH). A second sample was removed for trichloroacetic acid
precipitation. In order to establish whether PCR-induced mutations had
any effect; mRNAs derived from independent PCR reactions were
translated and the cross-linking products were compared. No differences
between independent experiments could be detected.
Denaturing immunoprecipitations were performed by heating the samples for 5 min at 95 °C in the presence of 1% SDS. Native immunoprecipitations were performed by releasing the ribosome from the nascent chain by incubation with 1 mM puromycin and 400 mM KCl for 10 min at 30 °C. Four volumes of immunoprecipitation buffer (10 mM Tris/HCl, pH 7.6, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100) were then added to all samples and aliquots were incubated overnight at 4 °C with the relevant antisera in the presence of 0.2 mg/ml phenylmethylsulfonyl fluoride and 1 mM methionine. Protein A-Sepharose was added for 2 h and samples processed as described previously (see Ref. 28). All samples were analyzed on 12% SDS-polyacrylamide gels and exposed overnight to a PhosphorImaging plate for visualization on a Fuji BAS-2000 PhosphorImaging system. Concanavalin A binding was performed as described by Krieg et al. (18).
In this study opsin "translocation intermediates" were used to investigate the insertion of a model multiple-spanning protein into the ER membrane using cross-linking techniques. A translocation intermediate is a truncated nascent chain which, due to the lack of a stop codon in the truncated mRNA, is not released from the ribosome and becomes trapped in the translocation machinery (24).
Prior to cross-linking analysis, the topology of the 155-amino acid
long opsin truncation (OP155cko) was established using a protease
protection assay (Fig. 1a). Nascent chains
protected against protease digestion were detected by
immunoprecipitation with an antibody specific for the N terminus of
opsin. Proteinase K digestion of OP155 after puromycin release resulted
in a large protected fragment which migrated slightly faster than the
undigested form (OP155-2CHO) (Fig. 1a, cf. lane 2, asterisk, with lane 1). This altered mobility is due to
cleavage of the exposed C terminus (cf. Fig. 1b).
Upon Endo Hf digestion, this large protected band shifted to migrate
with a mobility similar to the nonglycosylated nascent chain (OP155)
(Fig. 1a, lane 3, asterisk). A fraction of the proteinase
K-treated material had a smaller molecular weight after Endo Hf
treatment (Fig. 1a, lane 3) indicating that there was some
cleavage within the cytosolic loop of OP155 (cf. Fig. 1b). Digestion with trypsin after release of the ribosome
also resulted in a large protected fragment (Fig. 1a, lane 5, asterisk). In this case no cleavage of the C terminus was apparent
and cleavage in the cytosolic loop was again relatively inefficient,
despite two potential cleavage sites for trypsin at lysine residues 66 and 67 (cf. Fig. 1b). However, trypsin digestion
of the intact translocation intermediate (i.e. in the
presence of the ribosome) significantly enhanced cleavage within the
cytosolic loop to produce the fragment OP67-2CHO (Fig. 1a, lane
6, asterisk). Endo Hf digestion resulted in a ~7-kDa fragment
corresponding to an N-terminal polypeptide of ~67 residues with both
carbohydrate side chains removed (OP67) (Fig. 1a, lane 7, asterisk). Thus, the first transmembrane domain of the opsin
translocation intermediate is fully integrated in the correct
orientation with the N terminus glycosylated and the cytosolic loop
region sensitive to protease. Nonglycosylated OP155 chains (see Fig.
1a, lane 1) are not protease resistant and are therefore not
membrane integrated but are most likely ribosome-bound material which
co-isolates with the membrane fraction (see later). The predicted
cytosolic loop between the first and second transmembrane domains is
particularly sensitive to trypsin when the nascent chain is still
ribosome bound suggesting that after release the polypeptide chain
assumes a different conformation (cf. Fig. 1b). Control digestions in the presence of Triton X-100 resulted in total
loss of detectable nascent chain (Fig. 1a, lanes 4 and
8). Similar analysis showed the 127-amino acid nascent chain
(OP127) is also correctly inserted with the N terminus glycosylated and protease protected (data not shown).
Sec61
To investigate the proteins adjacent to membrane
inserting opsin nascent chains the heterobifunctional cross-linking
reagent m-maleimidobenzoyl-N-hydroxysuccinimide
ester (MBS) (reacting principally with cysteine and lysine residues,
see Ref. 13) was added to opsin translocation intermediates. The two
shortest chains analyzed were 106 and 137 amino acids in length (OP106 and OP137, respectively), these translocation intermediates have pronounced peptidyl-tRNA species of ~32 and ~37 kDa as previously reported for other nascent chains (7, 29). These peptidyl-tRNA species
are lost upon puromycin treatment of the samples (compare Fig.
2a, lanes 9 and 10 with Fig. 5,
lanes 1 and 2). Both OP106 and OP137 were only
found to be cross-linked to SRP54 (Fig. 2a, lane 4 and
12, open arrow). Only when the nascent chain was 155 amino
acids in length (OP155cko) was cross-linking to Sec61 observed (Fig.
2a, lane 21, closed arrow). Sec61
was visible as a
doublet within the molecular mass range 46-60 kDa (Fig. 2a,
lanes 18, and 21, closed arrow) similar to
cross-linking products previously identified using bifunctional
reagents (see Refs. 10 and 12). This doublet probably reflects
cross-linking of the nascent chain to different sites within
Sec61
.2 No cross-linking of Sec61
alone was visible (cf. Fig. 3), however, a
high molecular weight product was specifically immunoprecipitated with
anti-Sec61
serum under denaturing conditions (Fig. 2a, lane 22, unfilled arrow). This product must therefore include the
nascent chain, Sec61
and at least one unidentified protein. A high
molecular weight product was also immunoprecipitated with antisera
recognizing TRAM (Fig. 2a, lane 23, broad band), again
suggesting multiple cross-linking.
Bifunctional reagents similar to MBS but with shorter
(N-hydroxysuccinimdyl iodoacetate) and longer (succinimidyl
4-(p-maleimidophenyl)butyrate) spacer arms were also used.
Both gave very similar results to MBS showing that the length of the
spacer arm did not affect the cross-linking partners detected (data not
shown). However, when S-MBS, a water soluble analogue of MBS was used,
fewer cross-linking products were visible (Fig. 2b). SRP54
was still a cross-linking partner of all three nascent chains (Fig.
2b, lanes 4, 12, and 20, open arrows), but with
OP155cko, only Sec61 was detected (Fig. 2b, lanes 18 and
21, closed arrow). None of the higher molecular weight
species immunoprecipitated by antisera against Sec61
and TRAM were
visible (Fig. 2b, lanes 22 and 23) suggesting
that penetration of the bilayer by the cross-linking reagent was
necessary to obtain these products, but not for cross-linking to
Sec61
.
Our principal conclusion from this initial analysis is that Sec61 is
only seen as a major cross-linking partner when the opsin nascent chain
is 155 amino acids in length. This result was unaffected by either the
length of the spacer arm, or the solubility of the reagent used. The
simplest interpretation of these results is that cross-linking of
nascent opsin to ER membrane components occurs primarily from cysteine
residue 110 of the nascent chain, and that this residue is only close
to Sec61
when it has entered the plane of the membrane
(cf. Fig. 1c).
To further investigate ER proteins adjacent to cysteine residue 110, two cysteine specific cross-linking reagents with different length spacer arms, bismaleimidopropane (BMP, 11 Å) and bismaleimidohexane (BMH, 16.1 Å), were used. All known components of the ER translocation site have one or more cysteine residues (5, 15, 17), therefore the use of cysteine-specific homobifunctional reagents does not in principle restrict the cross-linking partner.
When opsin translocation intermediates were isolated and incubated with
the cross-linking reagent BMP, specific cross-linking products were
observed dependent upon the length of the translocation intermediate
(Fig. 3). OP106 contained no cysteine residue, and no cross-linking
products were observed (Fig. 3, lanes 1-8). OP127, OP132,
and OP137 all formed cross-linking products of apparent molecular mass
~42 kDa. Subtracting the contribution of the nascent chain, this
indicated each opsin translocation intermediate was cross-linked to a
protein of ~21 kDa (Fig. 3, lanes 10, 18, and 26, asterisk) which was not a known component of the ER targeting or
translocation machinery. In contrast, OP137, OP150cko, and OP155cko
all showed discrete cross-linking products with Sec61 (Fig. 3,
lanes 30, 38, and 46, diamond). The first obvious
evidence of Sec61
cross-linking products was only obtained when
OP155cko was used (Fig. 3, lane 45, closed arrow). The
Sec61
cross-linking product was a broad doublet similar to that seen
with MBS and S-MBS. In addition to the nascent chain-Sec61
adduct, a
high molecular weight product identical to that observed with MBS was also seen (Fig. 3, lane 46, unfilled arrow, cf. Fig.
2a, lane 22). Analysis using BMH gave similar results, again
showing that the cross-linking partners were not limited by the spacer
arm length of the cross-linking reagent used (data not shown).
A number of opsin translocation intermediates are cross-linked to SRP54
(Figs. 2, a and b, and 3). Using a floatation
assay (30) to separate membrane targeted nascent chains from
non-targeted chains, we analyzed the OP155-SRP54 adducts and found
these products fractionated with non-targeted nascent chains. In
contrast, the Sec61 and Sec61
cross-linking products were present
in the membrane fraction (data not shown). We conclude that the SRP54
adducts arise from ribosome bound opsin chains which are pelleted with the membrane fraction prior to cross-linking, but which have not interacted productively with the ER microsomes used (see also results
of protease protection above). Such incomplete release of SRP54 by
canine pancreatic microsomes has been previously reported (10).
The reticulocyte lysate translation system has been reported to
generate cross-linker-independent adducts under some circumstances (31). When control immunoprecipitations were performed without adding
cross-linking reagents, a weakly labeled OP155-SRP54 adduct was
observed (data not shown). No such products were immunoprecipitated with any of the other antisera used in this study confirming that the
21-kDa protein, Sec61 and Sec61
are all bona fide cross-linking partners.
Cross-linking of OP127,
OP132, and OP137 with BMP resulted in a single major cross-linking
partner of 21 kDa (Fig. 3, lanes 10, 18, and 26, asterisk) which was not a known component of the ER insertion
machinery (see Fig. 3, lanes 11-16, 19-24, and
27-32) but was immunoprecipitated with a monoclonal antibody
specific for opsin (data not shown). Cysteine residue 110 of OP127 is
predicted to be deeply buried in the ribosome (Fig. 1c) and
if the first transmembrane domain of OP127 is correctly inserted into
the membrane the nascent chain should be glycosylated and hence any
cross-linking products sensitive to Endo Hf digestion (Fig.
4). Endo Hf digestion does indeed increase the mobility
of both the OP127 cross-linking product (Fig. 4, lane 4, asterisk) and the diglycosylated nascent chain (indicated with a
dot). Thus, a correctly inserted and glycosylated OP127
translocation intermediate is cross-linked to a 21-kDa protein. The
21-kDa component was still a cross-linking partner in the absence of
membranes (Fig. 4, lane 6), indicating that the 21-kDa protein is of ribosomal origin. The cross-linking product was not
immunoprecipitated by antibodies recognizing the subunit of the
nascent polypeptide-associated complex, a previously identified 21-kDa ribosome-associated protein (30, 32).
The Sec61
Cross-linking of OP137 with
homobifunctional cysteine-specific reagents resulted in a discrete
Sec61-OP137 cross-linking product (Fig. 3, lane 30, and
data not shown). This product could be immunoprecipitated with antisera
against both opsin (Fig. 5, lane 2, diamond)
and Sec61
(Fig. 5, lane 3). The cross-linking product
bound to concanavalin A-Sepharose (Fig. 5, lane 4) and was
specifically eluted by 0.5 M
-methyl-D-mannoside (data not shown) indicating the
presence of a carbohydrate moiety. Since canine Sec61
does not
contain any potential N-linked glycosylation sites (15), the
carbohydrate present on the cross-linking product is from the opsin
nascent chain. Sec61
is therefore cross-linked to a correctly
inserted OP137 translocation intermediate. The OP137-Sec61
cross-linking product was also found to be sensitive to Endo Hf
digestion (data not shown).
The truncated opsin nascent chains used in this study were shown to insert into canine pancreatic microsomes and form bona fide translocation intermediates. Once the nascent chain was trapped in the translocation machinery it could be cross-linked to adjacent proteins.
Using heterobifunctional reagents, and nascent polypeptides ranging
from 106 to 155 amino acid residues in length, SRP54 adducts were
observed with all chain lengths. These products represent SRP-bound
nascent chains which have failed to target to the membrane. In striking
contrast, strong cross-linking of membrane translocation intermediates
to Sec61 was only observed with OP155cko, the longest of the nascent
chains studied. This result was independent of the spacer arm length
and solubility of the heterobifunctional reagents used suggesting that
the majority of cross-linking products obtained are from the -SH group
of cysteine residue 110 in opsin to -NH2 groups in the
adjacent ER protein (see Fig. 1c). We conclude that cysteine
110 is only sufficiently close to Sec61
for cross-linking when it is
45 amino acid residues from the peptidyl transferase site of the
ribosome, i.e. within the plane of the membrane. This is
consistent with Sec61
being the major protein constituent of the ER
insertion and translocation site (4-7, 10, 12-14, 16).
A comparison of membrane permeable and membrane impermeable (water
soluble) cross-linking reagents revealed a clear difference. Membrane
permeable reagents generated discrete high molecular weight products
specifically immunoprecipitated with antisera against Sec61 and
TRAM. These "multiple" cross-linking products must represent
OP155cko-Sec61
and OP155cko-TRAM products that are in turn
cross-linked to other components that remain to be identified (see
below). Such products were not seen when S-MBS was used although
cross-linking to Sec61
was maintained. Hence, penetration of the
bilayer is necessary to obtain the high molecular weight Sec61
and
TRAM products. Thus, analysis with heterobifunctional reagents fully
supports the view that Sec61
is the major protein component of an
aqueous channel (7, 33, 34) through which the nascent opsin chain is
inserted into the ER membrane. Sec61
and TRAM are adjacent to both
the nascent chain and additional, as yet unidentified, proteins.
To investigate the environment of a discrete region of the nascent
chain, the cross-linking partners of a single cysteine residue were
identified at different stages of the translocation process (Fig.
1c) using BMP and BMH. The first detectable cross-linking partner of the opsin nascent chain was a 21-kDa putative ribosomal protein. The protein is not the subunit of the nascent
polypeptide-associated complex, and may be identical to a previously
described ribosomal component detected as a photocross-linking partner
of short nascent luciferase chains (32).
When the chain length of the translocation intermediate was increased
to 137 amino acids, cross-linking to Sec61 was observed. Sec61
is
part of the Sec61 complex composed of Sec61
, Sec61
, and Sec61
(14) and is probably a tail-anchored membrane protein with a single
cysteine present in the cytoplasmic N terminus (15). This is the first
direct evidence that a membrane inserting nascent chain is adjacent to
Sec61
. Cross-linking to Sec61
was shown to be from glycosylated,
membrane inserted, translocation intermediates. Since the single
cysteine residue present in OP137 is probably still within the ribosome
(Fig. 1c) this suggests that the cytoplasmic domain of
Sec61
extends into the channel of the large ribosomal subunit.
After initial cross-linking of the nascent chain to Sec61 alone,
increasing the nascent chain length to 155 residues (OP155cko) allows
cross-linking to both Sec61
and Sec61
. Sec61
is proposed to be
the major protein component of the ER translocation site (5-7, 10, 12,
13). Thus, these data support the view that the integration of
multiple-spanning membrane proteins occurs at a general ER
translocation site, very similar to that which promotes the insertion
of single-spanning membrane proteins and the translocation of secretory
proteins (4, 35).
In addition to adducts between OP155cko and Sec61 alone, more
complex high molecular weight products were observed with all cross-linking reagents used. These cross-linking products are not
immunoprecipitated with antisera specific for Sec61
and therefore contain additional unidentified protein(s) which may be ribosomal (see
above).
These results are consistent with the ribosome and the ER translocation
site forming a continuous channel (33) where some ER protein components
extend into the ribosomal channel (7, 36). Sec61 appears to be the
major constituent of this membrane channel, our data suggest that
Sec61
may also contribute to the environment. Alternatively,
Sec61
may play a distinct role such as regulating the lateral exit
of transmembrane domains from the ER translocation site into the lipid
bilayer (4). Recent evidence has shown this to be a complex process
which may involve TRAM (8).
The approach used in this study only enables the detection of proteins
adjacent to the inserting opsin nascent chains. Recent photocross-linking analysis suggests that the translocation site is
partly composed of phospholipid, perhaps promoting the lateral exit of
hydrophobic transmembrane domains into the bilayer (20). Our results
suggest that both Sec61 and Sec61
are significant components of
the translocation channel, however, they do not preclude the
possibility that lipid also makes a significant contribution to the
site of opsin integration at the ER membrane. We are presently attempting to address this question.