| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(Received for publication, July 5, 1995) From the
Microsomal cytochrome P450 is inserted into the membrane of the
endoplasmic reticulum (ER) by its N-terminal signal/anchor sequence
which also functions as an ER retention signal. To analyze further
potential retention signals of cytochrome P450, topological domains of
cytochrome P450 2C1 or 2C2, epidermal growth factor receptor, a plasma
membrane protein, and bacterial alkaline phosphatase, a secreted
protein were exchanged. The N-terminal signal/anchor of cytochrome P450
2C1 functioned as an ER retention signal when placed at the N terminus
of several reporter proteins but not when fused at the C terminus of
the extracellular domain of epidermal growth factor receptor, with or
without a heterologous cytoplasmic domain. Chimeric proteins in which
the cytoplasmic domain of cytochrome P450 2C2 was substituted for that
of epidermal growth factor receptor were retained in the ER indicating
that an independent retention signal is present in the cytoplasmic part
of cytochrome P450 2C2. These chimeras were enzymatically active which
argues against misfolding as the primary cause of retention. The ER
retention signal of the cytoplasmic domain could not be localized to a
single amino acid segment by deletion analysis. These results show that
cytochrome P450 2C2 contains redundant, complex ER retention signals in
its cytoplasmic and N-terminal hydrophobic domains and that the
function of the N-terminal signal is context-dependent.
In eukaryotic cells, the endoplasmic reticulum (ER) ( In general, ER proteins can achieve their specific
localization in two ways: by direct retention or by retrieval from
distal compartments in the pathway. Best studied is the mechanism of
retention of soluble luminal ER proteins, for which a C-terminal KDEL
(HDEL for yeast proteins) sequence has been shown to function as an ER
retention signal (2) . Since the receptor recognizing this
signal is located in the intermediate compartment and Golgi, it is
believed that ER retention of KDEL-containing proteins is achieved by
their retrieval from early Golgi(3, 4) . New studies
suggest that, at least for some proteins, the KDEL sequence is not
sufficient to ensure ER retention and additional structural motifs may
play a role in ER localization(5, 6, 7) . Much less information is available about the mechanism of ER
retention of integral membrane proteins. For some of the ER membrane
proteins, a C-terminal sequence of KKXX or KXKXX has been shown to serve as an ER retention signal in a manner
similar to the KDEL signal, i.e. it also functions in
retrieval of these proteins from the early Golgi
compartment(6, 8, 9) . However, in some
proteins this sequence seems to be a non-essential, redundant signal,
and primary ER retention is achieved by a different mechanism (10) . It is not known how ER retention of some of its most
crucial membrane components is achieved. Typical ER membrane proteins,
such as ribophorins, cytochromes P450 (P450) and their reductase, and
cytochrome b The hydrophobic N-terminal
region of P450 is thought to be inserted in the ER membrane whereas the
remaining part of the protein is on the cytoplasmic side of the
membrane(13, 14, 15, 16) . In
addition to the N terminus, some other sequences of the protein may
also interact with, but not span, the
membrane(17, 18) . The 29 N-terminal amino acids of
P450 2C1 and 2C2 can induce ER retention when fused to the N terminus
of reporter proteins that normally are cytoplasmic or
secreted(19) . Similar conclusions, mapping the ER retention
signal to the N terminus of P450 have been reached by Murakami et
al.(20) . These studies with chimeric proteins indicated
that the cytoplasmic domains are not required for ER retention but did
not test whether this domain might also contribute to ER retention.
P450 is probably restricted to the ER in a manner that excludes this
protein from the recycling pathway and its large, bulky cytoplasmic
domain was suggested as a possible reason for exclusion from the
transport vesicles(11, 20) . It was, therefore, of
interest to further analyze whether the cytoplasmic domain, in the
absence of the N-terminal ER retention signal, can be transported out
of the ER. We now report that both the N-terminal and the cytoplasmic
domains of P450 2C1 and 2C2 contain independent redundant signals for
retention in the ER.
Figure 1:
Construction of plasmids encoding the
chimeric proteins. The names of the plasmids used for the isolation of
desired DNA fragments are given below each line representing the
constructions, which are labeled on the right side. The
following restriction enzymes were used to isolate DNA fragments: AccI (AC), BstXI (BS), NcoI (NC), SmaI (SM), KpnI (KP), HindIII (HD), DraIII (DR), BamHI (BM), PvuII (PV), EcoRI (R1), XbaI (XB), and SphI (SP). When needed, blunt-end
DNA fragments were obtained by treatment with, either, T4 DNA
polymerase (T) or Klenow fragment of DNA polymerase (K). PCR indicates DNA fragments prepared by
polymerase chain reaction amplification using oligonucleotide primers
described under ``Experimental Procedures'' and the indicated
plasmid DNA as template. The length of the lines for each fragment is
only illustrative and the actual size, in kilobases, of the fragments
is indicated by the numbers above the
lines.
Chimera ECC contains the extracellular domain
of EGFR attached to full-length P450 2C1. Since P450 2C1 has only
10-20% of the lauric acid hydroxylase activity of P450
2C2(21) , we subsequently exchanged the cytoplasmic domain of
P450 2C1 in ECC with the corresponding domain of P450 2C2, as shown in Fig. 1, to create ECC`. To create chimera EEC, the
extracellular and transmembrane domain of EGFR was attached to the
cytoplasmic domain of P450 2C2, beginning with residue 29. Since the
presence of P450 2C2 amino acids 21-28 seems to be important for
its enzymatic activity, ( Following are the pairs of oligonucleotide
primers used for PCR in various constructions: pCMV-OEE, KpnTM,
5`-GTAGGTACCATGATCGCCACTGGGATGGT-3`, and the M13 universal primer;
pCMV-OEC, KpnTM, and
5`-AGGAAGCTTCCCTCCCCCATGGCTCTGTTTTCGCATGAAGAGGCCGATCCC-3`; pCMV-EECI1,
which encodes a chimera with P450 2C2 amino acids 164-198
deleted, 5`-GCCAGCTGGAAAGTTCAATGAA-3` and c2END,
5`-GCGGATCCAAACTGTCCA-3`; pCMV-EECI2, which encodes a chimera with P450
2C2 residues 208-348 deleted, 5`-GCGAATTCACGCATGCCCTACACGGAT-3`
and c2END; pCMV-EECNC1, in which both, N- and C-terminal sequences of
P450 2C2 are deleted and the internal P450 region (residues
154-410) was amplified, 5`-GCAGGTACCATCATGGTGGAGGAGCTGAG-3` and
5`-GCTCTAGAAGTGGCCAGGGTCAAAC-3`; pCMV-EECN2, which encodes a deletion
of 200 residues from the N terminus of P450 2C2,
5`-GCCAGCTGGAAAGTTCAATGAA-3` and c2END; and pCMV-EECN3, which encodes a
deletion of 270 residues from the P450 N terminus,
5`-GCCAGCTGAACAGGAAAAGTGT-3` and c2END.
Figure 2:
Schematic structure of natural and
chimeric proteins. The three letters represent the luminal,
transmembrane, and cytoplasmic domain, respectively, and E refers to EGFR, C to P450, P to alkaline
phosphatase, and O indicates lack of any domain. The number below each box (stippled for P450, hatched for alkaline phosphatase, and open for EGFR)
indicates the number of amino acids in each domain. In EEP and ECP, the
intracellular domain is alkaline
phosphatase.
In agreement with our previous observations, a chimeric protein,
OCE, containing a hydrophobic signal/anchor peptide of P450 at the N
terminus is retained in the ER, as indicated by immunofluorescent
staining of reticular intracellular membranes (Fig. 3A)
while the analogous protein in which the luminal domain of EGFR was
deleted (OEE) was present in the plasma membrane. Interestingly, OEC, a
chimera with only the cytoplasmic domain of P450 fused to the
transmembrane domain of EGFR also exhibited a reticular pattern by
immunofluorescence staining suggesting retention in the ER. These
results were confirmed by cellular fractionation studies (Fig. 3B). After a 30-min pulse and 4 h of chase, OCE
was restricted to the ER fraction while substantial amounts of OEE were
in the plasma membrane fraction. OEC was also restricted primarily to
fractions containing ER membranes with no protein present in the plasma
membrane. These results suggest that the cytoplasmic domain as well as
the N-terminal sequence have ER retention properties. The reason for
the differences in the fractionation pattern of OCE and OEC is not
clear but may reflect differential distribution into rough or smooth
ER, the latter being the normal site of P450 in the liver.
Figure 3:
Subcellular localization of chimeric
proteins analyzed by immunofluorescence (panel A) and
fractionation of transfected cells (panel B). A, COS1
cells transfected with plasmids encoding OCE, OEE, and OEC were fixed,
permeabilized, and stained with the antibody against the intracellular
domain of EGFR which does not cross-react with endogenous EGFR (for OCE
and OEE) or anti-P450 2C3 antibody (for OEC) and, subsequently, with
FITC-conjugated IgG. B, COS1 cells transfected with plasmids
encoding OCE, OEE, and OEC were labeled for 30 min and subsequently
chased for 4 h. Cellular homogenates were centrifuged through a
discontinuous sucrose gradient, and each of 12 collected fractions was
immunoprecipitated with the corresponding antibody, as described in panel A. Localization of marker enzymes (NADPH-cytochrome c reductase for the ER, galactosyltransferase for the Golgi,
and 5`-nucleotidase for the plasma membrane) is indicated above the
autoradiogram.
To
further examine whether the cytoplasmic domain of P450 has ER retention
properties, chimeras which contain the glycosylated extracellular
domain of EGFR were studied (Fig. 4). Transfected COS1 cells
were pulse-labeled with Tran
Figure 4:
Endoglycosidase H sensitivity of chimeric
proteins synthesized in COS1 (panel A) and CHO (panel
B) cells. Cells transfected with plasmids encoding chimeras ECC,
EEC, ECO, and EEO were labeled with Tran
Since
EGFR is expressed in COS1 cells, the endogenous EGFR might interact
with the chimeric proteins forming unnatural dimers that were retained
in the ER. To exclude this possibility, we also expressed these
chimeras in CHO cells that do not express detectable amounts of EGFR.
In these cells, both ECC and EEC were sensitive to endo H digestion
after a 30-min labeling pulse and remained sensitive after a chase of 4
h (Fig. 4B). EGFR expressed in these cells became
completely resistant to endo H after a 4-h chase (not shown). In
contrast, analogous chimeras which did not contain the cytoplasmic
domain of P450 were not retained in the ER. Both ECO and EEO, which
have the cytoplasmic domain deleted, became resistant to endo H (Fig. 4A), and we have shown that a chimera with a
substitution of alkaline phosphatase for the EGFR cytoplasmic domain
(EEP) is also transported to the plasma membrane(19) . These
data demonstrate that the cytoplasmic domain of P450 is responsible for
the retention of EEC in the ER which suggests that an efficient ER
retention function exists in the cytoplasmic part of P450 that is
independent of the N-terminal transmembrane domain.
Figure 5:
Endoglycosidase H sensitivity of deletion
mutants. A, schematic localization of deletions introduced
into the cytoplasmic domain of P450. The single line represents the deleted fragment, whereas the bar stands
for the P450 fragment(s) remaining in the chimera EEC. The top bar represents full-length P450 (490 amino acids) in which first 28
amino acids (membrane-binding domain), that are deleted in chimera EEC,
are shown by a single line. B, COS1 cells,
transfected with the corresponding deletion mutants, were processed as
described in legend to Fig. 2. The expressed proteins were
immunoprecipitated with the antibody against P450 2C3 (for EECI1 and
EECI2) or the extracellular domain of EGFR (for all remaining chimeras)
and digested with endo H.
More extensive
deletion of the internal sequences of the cytoplasmic part of P450,
EECI2, also did not affect sensitivity to endo H digestion. On the
other hand, deletion of the C-terminal 152 amino acids, as in chimera
EECC1, caused partial release of ER retention as indicated by partial
resistance to endo H. However, a significant fraction of this mutant is
efficiently retained in the ER, since endo H sensitivity could be
observed even after prolonged chase (not shown). Since in this mutant
the deleted region contains the Cys which is the fifth ligand for the
heme, it is clearly inactive enzymatically. The loss of ER retention
function as a result of C-terminal deletions was more dramatic if
combined with an N-terminal deletion. Deletion of only 78 C-terminal
amino acids from EECN1, which was sensitive to endo H, resulted in a
chimera (EECNC1) completely resistant to endo H. These results indicate
that the ER retention mediated by the cytoplasmic domain of P450 is not
encoded by a single primary sequence and may be the result of either a
specific three-dimensional structure of the folded protein or
interaction of specific segments localized in the N-terminal and
C-terminal part of this domain.
This
possible position effect was further analyzed using chimera ECE, EEP,
and ECP. Chimera ECE is EGFR with its transmembrane segment replaced by
the P450 N-terminal 29 amino acids and ECP is similar except that
alkaline phosphatase is substituted for the EGFR intracellular domain.
EEP, which is transported to the plasma membrane (19) , was
used as a control. The chimeras and EGFR all were resistant to endo H
digestion, consistent with their transport out of the ER (Fig. 6). Since the antibody used cross-reacts with endogenous
EGFR in COS1 cells, which is the same size as ECE, ECE was also
expressed in CHO cells, which are devoid of an endogenous EGFR.
Immunofluorescent staining of the surface of non-permeabilized cells
transfected with chimera ECE provides additional evidence that this
protein is transported to the plasma membrane (Fig. 7). These
results indicate that the loss of ER retention function of the P450
N-terminal signal in ECO is not the result of its extreme C-terminal
location, since addition of cytoplasmic domains did not affect its
function. Consequently, the fusion of the extracellular domain of EGFR
at the N terminus is responsible for the loss of this function.
Figure 6:
Endoglycosidase H sensitivity of chimeric
proteins EEP, ECP, ECE, and native EGFR. Transfected COS1 cells were
processed as described in legend to Fig. 2. For
immunoprecipitation of the expressed proteins, the antibody against the
extracellular domain of EGFR was used for EGFR, ECE, and Mock, whereas
EEP and ECP were immunoprecipitated with anti-alkaline phophatase
antibody.
Figure 7:
Immunofluorescence localization of EGFR
and ECE in non-permeabilized CHO cells. CHO cells, transfected with
plasmids encoding EGFR or ECE, were fixed and stained with the antibody
against the extracellular domain of EGFR, followed by FITC-conjugated
IgG.
Studies on the subcellular localization of P450 2C2 indicate
that it is directly retained in the ER and does not undergo recycling
from the intermediate or Golgi compartment(11) . The
hydrophobic N-terminal signal/anchor sequence of P450 has been shown to
be sufficient to restrict localization of several reporter proteins to
the ER membrane (19, 20) . Murakami et al.(20) have shown that the ER retention of a chimera
mediated by the P450 (M1) N-terminal signal also does not involve
recycling. Thus, it would appear that the exclusion of P450 from the
recycling pathway is mediated by its N-terminal ER retention signal.
The location of this signal in the N-terminal transmembrane domain is
consistent with the transmembrane location of targeting signals for
Golgi specific proteins(25, 26, 27) . The
double lysine ER retention motif of some type 1 ER membrane proteins is
also located close to the transmembrane region(8) . Membrane
properties or proteins specific to an organelle may mediate the
retention function of such membrane-localized signals. The binding of
membrane-associated coatomers to a double-lysine motif may be an
example of such an interaction(28, 29) . In spite of
the experimental evidence for a retention signal in the N-terminal
region of P450 and these mechanistic considerations, the data presented
indicate that P450 2C1 and 2C2 contain a second ER retention signal in
the cytoplasmic domain which is able to redirect the localization of a
plasma membrane protein to the ER independent of the N-terminal region. Since artificial fusion proteins are used to demonstrate the ER
retention function of the cytoplasmic domain, it is possible that the
retention is artifactually induced by misfolding of the chimeric
protein. Misfolded luminal and transmembrane proteins are retained in
the ER(30) . However, the ER retained chimeric proteins, ECC`,
EEC`, and OEC, retain enzymatic activity similar to that of P450 2C2
and, therefore, the P450 domain in most of these molecules must be
correctly folded. The transport of chimera like EECN2 from the ER
indicate that the EGFR domain is also correctly folded and,
parenthetically, that a presumably incorrectly folded P450 fragment
does not trap the protein in the ER. In ECC, the large luminal domain
should force a luminal orientation of the N terminus of the P450
transmembrane region. Since this orientation is compatible with
activity of the enzyme, this result provides additional evidence for
the luminal orientation of the N terminus of P450 (11) which
has been a matter of some debate(16) . There are several
potential mechanisms by which the cytoplasmic domain of P450 could
mediate ER retention. ER retention signals of soluble luminal proteins
and some type 1 membrane proteins are simple linear sequences which
interact with receptors. Retention in the ER by the cytoplasmic domain
of P450 probably involves a different mechanism. First, P450 appears to
be restricted to the ER without recycling. Second, deletions within the
P450 domain of EEC suggest that either the retention signal is encoded
by two separate sequences (one in the region 160-200 and the
other in the C-terminal half of the protein) or that it is a
``patch'' signal, made of several interacting regions. The
160-200 and the C-terminal regions contain two segments of high
hydrophobicity within P450 so that peripheral interactions with the
membrane may be important in the retention. This view is consistent
with evidence that regions in the cytoplasmic domain of P450s are
associated with the membrane, in addition to the N-terminal
transmembrane domain(17, 18) . A second mechanism for
retention of ER proteins may be the formation of networks that are
excluded from the transport vesicles(10, 12) .
Oligomerization as a mechanism for retention has been suggested for
some Golgi proteins (31, 32) and for p63, a protein
localized in cis-Golgi network(33) . P450 is known to
interact with other ER membrane proteins, such as P450 reductase, as
part of its enzymatic activity, and it has been suggested that
microsomal P450s form oligomeric complexes, possibly
hexamers(34, 35) . The retention of EECN1 and EECC1
which are presumably inactive molecules, missing 163 N-terminal amino
acids and the Cys that is the fifth ligand for heme, respectively,
eliminates normal interactions in the enzymatic process as part of the
retention mechanism. Self-aggregation or interaction with other
membrane or cytoplasmic proteins to form a network or a
receptor-mediated process remains possible. The ER retention
function of the N terminus of P450 seems to be dependent on its context
in the chimeras. When at the N terminus of the protein, which is its
normal position in P450 2C1, it causes the retention of several
reporter proteins(19) . However, when the EGFR luminal domain
is fused to the beginning of the N-terminal signal sequence, as in ECE,
ECP, or ECO, the chimeric proteins are transported from the ER to the
plasma membrane. In contrast, a relatively short sequence of 29 amino
acids containing a glycosylation site preceding this signal did not
interfere with retention function(19) . The N-terminal
retention signal of P450 (M1) was also functional in retention when
placed at the C terminus of a chimera(20) . These results
suggest that properties of the protein fused at the N terminus of the
retention signal may determine whether it is functional or not. One
possibility is that the EGFR extracellular domain has positive signals
for transport out of the ER which override the retention signal in the
P450 N-terminal region. This seems unlikely since EEC and ECC are
retained in the ER, although if such a positive signal was dominant to
the N-terminal but not cytoplasmic retention signals it would be
possible. Further studies will be required to determine definitively
the reason for the different effects of protein sequence fused at the
beginning of the N-terminal retention signal on its function. The
context-dependent nature of the N-terminal signal also suggests that
the length of the hydrophobic core of the transmembrane domain is not
critical to its retention function. It has been postulated that the
main difference between transmembrane sequences of the ER, Golgi, and
plasma membrane proteins is the length of the hydrophobic core, which
is related to the difference between these membranes' content of
cholesterol and resulting thickness of respective
bilayers(36) . According to this model, the hydrophobic
transmembrane segment of P450, which is shorter than the
membrane-spanning peptide of EGFR, would retain a reporter protein in
the ER, whereas the transmembrane segment of EGFR would permit
transport of the same reporter to the plasma membrane. Consistent with
this prediction, OCP (19) and OCE are retained in the ER, and
OEE is transported to the plasma membrane. Therefore, a transmembrane
domain derived from a plasma membrane protein (EGFR) allows the chimera
to reach the plasma membrane whereas chimera inserted into the membrane
by a P450 ER-specific signal is retained in the ER. However, contrary
to these predictions, chimeras ECO and EEO, anchored in the membrane by
C terminally located transmembrane domains, are both transported to the
plasma membrane as are EEP and ECP that have a cytoplasmic domain of
alkaline phosphatase. Thus, the localization of a protein cannot be
predicted simply from the origin and the length of a transmembrane
sequence. Clearly, other properties of such a sequence and its context
in the protein mediate the retention in a specific compartment. Redundant signals for organelle targeting and retention may be a
general phenomenon. Although the KDEL signal and retrieval mechanism
has been clearly established, it has been shown that deletion of the
KDEL signal from calreticulin does not release this protein from the ER (5) . Two independent mechanisms seem to be operating:
KDEL-based retrieval and direct retention mediated by a calcium-binding
domain. Similarly, a subunit of the N-oligosaccharyl transferase
complex, an ER membrane protein, is not released from the ER when its
C-terminal double-lysine retrieval signal is mutagenized, also pointing
to the existence in its structure of an additional, independent
retention signal(10) . The presence of two distinct retention
signals organized similarly to that of P450 2C2 has been recently shown
for a cis-Golgi network-specific protein, Sed5(37) .
This protein's targeting and retention is mediated by its
N-terminal transmembrane domain and its cytoplasmic domain, in which no
discrete signal could be mapped. This and other studies (33, 38) suggest that the organelle-specific retention
of a protein may involve the action of more than one signal.
Volume 270,
Number 41,
Issue of October 13, 1995 pp. 24327-24333
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)is the first compartment encountered by proteins destined
for localization in membranous organelles or secretion. In order to
maintain the unique compartmental composition of the organelles in this
pathway, it is important that proteins with different destinations be
efficiently sorted. It is widely accepted that proteins are transported
by a bulk flow of vesicles, and their final localization is specified
by organelle-specific retention signals (1) . Thus, ER-specific
proteins must be sequestered from proteins destined for secretion or
other organelles.
, do not contain KKXX-like
signals. Their retention also could be mediated either by constant
retrieval from the early Golgi or by primary retention, by being
excluded from the transport vesicles. Some of these proteins, including
P450, seem to be restricted to the ER and do not undergo recycling
through the early Golgi(10, 11, 12) . It is
not known whether this retention results from the exclusion of an
ER-restricted protein from the vesicle budding area or its structural
incompatibility with packaging in vesicles. It has been suggested that
some ER membrane proteins may form a network of interacting
supramolecular complexes that would prevent their incorporation into
vesicles(10, 12) .
Materials
Tran S-label was from ICN
Radiochemicals, endoglycosidase H from Boehringer Mannheim, and protein
A-Sepharose from Pharmacia (Uppsala, Sweden). Cell culture media and
antibiotics were from Life Technologies, calf serum from Sigma.
Monoclonal antibody against the external domain of human epidermal
growth factor receptor was from Upstate Biotechnology Inc. (Lake
Placid, NY), FITC-conjugated goat anti-mouse IgG was from Tago
(Burlingame, CA), polyclonal antiserum against P450 2C3 was a gift from
Dr. Eric F. Johnson (Scripps Clinic, La Jolla, CA), and polyclonal
antiserum against alkaline phosphatase was a gift from Dr. Robert
Gennis (University of Illinois, Urbana, IL).
Plasmid Constructions
Construction of plasmids
pTZC2, pTZC1, pTZEGFR, pCMVC2, pCMVEGFR, pEGFRPHOA, and pC1PHOA has
been described(11, 15, 19) . The remaining
plasmids, encoding chimeric proteins, were constructed as shown in Fig. 1. Respective DNA fragments were either obtained by
restriction digestion or PCR amplification. Chimeric proteins are
designated with three letters, which refer to the luminal
(extracellular), transmembrane, and cytoplasmic domains, respectively.
E, C, P, and O refer to EGFR, P450, alkaline phosphatase, and no
domain, respectively.
)we constructed chimera EEC`, which
includes these residues (Fig. 1). The chimera OEC also contains
residues 21-28.Expression in COS1 and CHO Cells
COS1 cells were
transfected with the expression plasmids as described
previously(11) , whereas CHO cells were transfected using the
calcium chloride coprecipitation method, as modified(22) .
Biosynthetic labeling of the transfected cells, subcellular
fractionation, immunoprecipitation, endo H treatment, and indirect
immunofluorescence were performed as
described(11, 19) . Samples were analyzed by
SDS-polyacrylamide gel electrophoresis. Enzymatic activities of the
proteins expressed in transfected COS1 cells were assayed as described
before(23) .
Analysis of the Intracellular Localization of P450/EGFR
Chimeras
In order to analyze whether the ER retention of P450 is
mediated only by its N-terminal peptide, we constructed a series of
chimeric molecules in which reciprocal structural domains of P450 and
EGFR, a plasma membrane protein, were exchanged (Fig. 2). In
some constructions, bacterial alkaline phosphatase was fused to
chimeric proteins as the cytoplasmic domain. The extracellular domain
of EGFR is heavily glycosylated, and most of the attached carbohydrate
chains acquire endo H resistance during transport to the plasma
membrane(24) . Therefore, for chimeric proteins with the
extracellular domain of EGFR, retention in the ER could be assessed by
sensitivity to endo H. Localization of chimeras devoid of the
extracellular domain of EGFR were analyzed by immunofluorescence in
whole cells and by subcellular fractionation. Chimera OEE and OEC
contain the EGFR transmembrane sequence, modified to contain an
initiating methionine, at their N terminus. This sequence efficiently
directed the chimeric proteins' insertion into the microsomal
membranes, without changing the original topology, when tested in an in vitro translation/translocation system (results not shown).
S-label for 30 min and then
chased for 4 h. During a chase of this length, all the endogenous EGFR,
which is slightly larger than the chimeric constructs, becomes
resistant to endo H (Fig. 4A). ECC, in which the
extracellular domain of EGFR is followed by a full-length P450, was
retained in the ER, as shown by its complete sensitivity to digestion
with endo H (Fig. 4A). Since ECC contains the P450
N-terminal membrane insertion sequence which functions as an ER
retention signal, this result was expected. Surprisingly, EEC, which
does not contain the P450 N-terminal sequences, also remained sensitive
to endo H digestion even after a prolonged chase time, indicating that
it was retained in the ER as well (Fig. 4A).
S-label for 30 min
and chased in complete medium for 4 h (panel A). CHO cells (panel B) were pulse-labeled for 30 min only (lanes
1, 2, 5, and 6) or additionally chased
for 4 h (lanes 3, 4, and 7-10). The
expressed proteins were immunoprecipitated with the antibody against
the extracellular domain of EGFR. This antibody cross-reacts with
endogenous EGFR of COS1 cells, indicated with a dot.
Immunoprecipitated proteins were digested with endo H for 18 h and
analyzed by SDS-polyacrylamide gel electrophoresis.
Enzymatic Activity of the Chimeric Proteins Retained in
the ER
Since ER retention can result from the misfolding of some
proteins, it was important to establish whether the cytoplasmic domain
of P450 present in the various chimeric constructions was able to
achieve its proper conformation. To test proper conformation, the
enzymatic activities of chimeric proteins were assayed. Cytochrome P450
2C2 has a lauric acid hydroxylase activity that can be easily detected
in transfected COS1 cells(23) . Since EEC, as originally made,
did not include P450 2C2 amino acids 21-28 which are important
for its activity, we subsequently included these residues
in chimera EEC` and OEC. The presence of this sequence has no effect on
the ER retention (data not shown). The chimeras ECC`, EEC`, and OEC all
have activities very similar to that of native P450 when expressed in
COS1 cells. Relative to the activity of P450 2C2 (100%), the means and
standard errors of the activities for the chimeric proteins in three
experiments were: 109 ± 2.7% for ECC`, 119 ± 8.7% for
OEC, and 111 ± 10.8% for EEC`. Immunoprecipitation of
radiolabeled proteins showed that the proteins were expressed at
similar levels (data not shown). The cytoplasmic domain of P450 in EEC`
and OEC must be properly folded and correctly inserted in the ER
membrane since it retains full enzymatic activity, thus, retention of
these chimeras in the ER is not an artifact of misfolding.Mapping the ER Retention Signal in the Cytoplasmic Domain
of P450
In order to map the localization of the potential ER
retention signal, we constructed a series of deletions in chimera EEC
in the cytoplasmic domain of cytochrome P450 which are illustrated in Fig. 5A. Mutant EECN1 with a deletion of 163 amino
acids from the N terminus of P450 2C2 remained sensitive to endo H
digestion while the larger endogenous EGFR became resistant (Fig. 5B). However, further deletion of 35 more
residues (EECN2) resulted in a protein with complete resistance to endo
H digestion, consistent with its transport out of the ER. Since this
additional 35 amino acid deletion altered localization of the chimera,
we tested whether its deletion would be sufficient for elimination of
ER retention function. As is evident from endo H sensitivity, the
chimera EECI1 was retained in the ER and, thus, deletion of only 35
internal residues, encoding amino acids 164-198, does not affect
ER retention function. We also tested whether the 35 amino acid peptide
encoding amino acids 164-198 can induce the ER retention when
fused to the truncated EGFR. This hybrid was transported from the ER
with the same kinetics as EGFR, indicating lack of ER retention
activity in the analyzed peptide (results not shown).
Fusion of the N-terminal Signal of P450 to the EGFR
Luminal Domain Suppresses the ER Retention Function
Chimeric
protein ECO was found to be resistant to endo H digestion (Fig. 3), indicating that it was not retained in the ER despite
the presence of the P450 N-terminal ER retention signal. In contrast,
other chimeric proteins, P450/
-galactosidase, P450/alkaline
phosphatase, and P450/EGFR (OCE) were retained in the ER by the P450
N-terminal signal. Since the most obvious difference between these
retained hybrids and ECO is the location of the P450-derived signal, it
raised the possibility that the ER retention signal of the P450 N
terminus functions only when located at the N terminus of the protein
and/or when it is followed by additional protein sequence.
))
We thank Dr. E. F. Johnson for P450 2C3 antibody and
Dr. R. B. Gennis for anti-alkaline phophatase antibody.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  H. K. Anandatheerthavarada, N. B. V. Sepuri, G. Biswas, and N. G. Avadhani An Unusual TOM20/TOM22 Bypass Mechanism for the Mitochondrial Targeting of Cytochrome P450 Proteins Containing N-terminal Chimeric Signals J. Biol. Chem., July 11, 2008; 283(28): 19769 - 19780. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Szczesna-Skorupa and B. Kemper BAP31 Is Involved in the Retention of Cytochrome P450 2C2 in the Endoplasmic Reticulum J. Biol. Chem., February 17, 2006; 281(7): 4142 - 4148. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Pottekat and A. K. Menon Subcellular Localization and Targeting of N-Acetylglucosaminyl Phosphatidylinositol De-N-acetylase, the Second Enzyme in the Glycosylphosphatidylinositol Biosynthetic Pathway J. Biol. Chem., April 16, 2004; 279(16): 15743 - 15751. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Szczesna-Skorupa, C.-D. Chen, H. Liu, and B. Kemper Gene Expression Changes Associated with the Endoplasmic Reticulum Stress Response Induced by Microsomal Cytochrome P450 Overproduction J. Biol. Chem., April 2, 2004; 279(14): 13953 - 13961. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Szczesna-Skorupa, B. Mallah, and B. Kemper Fluorescence Resonance Energy Transfer Analysis of Cytochromes P450 2C2 and 2E1 Molecular Interactions in Living Cells J. Biol. Chem., August 15, 2003; 278(33): 31269 - 31276. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Petit, A. Lescure, M. Rederstorff, A. Krol, B. Moghadaszadeh, U. M. Wewer, and P. Guicheney Selenoprotein N: an endoplasmic reticulum glycoprotein with an early developmental expression pattern Hum. Mol. Genet., May 1, 2003; 12(9): 1045 - 1053. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Lee, B. Park, and K. Ahn Determinant for Endoplasmic Reticulum Retention in the Luminal Domain of the Human Cytomegalovirus US3 Glycoprotein J. Virol., February 1, 2003; 77(3): 2147 - 2156. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Charloteaux, L. Lins, H. Moereels, and R. Brasseur Analysis of the C-Terminal Membrane Anchor Domains of Hepatitis C Virus Glycoproteins E1 and E2: toward a Topological Model J. Virol., February 15, 2002; 76(4): 1944 - 1958. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Kida, M. Sakaguchi, M. Fukuda, K. Mikoshiba, and K. Mihara Membrane Topogenesis of a Type I Signal-Anchor Protein, Mouse Synaptotagmin II, on the Endoplasmic Reticulum J. Cell Biol., August 21, 2000; 150(4): 719 - 730. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Greenfield and S High The Sec61 complex is located in both the ER and the ER-Golgi intermediate compartment J. Cell Sci., January 5, 1999; 112(10): 1477 - 1486. [Abstract] [PDF]
|
|
|
|
 |
|
 M. Shenkman, M. Ayalon, and G. Z. Lederkremer Endoplasmic reticulum quality control of asialoglycoprotein receptor H2a involves a determinant for retention and not retrieval PNAS, October 14, 1997; 94(21): 11363 - 11368. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-D. Chen, B. Doray, and B. Kemper Efficient Assembly of Functional Cytochrome P450 2C2 Requires a Spacer Sequence between the N-terminal Signal Anchor and Catalytic Domains J. Biol. Chem., September 5, 1997; 272(36): 22891 - 22897. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Masaki, A. Yamamoto, and Y. Tashiro Membrane Topology and Retention of Microsomal Aldehyde Dehydrogenase in the Endoplasmic Reticulum J. Biol. Chem., July 12, 1996; 271(28): 16939 - 16944. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Naray-Fejes-Toth and G. Fejes-Toth Subcellular Localization of the Type 211beta -Hydroxysteroid Dehydrogenase. A GREEN FLUORESCENT PROTEIN STUDY J. Biol. Chem., June 28, 1996; 271(26): 15436 - 15442. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Szczesna-Skorupa and B. Kemper Endoplasmic Reticulum Retention Determinants in the Transmembrane and Linker Domains of Cytochrome P450 2C1 J. Biol. Chem., June 16, 2000; 275(25): 19409 - 19415. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Szczesna-Skorupa and B. Kemper The Juxtamembrane Sequence of Cytochrome P-450 2C1 Contains an Endoplasmic Reticulum Retention Signal J. Biol. Chem., November 21, 2001; 276(48): 45009 - 45014. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Mottola, N. Jourdan, G. Castaldo, N. Malagolini, A. Lahm, F. Serafini-Cessi, G. Migliaccio, and S. Bonatti A New Determinant of Endoplasmic Reticulum Localization Is Contained in the Juxtamembrane Region of the Ectodomain of Hepatitis C Virus Glycoprotein E1 J. Biol. Chem., July 28, 2000; 275(31): 24070 - 24079. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
